A cross-disorder dosage sensitivity map of the human genome.

Rare copy-number variants (rCNVs) include deletions and duplications that occur infrequently in the global human population and can confer substantial risk for disease. In this study, we aimed to quantify the properties of haploinsufficiency (i.e., deletion intolerance) and triplosensitivity (i.e., duplication intolerance) throughout the human genome. We harmonized and meta-analyzed rCNVs from nearly one million individuals to construct a genome-wide catalog of dosage sensitivity across 54 disorders, which defined 163 dosage sensitive segments associated with at least one disorder. These segments were typically gene dense and often harbored dominant dosage sensitive driver genes, which we were able to prioritize using statistical fine-mapping. Finally, we designed an ensemble machine-learning model to predict probabilities of dosage sensitivity (pHaplo & pTriplo) for all autosomal genes, which identified 2,987 haploinsufficient and 1,559 triplosensitive genes, including 648 that were uniquely triplosensitive. This dosage sensitivity resource will provide broad utility for human disease research and clinical genetics.

Genetic variants regulating immune cell levels in health and disease.

The complex network of specialized cells and molecules in the immune system has evolved to defend against pathogens, but inadvertent immune system attacks on "self" result in autoimmune disease. Both genetic regulation of immune cell levels and their relationships with autoimmunity are largely undetermined. Here, we report genetic contributions to quantitative levels of 95 cell types encompassing 272 immune traits, in a cohort of 1,629 individuals from four clustered Sardinian villages. We first estimated trait heritability, showing that it can be substantial, accounting for up to 87% of the variance (mean 41%). Next, by assessing ∼8.2 million variants that we identified and confirmed in an extended set of 2,870 individuals, 23 independent variants at 13 loci associated with at least one trait. Notably, variants at three loci (HLA, IL2RA, and SH2B3/ATXN2) overlap with known autoimmune disease associations. These results connect specific cellular phenotypes to specific genetic variants, helping to explicate their involvement in disease.

The individual and global impact of copy-number variants on complex human traits.

The impact of copy-number variations (CNVs) on complex human traits remains understudied. We called CNVs in 331,522 UK Biobank participants and performed genome-wide association studies (GWASs) between the copy number of CNV-proxy probes and 57 continuous traits, revealing 131 signals spanning 47 phenotypes. Our analysis recapitulated well-known associations (e.g., 1q21 and height), revealed the pleiotropy of recurrent CNVs (e.g., 26 and 16 traits for 16p11.2-BP4-BP5 and 22q11.21, respectively), and suggested gene functionalities (e.g., MARF1 in female reproduction). Forty-eight CNV signals (38%) overlapped with single-nucleotide polymorphism (SNP)-GWASs signals for the same trait. For instance, deletion of PDZK1, which encodes a urate transporter scaffold protein, decreased serum urate levels, while deletion of RHD, which encodes the Rhesus blood group D antigen, associated with hematological traits. Other signals overlapped Mendelian disorder regions, suggesting variable expressivity and broad impact of these loci, as illustrated by signals mapping to Rotor syndrome (SLCO1B1/3), renal cysts and diabetes syndrome (HNF1B), or Charcot-Marie-Tooth (PMP22) loci. Total CNV burden negatively impacted 35 traits, leading to increased adiposity, liver/kidney damage, and decreased intelligence and physical capacity. Thirty traits remained burden associated after correcting for CNV-GWAS signals, pointing to a polygenic CNV architecture. The burden negatively correlated with socio-economic indicators, parental lifespan, and age (survivorship proxy), suggesting a contribution to decreased longevity. Together, our results showcase how studying CNVs can expand biological insights, emphasizing the critical role of this mutational class in shaping human traits and arguing in favor of a continuum between Mendelian and complex diseases.

BEYOND: a randomized controlled trial comparing efficacy and safety of individualized follitropin delta dosing in a GnRH agonist versus antagonist protocol during the first ovarian stimulation cycle.

