Presence of G84E Allelic Heterogeneity of the European Prostate Cancer SNP Mutation of HOX13B-G84E Associated with the European R-Haplogroup of Y-Chromosome and Absence of Gene Flow into Moroccans Patients

SNP mutations in the HOXB13 gene associated with prostate cancer were determined in Moroccans prostate cancer patients (PCa). All PCa SNP mutations were new and belong to the SNP point-mutations located on the stop codon of HOXB13 exon 1 and 2 located in chromosome 17. The five mutations and their frequencies were as follows: rs1197613952 (12%), rs1597934612 (4%), rs1597933874 (4%), rs1597933837 (4%) and rs867793282 (4%). The European HOXB13-G84E (rs138213197) PCa mutation was not detected among Moroccan patients. The Y-chromosome genealogical haplotypes of the Western European (R1b1b2-M2G9) and the Eastern European (R191a-M-17) were not observed in Moroccans PCa patients. The patients have their own haplotypes E1b1 and J with a frequency of 55 and 35%, respectively. The results of the SNP mutations in the HOXB13, the absence of the HOXB13-G84E of the European in the Moroccans PCa patients, the absence of the Euro-pean-lineage haplogroups (R1a1a-M17 and R1b1b2-M269) and the presence of E1b1b and J in Moroccans PCa patients would clearly indicate the absence of gene flow from European to Moroccans gene pool.


Introduction
Recently, there has been a growing global research interest on profiling of the prostate cancer HOXB13 mutations, which is population-based and associated with the males' genealogy in different population [1]- [9]. Prostate cancer is the second most prevalent cancer in males, and the sixth cause of cancer-related deaths since it was first reported, and before the outset of the prevalence of the HOXB13-G84E mutation [10]. Furthermore, the germline HOXB13-G84E mutation has consistently been associated with prostate cancer (PCa) risk among European pedigree [7] [10] [11] [12] [13] [14]. More recently, in a wide-range population study that included results from the UK Biobank, it was documented that the G84E mutation was found in 1545 (0.34%) subjects out of 460,224 participants of European pedigree [15]. On the contrary, further evidence to buttress and substantiate this assertion showed that the G84E mutation was not found among prostate cancer males of both African and Asian descent [12] [16].
For several years, our research outcomes on the Y-chromosome haplogroups of Arabs genealogical-lineages as determined by mutations of the Short Tandem Repeats (STRs) of the non-recombined region of the Y-chromosome showed that the epidemiology of cancers correlates with the genealogy and ethnicity of the population clusters [9] [17] [18] [19] [20] [21]. This has prompted the scientific question on whether the incidence of such ancestral-based mutation of prostate cancer in males is associated with or can be predicted by the gene flow in males' haplogroups and their genealogical-lineages vis-à-vis profiling of the Y-chromosome among different population. Consequently, we hypothesized the presence of allelic heterogeneity in prostate cancer mutation among Moroccan prostate cancer patients and European males. Our objectives were to determine and profile the gene flow of HOX13B-G84E and the Y-chromosome haplogroups (R1b1b2-M269 and R1a1a-M17) from European to Moroccan males taking into cognizance the fact that both populations are located on the very busy crossing route of human historical migration between the two ancient continents of Europe and Africa. Morocco was our preferred choice for this study, because it is located in the northwestern part of Africa, and has the closest proximity (with just 14 km away from Spain across the Gibraltar strait and the Atlantic Ocean to the west) for any possible reciprocal human migration to and from Europe. Additionally, Morocco represents a unique population cluster for testing our hypothesis, because it presented a vehicle to determine if there was a gene flow profile of the HOXB13-G84E mutation of prostate cancer associated with the European genealogy gene flow (if any) of the R1b1b2-M269 and R1a1a-M17 Y-chromosome haplogroups from Europe into Morocco.

Genotyping of the Single Nucleotide Polymorphism (SNP) and PCR of HOXB13 Gene Cluster on Chromosome 17q21-22
A total of 50 fresh prostate biopsies were collected from 50 men undergoing

YHRD GeneBank Quality Assurance of Genotyping and Data Deposition
In accordance with the request of the Y-STR Haplotype Reference Database

Results
The profile of the HOXB13 mutations and the coding sequence (CDS) changes among PCa Moroccan patients is presented in Table 1     ranged between (0.8% -2.7%) and these were: 1.5 %, 1.8%, 2.7%, 0.6%, 2.6% and 0.8% for the haplogroups C, G, I, L, T, and Q respectively.
The prevalence of the SNP mutations among Moroccan PCa patients, which ranged from a minimum of (4%) to a maximum of (12%) was higher than that Western Europe and North America, respectively, as well as [11] and in Asian pastoral Nomadic population [25]. More recently, in a wide-ranging population sampling that included results from the UK Biobank, it was documented that the G84E mutation was found in 1545 (0.34%) subjects out of 460,224 participants of European descent [15]. The observed high rate of frequency among Moroccan PCa patients was due to the high rate of consanguinity (40%) among Moroccan population particularly the Bedouins [22]. Over centuries, this has led to more pathogenicity of these PCa mutations from one generation to another. The most prevalent mutation was the change in SNP of (C > A), which could be employed for prediction of PCa SNP mutations among Moroccan patients, and as a baseline study for any gene therapy innovative research in the future. This obviously will lead to better treatment and management outcomes worldwide.
The less frequent haplogroups C, G, I, L, T, and Q with frequency of 1.5 %, 1.8%, 2.7%, 0.6%, 2.6% and 0.8%, respectively, were not included in the study design because we already knew that are minimal as reported by several thesis of our MSc students and they are irrelevant to both European as well as Moroccans genealogical lineages which we tested.

Conclusion
This study provides a new approach in deciphering the association between PCa These results have potential clinical applications on PCa profiling among different population ancestry, which could be inferred and predicted using its association with different genealogical lineages of the Y-chromosome haplogroups.

Data Deposition and Availability
All sequences of the determined mutation of the HOXB13 were deposited in NCBI GeneBank in accordance with each respective accession number, and have been made available on the public domain of the NCBI (http://www.ncbi.nlm.nih.gov/).
Our results of the Y-chromosome were registered and deposited in the International Y-chromosome Haplotype Reference Database (YHRD) under the Accession Numbers YA003522 and YA003523 (http://www.yhrd.org/).