Scrutinizing the Profile and Risk Factors of Suicide: A Perspective from a Case-Control Study Focused on a Northern Region of Spain.

Suicide is a major public health problem the prevention of which has become a priority, and, to this end, knowledge of its risk factors is essential. This study aims to evaluate the impact of some social, medico-legal, and clinical issues on suicide deaths. A total of 135 cases were identified as suicides that occurred in a region of northern Spain between 2018 and 2020. Controls (three for each case) were matched by age, sex, and urban-rural areas. The information was collected retrospectively through electronic health record systems. A binary logistic regression analysis was performed to study the association between individual risk factors and suicide. Being male (78.5%), between 40 and 60 years of age, unmarried (70.9%), and unemployed (85%) were associated with suicide deaths. Although the existence of a previous self-harm attempt is presented as the most robust risk factor (OR 22.121 [8.997-54.389]), the presence of a psychiatric diagnosis (OR 12.583 [7.686-20.601]) and cancer (OR 3.729 [1.845-7.536]) also showed a significant relationship with suicide (p < 0.05). Defining and knowing the risk factors for suicide helps to better understand the profiles of those individuals who are vulnerable, and enables prevention actions to be taken in both social and medical spheres.

Forensic implications of the presence of chimerism after hematopoietic stem cell transplantation.

Biological vestiges are used in forensic science to resolve a large number of cases by typing the genetic profile and identifying the individual to whom it belongs. However, chimeric persons that possess cells with two or more different DNA make these types of analyses difficult. This situation can occur naturally, by errors in the fertilization or early embryogenesis, or in an artificial way, for example after hematopoietic stem cell transplantation (HSCT), when host and donor cells coexist in the patient. In this paper, we will specially focus on the latter. The vestiges from transplant patients represent a challenge from a forensic perspective since the interpretation of the genetic fingerprint can be misleading because of the presence of chimerism. Due to the high number of transplant patients (and their increase over the years) and the existence of natural chimeras (probably many of them hidden), it is necessary to consider whether we are facing a possible chimeric person or someone who has been a donor of hematopoietic stem cells in a forensic context. In this review, the presence of donor bone marrow derived cells in some tissues of forensic interest will be discussed. Finally, to emphasize the importance of chimerism after HSCT in forensic genetics, some real-life cases will be examined.

The genetic profile of bone marrow transplant patients in different samples of forensic interest.

Humans constantly lose epithelial cells, and these biological traces are frequently studied in the context of criminal investigations. The objective of this work was to examine the genetic profile in samples of forensic interest (nail and skin epithelial cells) of bone marrow transplant patients and discuss its forensic and clinical implications. The genetic profile of nail, epidermal cells and blood samples of patients receiving HSCT was analyzed by the amplification and sequencing of 38 insertion/deletion polymorphisms and 15 short tandem repeat polymorphisms. In this analysis, the age of patients and donors, the time elapsed from the transplant, the type of conditioning prior to the transplant and whether the patient suffered graft-versus-host disease were considered. Donor chimerism can be detected in the DNA extracted from nail and skin epithelial cells of transplant patients. No statistically significant correlation was found between the type of conditioning and the percentage of donor DNA in nail (p > 0.05). A positive correlation, without statistical significance, was encountered when we analyzed the relationship between the time elapsed from the transplant with the percent donor chimerism found in epithelial cells of the epidermis and in nails. We conclude that within a judicial context (e.g. when testifying as an expert witness) it is necessary to consider whether we are facing a possible transplant patient or a person who has been a bone marrow donor.

Genetic DNA profile in urine and hair follicles from patients who have undergone allogeneic hematopoietic stem cell transplantation.

Biological samples from patients who have undergone allogeneic hematopoietic stem cell transplantation (HSCT) constitute a challenge for individual identification. In this study we analyzed the genetic profiles (by the amplification of 15 autosomic STRs) of HSCT patients found in different types of samples (blood, hair and urine) that may be the source of DNA in civil or criminal forensic cases. Our results show that while in hair follicles the donor component was not detected in any patient, thus being a reliable source of biological material for forensic identification, mixed chimerism was detected in urine samples from all patient, and no correlation was found between the time elapsed from the transplant and the percentage of chimerism. These results certainly have practical implications if the urine is being considered as a source of DNA for identification purposes in HSTC patients. Moreover, taking into consideration that chimerism was found not only in patients with leukocyturia (given the hematopoietic origin of leukocytes, this was expected), but also in those without observable leukocytes in the sediment, we conclude that an alternative source or sources of donor DNA must be implicated.

Indel analysis by droplet digital PCR: a sensitive method for DNA mixture detection and chimerism analysis.