STUDY QUESTION: How does a gonadotrophin-releasing hormone (GnRH) agonist versus a GnRH antagonist protocol affect ovarian response when using an individualized fixed daily dose of follitropin delta for ovarian stimulation? SUMMARY ANSWER: The BEYOND trial data demonstrate thatindividualized fixed-dose follitropin delta is effective when used in a GnRH agonist protocol, compared with a GnRH antagonist protocol, in women with anti-Müllerian hormone (AMH) ≤35 pmol/l and no increased risk of ovarian hyperstimulation syndrome (OHSS). WHAT IS KNOWN ALREADY: The efficacy and safety of an individualized fixed daily dose of follitropin delta (based on body weight and AMH) have been established in randomized controlled trials (RCTs) using a GnRH antagonist protocol. Preliminary study data indicate that individualized follitropin delta is also efficacious in a GnRH agonist protocol (RAINBOW trial, NCT03564509). There are no prospective comparative data using individualized follitropin delta for ovarian stimulation in a GnRH agonist versus a GnRH antagonist protocol. STUDY DESIGN, SIZE, DURATION: This is the first randomized, controlled, open-label, multi-centre trial exploring efficacy and safety of individualized follitropin delta dosing in a GnRH agonist versus a GnRH antagonist protocol in participants undergoing their first ovarian stimulation cycle for IVF/ICSI. A total of 437 participants were randomized centrally and stratified by centre and age. The primary endpoint was the number of oocytes retrieved. Secondary endpoints included ongoing pregnancy rates, adverse drug reactions (including OHSS), live births, and neonatal outcomes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants (18-40 years; AMH ≤35 pmol/l) were enrolled at specialist reproductive health clinics in Austria, Denmark, Israel, Italy, the Netherlands, Norway, and Switzerland. The mean number of oocytes retrieved was compared between the GnRH agonist and antagonist protocols using a negative binomial regression model with age and AMH at screening as factors. Analyses were based on all randomized subjects, using a multiple imputation method for randomized subjects withdrawing before the start of stimulation. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 437 randomized subjects, 221 were randomized to the GnRH agonist, and 216 were randomized to the GnRH antagonist protocol. The participants had a mean age of 32.3 ± 4.3 years and a mean serum AMH of 16.6 ± 7.8 pmol/l. A total of 202 and 204 participants started ovarian stimulation with follitropin delta in the GnRH agonist and antagonist groups, respectively. The mean number of oocytes retrieved was statistically significantly higher in the agonist group (11.1 ± 5.9) versus the antagonist group (9.6 ± 5.5), with an estimated mean difference of 1.31 oocytes (95% CI: 0.22; 2.40, P = 0.0185). The difference in number of oocytes retrieved was influenced by the patients' age and ovarian reserve, with a greater difference observed in patients aged <35 years and in patients with high ovarian reserve (AMH >15 pmol/l). Both the GnRH agonist and antagonist groups had a similar proportion of cycle cancellations (2.0% [4/202] versus 3.4% [7/204]) and fresh blastocyst transfer cancellations (13.4% [27/202] versus 14.7% [30/204]). The estimated ongoing pregnancy rate per started cycle was numerically higher in the GnRH agonist group (36.9% versus 29.1%; difference: 7.74% [95% CI: -1.49; 16.97, P = 0.1002]). The most commonly reported adverse events (≥1% in either group; headache, OHSS, nausea, pelvic pain, or discomfort and abdominal pain) were similar in both groups. The incidence of early moderate/severe OHSS was low (1.5% for the agonist group versus 2.5% for antagonist groups). Estimated live birth rates per started cycle were 35.8% and 28.7% in the GnRH agonist and antagonist groups, respectively (treatment difference 7.15%; 95% CI: -2.02; 16.31; P = 0.1265). The two treatment groups were comparable with respect to neonatal health data for singletons and twins and for incidence of congenital malformations (2.7% and 3.3% for the GnRH agonist versus antagonist groups, respectively). LIMITATIONS, REASONS FOR CAUTION: All participants had AMH ≤35 pmol/l and were ≤40 years old. Clinicians should remain cautious when using a GnRH agonist protocol in patients with AMH >35 pmol/l (i.e. those with an increased OHSS risk). The incidence of OHSS in the GnRH antagonist group may have been lower if a GnRH agonist trigger had been allowed. Outcomes of transfers with cryopreserved blastocysts were not followed up, therefore the cumulative live birth rates and neonatal outcomes after cryotransfer are unknown. WIDER IMPLICATIONS OF THE FINDINGS: In women with AMH ≤35 pmol/l, an individualized fixed daily dose of follitropin delta resulted in a significantly higher number of oocytes retrieved when used in a GnRH agonist protocol compared with a GnRH antagonist protocol, with no additional safety signals observed and no additional risk of OHSS. Live birth rates following ovarian stimulation with individualized follitropin delta were not statistically different between the GnRH protocols; however, the trial was not powered to assess this endpoint. There were no safety concerns with respect to neonatal health after ovarian stimulation with follitropin delta in either protocol. STUDY FUNDING/COMPETING INTEREST(S): The trial was funded by Ferring Pharmaceuticals. EE, EP, and MS have no competing interests. AP has received research support from Ferring, and Gedeon Richter, and honoraria or consultation fees from Preglem, Novo Nordisk, Ferring, Gedeon Richter, Cryos, Merck A/S. BC has received consulting fees from Ferring and Merck, and his department received fees from Ferring to cover the costs of patient enrolment. MBS has received support to attend meetings and/or travel from Ferring, and was a board member for FertiPROTEKT e.V. until 2023. JS has received honoraria or consultation fees from Ferring and Merck, and support for attending meetings and/or travel from Ferring, Merck, and GoodLife. TS has received support/travel expenses from Ferring for attending a congress meeting, and participated in an advisory board for Merck. YS has received grants/research support from Ferring and support to attend a professional society congress meeting from Merck. RL and PP are employees of Ferring Pharmaceuticals. PP is a BOD member of PharmaBiome and owns stocks of Takeda Pharmaceuticals. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov identifier NCT03809429; EudraCT Number 2017-002783-40. TRIAL REGISTRATION DATE: 7 April 2019. DATE OF FIRST PATIENT'S ENROLMENT: 2 May 2019.

