Rapidly mutating Y-STRs in rapidly expanding populations: Discrimination power of the Yfiler Plus multiplex in northern Africa

The male-specific northern African genetic pool is characterised by a high frequency of the E-M81 haplogroup, which expanded in very recent times (2-3 kiloyears ago). As a consequence of their recent coalescence, E-M81 chromosomes often cannot be completely distinguished on the basis of their Y-STR profiles, unless rapidly-mutating Y-STRs (RM Y-STRs) are analysed. In this study, we used the Yfiler® Plus kit, which includes 7 RM Y-STRs and 20 standard Y-STR, to analyse 477 unrelated males coming from 11 northern African populations sampled from Morocco, Algeria, Libya and Egypt. The Y chromosomes were assigned to monophyletic lineages after the analysis of 72 stable biallelic polymorphisms and, as expected, we found a high proportion of E-M81 subjects (about 46%), with frequencies decreasing from west to east. We found low intra-population diversity indexes, in particular in the populations that experienced long-term isolation. The AMOVA analysis showed significant differences between the countries and between most of the 11 populations, with a rough differentiation between northwestern Africa and northeastern Africa, where the Egyptians Berbers from Siwa represented an outlier population. The comparison between the Yfiler® and the Yfiler® Plus network of the E-M81 Y chromosomes confirmed the high power of discrimination of the latter kit, thanks to higher variability of the RM Y-STRs: indeed, the number of chromosomes sharing the same haplotype was drastically reduced from 201 to 81 and limited, in the latter case, to subjects from the same population

Identification and molecular characterisation of an AMEL-X null allele due to an Alu insertion

The amelogenin locus is used in most forensic STR multiplex kits as sex-typing marker. In the present work, we performed the molecular characterisation of an AMEL-X null allele, observed in an Eritrean male profile when using either the PowerPlex® Fusion 6C or the AmpFLSTR™ NGM SElect™ multiplexes. After X-specific PCR amplification of the amelogenin locus using an in-house primer pair and Sanger sequencing, we found that the AMEL-X amplification failure was due to an insertion of a 370 bp Alu element. We recognised all the hallmarks of the Alu elements, i.e. the target specific duplications (TSDs) at the boundaries of the insertion, the 3’ A-tail and the LINE1 endonuclease cleavage site, and we used these elements to precisely identify the insertion point of the retro-transposon. We found that the Alu insertion did not disrupt the primer binding sites of the AMEL-X neither in the PowerPlex® Fusion 6C nor in the AmpFLSTR™ NGM SElect™ system. Instead, the amplification failure of the X-specific amelogenin marker could be explained by the competition of the Y-specific amelogenin locus for the same primer pair, which lead to the sub-optimal amplification of the longer AMEL-X. The analysis of 145 additional eastern African males did not reveal other AMEL-X null alleles. To our knowledge, this is the first reported case of an Alu insertion causing an AMEL-X dropout, which is usually due to point mutations at the primer binding sites

IDENTIFICATION AND MOLECULAR CHARACTERISATION OF AN AMEL-X NULL ALLELE DUE TO AN ALU INSERTION.

The amelogenin locus is used in most forensic STR multiplex kits as sex-typing marker. In the present work, we performed the molecular characterisation of an AMEL-X null allele, observed in a male sample from Eritrea. The STR profile of this subject was obtained using both the AmpFLSTR™ NGM SElect™ and the PowerPlex® Fusion 6C, obtaining the same result at the amelogenin locus. After X-specific PCR amplification of the amelogenin locus using an in-house primer pair and Sanger sequencing of the AMEL-X null sample, we found that the AMEL-X amplification failure was due to the insertion of an Alu element of 370 bp. We recognised all the hallmarks of the Alu elements, i.e. the TSDs at the boundaries of the inserted element, the 3’ A-tail and the LINE1 endonuclease cleavage site, and we used these elements to precisely identify the insertion point of the Alu. Using this approach, we found that the Alu insertion did not disrupt the primer binding sites of the AMEL-X neither in the AmpFLSTR™ NGM SElect™ nor in the PowerPlex® Fusion 6C system. Instead, the amplification failure of the X-specific amelogenin marker could be explained by the competition of the Yspecific amelogenin locus for the same primers and, consequently, by the sub-optimal amplification of the longer AMEL-X. The insertion allele was not observed in additional 145 eastern African males, 88 genotyped with the PowerPlex® Fusion 6C and 57 analysed by AMEL-X specific PCR assay. To our knowledge, this is the first reported case of an Alu insertion causing an AMEL-X dropout, which is usually consequence of point mutations at the primer binding sites

TOWARDS A FULLY IMPLANTABLE AUTONOMOUS ARTIFICIAL PANCREAS

Abstracts from the 44th ESAO and 7th IFAO Congress, 6-9 September 2017, Vienna, Austria