Several methods have been developed to determinate genetic profiles from a mixed samples and chimerism analysis in transplanted patients. The aim of this study was to explore the effectiveness of using the droplet digital PCR (ddPCR) for mixed chimerism detection (a mixture of genetic profiles resulting after allogeneic hematopoietic stem cell transplantation (HSCT)). We analyzed 25 DNA samples from patients who had undergone HSCT and compared the performance of ddPCR and two established methods for chimerism detection, based upon the Indel and STRs analysis, respectively. Additionally, eight artificial mixture DNA samples were created to evaluate the sensibility of ddPCR. Our results show that the chimerism percentages estimated by the analysis of a single Indel using ddPCR were very similar to those calculated by the amplification of 15 STRs (r 2 = 0.970) and with the results obtained by the amplification of 38 Indels (r 2 = 0.975). Moreover, the amplification of a single Indel by ddPCR was sensitive enough to detect a minor DNA contributor comprising down to 0.5 % of the sample. We conclude that ddPCR can be a powerful tool for the determination of a genetic profile of forensic mixtures and clinical chimerism analysis when traditional techniques are not sensitive enough.

Osterix and RUNX2 are Transcriptional Regulators of Sclerostin in Human Bone.

Sclerostin, encoded by the SOST gene, works as an inhibitor of the Wnt pathway and therefore is an important regulator of bone homeostasis. Due to its potent action as an inhibitor of bone formation, blocking sclerostin activity is the purpose of recently developed anti-osteoporotic treatments. Two bone-specific transcription factors, RUNX2 and OSX, have been shown to interact and co-ordinately regulate the expression of bone-specific genes. Although it has been recently shown that sclerostin is targeted by OSX in mice, there is currently no information of whether this is also the case in human cells. We have identified SP-protein family and AML1 consensus binding sequences at the human SOST promoter and have shown that OSX, together with RUNX2, binds to a specific region close to the transcription start site. Furthermore, we show that OSX and RUNX2 activate SOST expression in a co-ordinated manner in vitro and that SOST expression levels show a significant positive correlation with OSX/RUNX2 expression levels in human bone. We also confirmed previous results showing an association of several SOST/RUNX2 polymorphisms with bone mineral density.

A Sclerostin super-producer cell line derived from the human cell line SaOS-2: a new tool for the study of the molecular mechanisms driving Sclerostin expression.

Sclerostin, the product of the SOST gene, is a key regulator of bone homeostasis. Sclerostin interferes with the Wnt signalling pathway and, therefore, has a negative effect on bone formation. Although the importance of sclerostin in bone homeostasis is well established, many aspects of its biology are still unknown. Due to its restricted pattern of expression, in vitro studies of SOST gene regulation are technically challenging. Furthermore, a more profound investigation of the molecular mechanism controlling sclerostin expression has been hampered by the lack of a good human in vitro model. Here, we describe two cell lines derived from the human osteosarcoma cell line SaOS-2 that produce elevated levels of sclerostin. Analysis of the super-producer cell lines showed that sclerostin levels were still reduced in response to parathyroid hormone treatment or in response to mechanical loading, indicating that these regulatory mechanisms were not affected in the presented cell lines. In addition, we did not find differences between the promoter or ECR5 sequences of our clones and the SaOS-2 parental line. However, the methylation of the proximal CpG island located at the SOST promoter was lower in the super-producer clones, in agreement with a higher level of SOST transcription. Although the underlying biological causes of the elevated levels of sclerostin production in this cell line are not yet clear, we believe that it could be an extremely useful tool to study the molecular mechanisms driving sclerostin expression in humans.

Haplotypes of intron 4 of the estrogen receptor alpha gene and hip fractures: a replication study in Caucasians.

BACKGROUND: Despite their great impact, few genetic association studies have used hip fractures as an endpoint. However, the association of two polymorphisms on intron 4 of estrogen receptor alpha (ESR1) with hip fractures was recently reported in a Chinese population. The aim of this study was to investigate whether such association is also present in Caucasians. METHODS: We analyzed those two SNPs and another neighbour SNP located on the exon 4 of ESR1 in 787 patients with hip fractures and 953 controls from Spain. RESULTS: The allelic frequencies differed markedly from those reported in Asian populations. Nevertheless, haplotypes including the rs3020314 and rs1884051 loci in intron 4 showed a significant association with hip fractures (omnibus test p = 0.006 in the whole group and 0.00005 in women). In the sex-stratified analysis, the association was significant in females, but not in males. In women, the CA haplotype appeared to have a protective influence, being present in 6.5% of the controls, but only in 3% of patients with fractures (odds ratio 0.39; 95% confidence interval 0.26-0.59; estimated population preventive fraction 3.5%). The inclusion of the rs1801132 SNP of exon 4 further increased the statistical significance of the association (odds ratio 0.17; 95% CI 0.08-0.37; p = 0.00001). Each SNP appeared to contribute independently to the association. No genotype-related differences in gene expression were found in 42 femoral bone samples. CONCLUSIONS: This study confirms the association of some polymorphisms in the region of exon 4/intron 4 of ESR1 and hip fractures in women. However, there are marked differences in allele frequencies between Asian and Caucasian populations.