Genome-wide association study of circulating interleukin 6 levels identifies novel loci.

Interleukin 6 (IL-6) is a multifunctional cytokine with both pro- and anti-inflammatory properties with a heritability estimate of up to 61%. The circulating levels of IL-6 in blood have been associated with an increased risk of complex disease pathogenesis. We conducted a two-staged, discovery and replication meta genome-wide association study (GWAS) of circulating serum IL-6 levels comprising up to 67 428 (ndiscovery = 52 654 and nreplication = 14 774) individuals of European ancestry. The inverse variance fixed effects based discovery meta-analysis, followed by replication led to the identification of two independent loci, IL1F10/IL1RN rs6734238 on chromosome (Chr) 2q14, (Pcombined = 1.8 × 10-11), HLA-DRB1/DRB5 rs660895 on Chr6p21 (Pcombined = 1.5 × 10-10) in the combined meta-analyses of all samples. We also replicated the IL6R rs4537545 locus on Chr1q21 (Pcombined = 1.2 × 10-122). Our study identifies novel loci for circulating IL-6 levels uncovering new immunological and inflammatory pathways that may influence IL-6 pathobiology.

The Human-Specific BOLA2 Duplication Modifies Iron Homeostasis and Anemia Predisposition in Chromosome 16p11.2 Autism Individuals.

Human-specific duplications at chromosome 16p11.2 mediate recurrent pathogenic 600 kbp BP4-BP5 copy-number variations, which are among the most common genetic causes of autism. These copy-number polymorphic duplications are under positive selection and include three to eight copies of BOLA2, a gene involved in the maturation of cytosolic iron-sulfur proteins. To investigate the potential advantage provided by the rapid expansion of BOLA2, we assessed hematological traits and anemia prevalence in 379,385 controls and individuals who have lost or gained copies of BOLA2: 89 chromosome 16p11.2 BP4-BP5 deletion carriers and 56 reciprocal duplication carriers in the UK Biobank. We found that the 16p11.2 deletion is associated with anemia (18/89 carriers, 20%, p = 4e-7, OR = 5), particularly iron-deficiency anemia. We observed similar enrichments in two clinical 16p11.2 deletion cohorts, which included 6/63 (10%) and 7/20 (35%) unrelated individuals with anemia, microcytosis, low serum iron, or low blood hemoglobin. Upon stratification by BOLA2 copy number, our data showed an association between low BOLA2 dosage and the above phenotypes (8/15 individuals with three copies, 53%, p = 1e-4). In parallel, we analyzed hematological traits in mice carrying the 16p11.2 orthologous deletion or duplication, as well as Bola2+/- and Bola2-/- animals. The Bola2-deficient mice and the mice carrying the deletion showed early evidence of iron deficiency, including a mild decrease in hemoglobin, lower plasma iron, microcytosis, and an increased red blood cell zinc-protoporphyrin-to-heme ratio. Our results indicate that BOLA2 participates in iron homeostasis in vivo, and its expansion has a potential adaptive role in protecting against iron deficiency.