Forensic data and microvariant sequence characterization of 27 Y-STR loci analyzed in four Eastern African countries

By using the recently introduced 6-dye Yfiler® Plus multiplex, we analyzed 462 males belonging to 20 ethnic groups from four eastern African countries (Eritrea, Ethiopia, Djibouti and Kenya). Through a Y-STR sequence analysis, combined with 62 SNP-based haplogroup information, we were able to classify observed microvariant alleles at four Y-STR loci as either monophyletic (DYF387S1 and DYS458) or recurrent (DYS449 and DYS627). We found evidence of non-allelic gene conversion among paralogous STRs of the two-copy locus DYF387S1. Twenty-two diallelic and triallelic patterns observed at 13 different loci were found to be significantly over-represented (p<10-6) among profiles obtained from cell lines compared to those from blood and saliva. Most of the diallelic/triallelic patterns from cell lines involved recurrent mutations at rapidly mutating loci (RM Y-STRs) included in the multiplex (p<10-2). At haplotype level, intra-population diversity indices were found to be among the lowest so far reported for the Yfiler® Plus, while statistically significant differences among countries and ethnic groups were detected when considering haplotype frequencies alone (FST) or by using molecular distances among haplotypes (ΦST). The strong population subdivision observed is probably the consequence of the patrilineal social organization of most eastern African ethnic groups, and suggests caution in the use of country-based haplotype frequency distributions for forensic inferences in this region

Rapidly mutating Y-STRs in rapidly expanding populations:discrimination power of the Yfiler Plus multiplex in northern Africa.

The male-specific northern African genetic pool is characterised by a high frequency of the E-M81 haplogroup, which expanded in very recent times (2-3 kiloyears ago). As a consequence of their recent coalescence, E-M81 chromosomes often cannot be completely distinguished on the basis of their Y-STR profiles, unless rapidly-mutating YSTRs (RM Y-STRs) are analysed. In this study, we used the Yfiler® Plus kit, which includes 7 RM Y-STRs, to analyse 477 unrelated males coming from 11 northern African populations sampled from Morocco, Algeria, Libya and Egypt. The Y chromosomes were assigned to monophyletic lineages after the analysis of 72 stable biallelic polymorphisms and, as expected, we found a high proportion of E-M81 subjects (about 46%), with frequencies decreasing from west to east. We found low intrapopulation diversity indexes, in particular in the populations that experienced long-term isolation. The AMOVA analysis showed significant differences between the countries and between most of the 11 populations, with a rough differentiation between northwestern Africa and northeastern Africa, where the Egyptians Berbers from Siwa represented an outlier population. The comparison between the Yfiler® and the Yfiler® Plus network of the EM81 Y chromosomes confirmed the high power of discrimination of the latter kit, thanks to higher variability of the RM Y-STRs: indeed, the number of chromosomes sharing the same haplotype was drastically reduced from 201 to 81 and limited, in the latter case, to subjects from the same population

The relative contribution of the decreasing tumour thickness trend to the 2010s increase in net survival from cutaneous malignant melanoma in Italy: a population-based investigation

Background: We determined the relative contribution of decreased tumour thickness to the favourable trend in survival from cutaneous malignant melanoma (CMM) in Italy. Methods: Eleven local cancer registries covering a population of 8 056 608 (13.4% of the Italian population in 2010) provided the records of primary CMM cases registered between 2003 and 2017. Age standardized 5-year net survival (NS) was calculated. Multivariate analysis of 5-year NS was done by calculating the relative excess risk of death (RER). The relative contribution of the decrease in tumour thickness to the RER was evaluated using a forward stepwise flexible parametric survival model including the available prognostic factors. Results: Over the study period, tumour thickness was inversely associated with 5-year NS and multivariate RER in both sexes. The median thickness was 0.90 mm in 2003-2007, 0.85 mm in 2008-2012 and 0.75 mm in 2013-2017 among men, and 0.78 mm, 0.77 mm and 0.68 mm among women, respectively. The 5-year NS was 86.8%, 89.2% and 93.2% (men), and 91.4%, 92.0%, and 93.4% (women). In 2013-2017, thus, men exhibited the same survival as women despite still having thicker lesions. For them, the increasing survival trend was more pronounced with increasing thickness, and the inclusion of thickness into the forward stepwise model made the RER in 2013-2017 versus 2003-2007 to increase from 0.64 (95% confidence interval, 0.51-0.80) to 0.70 (0.57-0.86). This indicates that thickness trend accounted for less than 20% of the survival increase. For women, the results were not significant but, with multiple imputation of missing thickness values, the RER rose from 0.74 (0.58-0.93) to 0.82 (0.66-1.02). Conclusions: Especially for men, the decrease in tumour thickness accounted for a lesser part of the improvement in survival observed in 2013-2017. The introduction of targeted therapies and immune checkpoint inhibitors in 2013 is most likely to account for the remaining component