Adiposity, estradiol, and genetic variants of steroid-metabolizing enzymes as determinants of bone mineral density.

OBJECTIVES: Bone mineral density (BMD) is a complex trait resulting from the interplay of genetic and acquired factors. The objective of this study was to explore the influence of several anthropometric, lifestyle, genetic, and hormonal factors on BMD and analyze the possible differences in men and women. METHODS: We studied 572 individuals over 50 years of age (381 postmenopausal women and 191 men). Lumbar spine and femoral neck BMD were measured by dual energy x-ray absorptiometry. The free estrogen index (FEI) was calculated as the ratio of serum estradiol to sex hormone binding globulin in 241 individuals. Three polymorphisms in the genes coding for 17-hydroxylase/liase, sulfotransferase, and 5alpha-reductase were studied in DNA isolated from blood cells. RESULTS: Body mass index was strongly correlated to spine and femoral BMD both in women and in men (r = 0.32-0.49; P < 0.001). FEI was also independently correlated with spine BMD in both sexes (r = 0.23 and 0.34, P < 0.01), and with femoral neck in women (r = 0.30). Women with G alleles of the sulfotransferase gene tended to have higher spine BMD than those with C alleles (P = 0.025). No other genotype-related differences in BMD were found. CONCLUSIONS: In conclusion, the results of this study point toward body weight and estradiol levels as major factors determining BMD both in women and in men. A common polymorphism of the sulfotransferase gene also appears to be associated to spine BMD in women.

Identification of an aromatase haplotype that is associated with gene expression and postmenopausal osteoporosis.

CONTEXT: Osteoporosis has a significant genetic component. The aromatase-dependent conversion of androgenic precursors is the main source of estrogens in postmenopausal women. OBJECTIVE: The objective of the investigation was to study the relationship of a set of single nucleotide polymorphisms (SNPs) of the aromatase gene with osteoporosis and determine their functional influence on gene transcription. DESIGN, PARTICIPANTS, AND METHODS: This was a case-control study including 135 women with vertebral fractures due to postmenopausal osteoporosis and 312 controls. Alleles at four SNPs situated between exons I.2 and 3 were determined by Taqman assays. Total aromatase RNA and differential allelic-specific expression were studied by RT-real time PCR in adipose tissue samples taken from 50 individuals. RESULTS: The SNPs studied were in strong linkage disequilibrium. A common haplotype, present in about half of the population, was identified as being associated with an increased risk of fractures (odds ratio 1.8, 95% confidence interval 1.2-2.8, P = 0.006). There was evidence of differential allelic expression. In heterozygous individuals, transcripts bearing T alleles at rs700518 SNP (which were included in the risk haplotype) were less abundant than those with the alternative C alleles (P < 0.001). Total aromatase expression was four times lower in fat samples from individuals who were homozygotes for the unfavorable alleles than in the opposite homozygotes (P = 0.007). CONCLUSIONS: A common haplotype of aromatase associated with gene expression is also associated with the risk of osteoporotic vertebral fractures in postmenopausal women. These data are in line with the hypothesis that the aromatase-dependent synthesis of estrogens plays an important role in bone homeostasis in postmenopausal women.

A gene-to-gene interaction between aromatase and estrogen receptors influences bone mineral density.