PRF1 mutation alters immune system activation, inflammation, and risk of autoimmunity.

BACKGROUND: Defective alleles within the PRF1 gene, encoding the pore-forming protein perforin, in combination with environmental factors, cause familial type 2 hemophagocytic lymphohistiocytosis (FHL2), a rare, severe autosomal recessive childhood disorder characterized by massive release of cytokines-cytokine storm. OBJECTIVE: The aim of this study was to determine the function of hypomorph PRF1:p.A91V g.72360387 G > A on multiple sclerosis (MS) and type 1 diabetes (T1D). METHODS: We cross-compare the association data for PRF1:p.A91V mutation derived from GWAS on adult MS and pediatric T1D in Sardinians. The novel association with T1D was replicated in metanalysis in 12,584 cases and 17,692 controls from Sardinia, the United Kingdom, and Scotland. To dissect this mutation function, we searched through the coincident association immunophenotypes in additional set of general population Sardinians. RESULTS: We report that PRF1:p.A91V, is associated with increase of lymphocyte levels, especially within the cytotoxic memory T-cells, at general population level with reduced interleukin 7 receptor expression on these cells. The minor allele increased risk of MS, in 2903 cases and 2880 controls from Sardinia p = 2.06 × 10-4, odds ratio OR = 1.29, replicating a previous finding, whereas it protects from T1D p = 1.04 × 10-5, OR = 0.82. CONCLUSION: Our results indicate opposing contributions of the cytotoxic T-cell compartment to MS and T1D pathogenesis.

High-security closed devices are efficient and safe to protect human oocytes from potential risk of viral contamination during vitrification and storage especially in the COVID-19 pandemic.

PURPOSE: The main purpose and research question of the study are to compare the efficacy of high-security closed versus open devices for human oocytes' vitrification. METHODS: A prospective randomized study was conducted. A total of 737 patients attending the Infertility and IVF Unit at S.Orsola University Hospital (Italy) between October 2015 and April 2020 were randomly assigned to two groups. A total of 368 patients were assigned to group 1 (High-Security Vitrification™ - HSV) and 369 to group 2 (Cryotop® open system). Oocyte survival, fertilization, cleavage, pregnancy, implantation, and miscarriage rate were compared between the two groups. RESULTS: No statistically significant differences were observed on survival rate (70.3% vs. 73.3%), fertilization rate (70.8% vs. 74.9%), cleavage rate (90.6% vs. 90.3%), pregnancy/transfer ratio (32.0% vs. 31.8%), implantation rate (19.7% vs. 19.9%), nor miscarriage rates (22.1% vs. 21.5%) between the two groups. Women's mean age in group 1 (36.18 ± 3.92) and group 2 (35.88 ± 3.88) was not significantly different (P = .297). A total of 4029 oocytes were vitrified (1980 and 2049 in groups 1 and 2 respectively). A total of 2564 were warmed (1469 and 1095 in groups 1 and 2 respectively). A total of 1386 morphologically eligible oocytes were inseminated by intracytoplasmic sperm injection (792 and 594 respectively, P = .304). CONCLUSIONS: The present study shows that the replacement of the open vitrification system by a closed one has no impact on in vitro and in vivo survival, development, pregnancy and implantation rate. Furthermore, to ensure safety, especially during the current COVID-19 pandemic, the use of the closed device eliminates the potential samples' contamination during vitrification and storage.

Overexpression of the Cytokine BAFF and Autoimmunity Risk.