Adolescent and Young Adult Cancer Survivors: Design and Characteristics of the First Nationwide Population-Based Cohort in Italy

Purpose: Adolescent and young adult (AYA, 15-39 years) cancer survivors (alive at least 5 years after cancer diagnosis) are less studied than younger and older cancer survivors and research on their late effects is limited. To facilitate research on long-term outcomes of AYA cancer survivors, we established, in Italy, a population-based AYA cancer survivors' cohort. This article describes the study design and main characteristics of this cohort.Methods: The cohort derives from population-based cancer registries (CRs). Each CR identified AYA cancer patients retrospectively. Treatment for first primary cancer and all health events from diagnosis to death can be traced through linkage with available health databases, such as hospital discharge records (HDRs), mortality files, and outpatient and pharmaceutical databases.Results: Thirty-four CRs participated to the cohort which overall includes 93,291 AYAs with cancer and 67,692 cancer survivors. First primary cancer distribution in AYA cancer survivors differs by sex and age groups because of the different cancer types diagnosed in AYAs. Almost 78% of AYA cancer survivors have HDRs and 14.8% also pharmaceutical and outpatient databases.Conclusion: This cohort will be used to study, for the first time in Italy, the pattern and excess risk of late effects in AYA cancer survivors. HDRs, outpatient and pharmaceutical databases will be used to define primary treatment to assess its impact on AYA cancer survivors' late effects. This cohort exploiting data sources already available at CRs, minimize the data collection effort and it will contribute to assess the feasibility of using administrative database to study cancer survivors' late effects

The descriptive epidemiology of melanoma in Italy has changed - for the better

: A recent research project using data from a total of 40 cancer registries has provided new epidemiologic insights into the results of efforts for melanoma control in Italy between the 1990s and the last decade. In this article, the authors present a summary and a commentary of their findings. Incidence increased significantly throughout the study period in both sexes. However, the rates showed a stabilization or a decrease in men and women aged below 35 years. The risk of disease increased for successive cohorts born until 1973 (women) and 1975 (men) while subsequently tending to decline. The trend towards decreasing tumor thickness and increasing survival has continued, but a novel favorable prognostic factor has emerged since 2013 for patients - particularly for males - with thick melanoma, most likely represented by molecular targeted therapies and immune checkpoint inhibitors. Due to this, the survival gap between males and females has been filled out. In the meanwhile, and despite the incidence increase, dermatologists have not lowered their threshold to perform skin biopsy. Skin biopsy rate has increased because of the increasingly greater volume of dermatologic office visits, but the proportion of skin biopsies out of dermatologic office visits has remained constant. In summary, an important breakthrough in melanoma control in Italy has taken place. Effective interventions have been implemented across the full scope of care, which involve many large local populations - virtually the whole national population. The strategies adopted during the last three decades represent a valuable basis for further steps ahead in melanoma control in Italy

Prescriptive adherence to GINA guidelines and asthma control: An Italian cross sectional study in general practice

Background: Although general practitioners (GPs) are frequently the first healthcare professionals whom asthma patients refer to for their symptoms, few studies have explored the extent of adherence to guidelines for asthma management based on data provided directly by GPs. Aims of the present study were to assess drug prescriptions for asthma by GPs and to evaluate prescriptive adherence to GINA guidelines (GL) and its relationship with disease control in real life. Methods: 995 asthmatic patients (45% males, mean age 43.3 ± 17.7 yrs) were enrolled by 107 Italian GPs distributed throughout the country. Data on diagnosis, disease severity, prescribed anti-asthmatic drugs and control were collected through questionnaires filled out by GPs taking into consideration the 2009 GINA Guidelines. Data on drug use and chronic sinusitis, nasal polyposis, chronic bronchitis, emphysema were reported by patients through a self-administered questionnaire. Results: The large majority of patients were classified by GPs as having intermittent (48.4%) or mild persistent asthma (25.3%); 61% had co-morbid allergic rhinitis (AR). The prevalent therapeutic regimen used by patients was a combination of inhaled corticosteroids (ICS) plus long-acting β2-agonists (LABA) (54.1%), even in the intermittent/mild persistent group. ICS as mono-therapy or in combination with other drugs but LABA, was the second most frequently adopted treatment (14.4%). In general, the GPs adherence to GL treatment indications was 28.8%, with a significant association with a good asthma control (OR 1.85, 95% CI 1.18–2.92). On the other hand, comorbidity (OR 0.52, 95% CI 0.32–0.84), moderate (0.44, 0.28–0.69) and severe (0.06, 0.02–0.20) persistent asthma showed significant negative effects on asthma control. Conclusions: Our results show that over-treatment of intermittent/mild persistent asthma is frequent in the GPs setting while therapeutic regimens are more appropriately applied for moderate/severe asthma. In general, we found low adherence to GINA GL treatment recommendations even if its relevance in asthma control was confirmed