OBJECTIVE: The aromatization of androgenic precursors is the main source of estrogens in postmenopausal women. We tested the hypothesis that allelic variants of the genes coding for aromatase and estrogen receptors (ER) could interact to determine the estrogenic signals on the bone tissue and, consequently, bone mineral density (BMD). DESIGN: Cross-sectional study including 331 postmenopausal women. METHODS: BMD was measured by dual energy x-ray absorptiometry. A CG polymorphism of the aromatase gene as well as three polymorphisms of ERalpha (a TA repeat in the promoter region, a C T single nucleotide polymorphism (SNP) in intron 1 and an AG SNP in exon 8) and a CA repeat polymorphism of ERbeta were studied. RESULTS: Age, body weight and the aromatase genotype were associated with BMD. Allelic variants of ERbeta and the exon 8 of ERalpha did not show a significant association with BMD. The polymorphisms located on the promoter and intron 1 of ERalpha interacted strongly with aromatase. Thus, in women TT homozygous for the ERalpha gene, there was a marked influence of aromatase genotypes on BMD: spine BMD was 0.724 +/- 0.027 g/cm2 in women with CC aromatase alleles and 0.926 +/- 0.032 g/cm2 in those with GG alleles (P < 0.001). Hip BMD in women with CC and GG aromatase genotypes was 0.722 +/- 0.020 and 0.842 +/- 0.026 g/cm2 respectively (P = 0.002). On the contrary, there were no aromatase-related differences in BMD in women with CT/CC alleles of ERalpha. Similarly, aromatase-related differences in BMD were found in women with short alleles at the promoter region of ERalpha, but not in those with long alleles. Both ERalpha polymorphisms were in strong linkage disequilibrium (P < 0.001). CONCLUSION: These results suggest that the interaction between polymorphisms of genes involved in estrogen synthesis and estrogen signaling exerts an important influence on BMD in postmenopausal women, thus helping to explain, in part, its heritable component. Nevertheless, further studies are warranted to confirm this gene-to-gene interaction in other populations.

Aromatase gene and osteoporosis: relationship of ten polymorphic loci with bone mineral density.

Aromatase activity appears to be important for bone homeostasis in postmenopausal women. In fact, therapy with aromatase inhibitors is associated with bone loss and fractures. A common biallelic A/G polymorphism in the 3'-untranslated region (UTR) of CYP19-aromatase gene has been associated with differences in gene transcription and the risk of estrogen-responsive tumors. We explored the relationship of such a polymorphism and other 9 polymorphisms situated within or near CYP19 gene with bone mass. The study group comprised 286 postmenopausal women. DNA was isolated from peripheral blood. Biallelic and insertion/deletion polymorphisms were analyzed with exonuclease assays using TaqMan probes. A microsatellite polymorphism in intron 4 was studied by capillary electrophoresis. Bone mineral density (BMD) was determined by DXA. In this cross-sectional study, the postmenopausal decrease in bone mass appeared to be slower in women with AA genotype in the 3'UTR, than in those with AG or GG genotypes. Consequently, there were significant genotype-related differences in BMD. In women after age of 60, hip T-scores were: AA -1.3 +/- 0.1, AG -1.3 +/- 0.2, GG -1.9 +/- 0.1 (P = 0.002). Lumbar spine T-scores were: AA -1.9 +/- 10.2, AG -2.2 +/- 0.1, GG -3.0 +/- 0.2 (P = 0.001). Moreover, GG genotype showed a trend for lower free estrogen levels. This polymorphism was strongly linked to a tetranucleotide repeat in intron 4, as well as to other biallelic polymorphisms situated between 3'UTR and I.2 promoter regions. They all were associated with BMD. However, biallelic polymorphisms in the extreme 5' region of CYP19 and two polymorphisms in neighbor genes were not associated with BMD. In conclusion, common variations of CYP19-aromatase are associated with differences in BMD that seem to be important from an individual as well as from a population perspective.

Age-related influence of common aromatase gene polymorphisms on bone mass of healthy men.

Androgens and estrogens are critical factors for bone homeostasis; hence, polymorphisms of genes involved in the metabolism and activity of sex steroids are likely candidates to influence bone mass. Therefore, we studied the association of two of those microsatellite polymorphisms, situated in intron 4 of CYP19-aromatase and exon 1 of androgen receptor, with bone mass in a group of 324 healthy men of a wide age range (mean age 49, range 22-75). CYP19 and androgen receptor alleles were typed by capillary electrophoresis after PCR amplification. Bone mass was measured by dual X-ray absorptiometry at the hip and the spine. No association was found between androgen receptor variation and bone mass. However, among the 184 subjects aged more than 45 years, a significant association was found between CYP19 alleles and bone mass at the lumbar spine (P = 0.001) and total hip (P = 0.01). Individuals with long alleles had higher bone mass, even after adjusting for body weight, height, or calcium intake. Mean spine Z scores were -0.1 (95% CI, -0.3 to 0.2), -0.1 (-0.4 to 0.2), and 0.6 (0.3 to 0.9) for individuals with short, intermediate, and long alleles, respectively. Total hip Z scores were 0.4 (0.2 to 0.6), 0.4 (0.2 to 0.6), and 0.8 (0.5 to 1.0), respectively. Longer CYP19 alleles were also associated with higher free estradiol index. These results suggest that common variations in CYP19-aromatase gene may have an important influence on the maintenance of male skeleton after peak bone mass is reached.