BACKGROUND: Genomewide association studies of autoimmune diseases have mapped hundreds of susceptibility regions in the genome. However, only for a few association signals has the causal gene been identified, and for even fewer have the causal variant and underlying mechanism been defined. Coincident associations of DNA variants affecting both the risk of autoimmune disease and quantitative immune variables provide an informative route to explore disease mechanisms and drug-targetable pathways. METHODS: Using case-control samples from Sardinia, Italy, we performed a genomewide association study in multiple sclerosis followed by TNFSF13B locus-specific association testing in systemic lupus erythematosus (SLE). Extensive phenotyping of quantitative immune variables, sequence-based fine mapping, cross-population and cross-phenotype analyses, and gene-expression studies were used to identify the causal variant and elucidate its mechanism of action. Signatures of positive selection were also investigated. RESULTS: A variant in TNFSF13B, encoding the cytokine and drug target B-cell activating factor (BAFF), was associated with multiple sclerosis as well as SLE. The disease-risk allele was also associated with up-regulated humoral immunity through increased levels of soluble BAFF, B lymphocytes, and immunoglobulins. The causal variant was identified: an insertion-deletion variant, GCTGT→A (in which A is the risk allele), yielded a shorter transcript that escaped microRNA inhibition and increased production of soluble BAFF, which in turn up-regulated humoral immunity. Population genetic signatures indicated that this autoimmunity variant has been evolutionarily advantageous, most likely by augmenting resistance to malaria. CONCLUSIONS: A TNFSF13B variant was associated with multiple sclerosis and SLE, and its effects were clarified at the population, cellular, and molecular levels. (Funded by the Italian Foundation for Multiple Sclerosis and others.).

Parent-of-origin-specific allelic associations among 106 genomic loci for age at menarche.

Age at menarche is a marker of timing of puberty in females. It varies widely between individuals, is a heritable trait and is associated with risks for obesity, type 2 diabetes, cardiovascular disease, breast cancer and all-cause mortality. Studies of rare human disorders of puberty and animal models point to a complex hypothalamic-pituitary-hormonal regulation, but the mechanisms that determine pubertal timing and underlie its links to disease risk remain unclear. Here, using genome-wide and custom-genotyping arrays in up to 182,416 women of European descent from 57 studies, we found robust evidence (P < 5 × 10(-8)) for 123 signals at 106 genomic loci associated with age at menarche. Many loci were associated with other pubertal traits in both sexes, and there was substantial overlap with genes implicated in body mass index and various diseases, including rare disorders of puberty. Menarche signals were enriched in imprinted regions, with three loci (DLK1-WDR25, MKRN3-MAGEL2 and KCNK9) demonstrating parent-of-origin-specific associations concordant with known parental expression patterns. Pathway analyses implicated nuclear hormone receptors, particularly retinoic acid and γ-aminobutyric acid-B2 receptor signalling, among novel mechanisms that regulate pubertal timing in humans. Our findings suggest a genetic architecture involving at least hundreds of common variants in the coordinated timing of the pubertal transition.

Impact of BRCA1 and BRCA2 mutations on ovarian reserve and fertility preservation outcomes in young women with breast cancer.

PURPOSE: To determine the impact of BRCA1 and BRCA2 mutations on ovarian reserve and fertility preservation outcome. The main purpose and research question of the study is to determine the impact of BRCA1 and BRCA2 mutations on ovarian reserve and fertility preservation outcomes. METHODS: Prospective study: 67 breast cancer patients between 18 and 40 years old, undergoing a fertility preservation by means of oocyte storage were considered. Inclusions criteria for the study were age between 18 and 40 years old, BMI between 18 and 28, breast cancer neoplasm stage I and II according to American Joint Committee on Cancer classification (2017) and no metastasis. EXCLUSION CRITERIA: age over 40 years old, BMI < 18 and > 28, breast cancer neoplasm stage III and IV and do not performed the BRCA test. A total of 21 patients had not performed the test and were excluded. Patients were divided into four groups: Group A was composed by 11 breast cancer patients with BRCA 1 mutations, Group B was composed by 11 breast cancer patients with BRCA 2 mutations, Group C was composed by 24 women with breast cancer without BRCA mutations, and Group D (control) was composed by 181 normal women. RESULTS: Group A showed significant lower AMH levels compared to Group C and D (1.2 ± 1.1 vs 4.5 ± 4.1 p < 0.05 and 1.2 ± 1.1 vs 3.8 ± 2.5 p < 0.05). BRCA1 mutated patients showed a significant lower rate of mature oocytes (MII) compared to Group C (3.1 ± 2.3 vs 7.2 ± 4.4 p < 0,05) and Group D (3.1 ± 2.3 vs 7.3 ± 3.4; p < 0,05). Breast cancer patients needed a higher dose of gonadotropins compared to controls (Group A 2206 ± 1392 Group B2047.5 ± 829.9 Group C 2106 ± 1336 Group D 1597 ± 709 p < 0,05). No significant differences were found among the groups considering basal FSH levels, duration of stimulation, number of developed follicles, and number of total retrieved oocytes. Regarding BRCA2 mutation, no effect on fertility was shown in this study. CONCLUSIONS: The study showed that BRCA1 patients had a higher risk of premature ovarian insufficiency (POI) confirmed by a diminished ovarian reserve and a lower number of mature oocytes suitable for cryopreservation.

Seventy-five genetic loci influencing the human red blood cell.

Anaemia is a chief determinant of global ill health, contributing to cognitive impairment, growth retardation and impaired physical capacity. To understand further the genetic factors influencing red blood cells, we carried out a genome-wide association study of haemoglobin concentration and related parameters in up to 135,367 individuals. Here we identify 75 independent genetic loci associated with one or more red blood cell phenotypes at P < 10(-8), which together explain 4-9% of the phenotypic variance per trait. Using expression quantitative trait loci and bioinformatic strategies, we identify 121 candidate genes enriched in functions relevant to red blood cell biology. The candidate genes are expressed preferentially in red blood cell precursors, and 43 have haematopoietic phenotypes in Mus musculus or Drosophila melanogaster. Through open-chromatin and coding-variant analyses we identify potential causal genetic variants at 41 loci. Our findings provide extensive new insights into genetic mechanisms and biological pathways controlling red blood cell formation and function.

New gene functions in megakaryopoiesis and platelet formation.

Platelets are the second most abundant cell type in blood and are essential for maintaining haemostasis. Their count and volume are tightly controlled within narrow physiological ranges, but there is only limited understanding of the molecular processes controlling both traits. Here we carried out a high-powered meta-analysis of genome-wide association studies (GWAS) in up to 66,867 individuals of European ancestry, followed by extensive biological and functional assessment. We identified 68 genomic loci reliably associated with platelet count and volume mapping to established and putative novel regulators of megakaryopoiesis and platelet formation. These genes show megakaryocyte-specific gene expression patterns and extensive network connectivity. Using gene silencing in Danio rerio and Drosophila melanogaster, we identified 11 of the genes as novel regulators of blood cell formation. Taken together, our findings advance understanding of novel gene functions controlling fate-determining events during megakaryopoiesis and platelet formation, providing a new example of successful translation of GWAS to function.

Identification of novel genetic Loci associated with thyroid peroxidase antibodies and clinical thyroid disease.

Autoimmune thyroid diseases (AITD) are common, affecting 2-5% of the general population. Individuals with positive thyroid peroxidase antibodies (TPOAbs) have an increased risk of autoimmune hypothyroidism (Hashimoto's thyroiditis), as well as autoimmune hyperthyroidism (Graves' disease). As the possible causative genes of TPOAbs and AITD remain largely unknown, we performed GWAS meta-analyses in 18,297 individuals for TPOAb-positivity (1769 TPOAb-positives and 16,528 TPOAb-negatives) and in 12,353 individuals for TPOAb serum levels, with replication in 8,990 individuals. Significant associations (P<5×10(-8)) were detected at TPO-rs11675434, ATXN2-rs653178, and BACH2-rs10944479 for TPOAb-positivity, and at TPO-rs11675434, MAGI3-rs1230666, and KALRN-rs2010099 for TPOAb levels. Individual and combined effects (genetic risk scores) of these variants on (subclinical) hypo- and hyperthyroidism, goiter and thyroid cancer were studied. Individuals with a high genetic risk score had, besides an increased risk of TPOAb-positivity (OR: 2.18, 95% CI 1.68-2.81, P = 8.1×10(-8)), a higher risk of increased thyroid-stimulating hormone levels (OR: 1.51, 95% CI 1.26-1.82, P = 2.9×10(-6)), as well as a decreased risk of goiter (OR: 0.77, 95% CI 0.66-0.89, P = 6.5×10(-4)). The MAGI3 and BACH2 variants were associated with an increased risk of hyperthyroidism, which was replicated in an independent cohort of patients with Graves' disease (OR: 1.37, 95% CI 1.22-1.54, P = 1.2×10(-7) and OR: 1.25, 95% CI 1.12-1.39, P = 6.2×10(-5)). The MAGI3 variant was also associated with an increased risk of hypothyroidism (OR: 1.57, 95% CI 1.18-2.10, P = 1.9×10(-3)). This first GWAS meta-analysis for TPOAbs identified five newly associated loci, three of which were also associated with clinical thyroid disease. With these markers we identified a large subgroup in the general population with a substantially increased risk of TPOAbs. The results provide insight into why individuals with thyroid autoimmunity do or do not eventually develop thyroid disease, and these markers may therefore predict which TPOAb-positives are particularly at risk of developing clinical thyroid dysfunction.

A meta-analysis of thyroid-related traits reveals novel loci and gender-specific differences in the regulation of thyroid function.

Thyroid hormone is essential for normal metabolism and development, and overt abnormalities in thyroid function lead to common endocrine disorders affecting approximately 10% of individuals over their life span. In addition, even mild alterations in thyroid function are associated with weight changes, atrial fibrillation, osteoporosis, and psychiatric disorders. To identify novel variants underlying thyroid function, we performed a large meta-analysis of genome-wide association studies for serum levels of the highly heritable thyroid function markers TSH and FT4, in up to 26,420 and 17,520 euthyroid subjects, respectively. Here we report 26 independent associations, including several novel loci for TSH (PDE10A, VEGFA, IGFBP5, NFIA, SOX9, PRDM11, FGF7, INSR, ABO, MIR1179, NRG1, MBIP, ITPK1, SASH1, GLIS3) and FT4 (LHX3, FOXE1, AADAT, NETO1/FBXO15, LPCAT2/CAPNS2). Notably, only limited overlap was detected between TSH and FT4 associated signals, in spite of the feedback regulation of their circulating levels by the hypothalamic-pituitary-thyroid axis. Five of the reported loci (PDE8B, PDE10A, MAF/LOC440389, NETO1/FBXO15, and LPCAT2/CAPNS2) show strong gender-specific differences, which offer clues for the known sexual dimorphism in thyroid function and related pathologies. Importantly, the TSH-associated loci contribute not only to variation within the normal range, but also to TSH values outside the reference range, suggesting that they may be involved in thyroid dysfunction. Overall, our findings explain, respectively, 5.64% and 2.30% of total TSH and FT4 trait variance, and they improve the current knowledge of the regulation of hypothalamic-pituitary-thyroid axis function and the consequences of genetic variation for hypo- or hyperthyroidism.

The association of thyroid peroxidase antibody risk loci with susceptibility to and phenotype of Graves' disease.

BACKGROUND: Despite great progress, the genetic basis of Graves' disease (GD) remains poorly understood. Recently, a population-based genomewide association study (GWAS) identified five novel loci (ATXN2/SH2B3, MAGI3, BACH2, TPO and KALRN) as significantly associated with the presence of thyroid peroxidase autoantibodies (TPOAbs), whereas several other loci showed suggestive association. METHODS: In this study, we investigated 16 single nucleotide polymorphisms (SNPs) associated with TPOAbs for the association with susceptibility to and phenotype of GD in a cohort of 647 patients with GD and 769 controls from a Polish Caucasian population. RESULTS: SNPs within/near HCP5 (rs3094228, P = 1·6 × 10(-12) , OR = 1·88), MAGI3 (rs1230666, P = 1·9 × 10(-5) , OR = 1·51) and ATXN2/SH2B3 (rs653178, P = 0·0015, OR = 1·28) loci were significantly associated with susceptibility to GD. Allele frequencies differed significantly in subgroups of patients with GD stratified by age of GD onset for HCP5 (P = 0·0014, OR = 1·50) and showed a suggestive difference for MAGI3 (P = 0·0035, OR = 1·50) SNPs. Although rs11675434 located near TPO showed no association with GD susceptibility, it was significantly associated with the presence of clinically evident Graves' ophthalmopathy (GO, P = 5·2 × 10(-5) , OR = 1·64), and this effect was independent from smoking status, age of GD onset and gender. CONCLUSIONS: This is the first study showing an association of the ATXN2/SH2B3 locus with susceptibility to GD. Furthermore, we observed a novel significant association within the HLA region at a SNP located near HCP5 and confirmed the association of the MAGI3 locus with GD susceptibility. HCP5 and MAGI3 SNPs were further correlated with age of GD onset. Finally, we identified TPO as a new susceptibility locus for GO.

Genome-wide association study of susceptibility loci for breast cancer in Sardinian population.

BACKGROUND: Despite progress in identifying genes associated with breast cancer, many more risk loci exist. Genome-wide association analyses in genetically-homogeneous populations, such as that of Sardinia (Italy), could represent an additional approach to detect low penetrance alleles. METHODS: We performed a genome-wide association study comparing 1431 Sardinian patients with non-familial, BRCA1/2-mutation-negative breast cancer to 2171 healthy Sardinian blood donors. DNA was genotyped using GeneChip Human Mapping 500 K Arrays or Genome-Wide Human SNP Arrays 6.0. To increase genomic coverage, genotypes of additional SNPs were imputed using data from HapMap Phase II. After quality control filtering of genotype data, 1367 cases (9 men) and 1658 controls (1156 men) were analyzed on a total of 2,067,645 SNPs. RESULTS: Overall, 33 genomic regions (67 candidate SNPs) were associated with breast cancer risk at the p < 0(-6) level. Twenty of these regions contained defined genes, including one already associated with breast cancer risk: TOX3. With a lower threshold for preliminary significance to p < 10(-5), we identified 11 additional SNPs in FGFR2, a well-established breast cancer-associated gene. Ten candidate SNPs were selected, excluding those already associated with breast cancer, for technical validation as well as replication in 1668 samples from the same population. Only SNP rs345299, located in intron 1 of VAV3, remained suggestively associated (p-value, 1.16 x 10(-5)), but it did not associate with breast cancer risk in pooled data from two large, mixed-population cohorts. CONCLUSIONS: This study indicated the role of TOX3 and FGFR2 as breast cancer susceptibility genes in BRCA1/2-wild-type breast cancer patients from Sardinian population.

The burden of multiple sclerosis variants in continental Italians and Sardinians.

BACKGROUND: Recent studies identified > 100 non-HLA (human leukocyte antigen) multiple sclerosis (MS) susceptibility variants in Northern European populations, but their role in Southern Europeans is largely unexplored. OBJECTIVE: We aimed to investigate the cumulative impact of those variants in two Mediterranean populations: Continental Italians and Sardinians. METHODS: We calculated four weighted Genetic Risk Scores (wGRS), using up to 102 non-HLA MS risk variants and 5 HLA MS susceptibility markers in 1691 patients and 2194 controls from continental Italy; and 2861 patients and 3034 controls from Sardinia. We then assessed the differences between populations using Nagelkerke's R(2) and the area under the Receiver Operating Characteristic (ROC) curves. RESULTS: As expected, the genetic burden (mean wGRS value) was significantly higher in MS patients than in controls, in both populations. Of note, the burden was significantly higher in Sardinians. Conversely, the proportion of variability explained and the predictive power were significantly higher in continental Italians. Notably, within the Sardinian patients, we also observed a significantly higher burden of non-HLA variants in individuals who do not carry HLA risk alleles. CONCLUSIONS: The observed differences in MS genetic burden between the two Mediterranean populations highlight the need for more genetic studies in South Europeans, to further expand the knowledge of MS genetics.

A genome-wide association scan on the levels of markers of inflammation in Sardinians reveals associations that underpin its complex regulation.

Identifying the genes that influence levels of pro-inflammatory molecules can help to elucidate the mechanisms underlying this process. We first conducted a two-stage genome-wide association scan (GWAS) for the key inflammatory biomarkers Interleukin-6 (IL-6), the general measure of inflammation erythrocyte sedimentation rate (ESR), monocyte chemotactic protein-1 (MCP-1), and high-sensitivity C-reactive protein (hsCRP) in a large cohort of individuals from the founder population of Sardinia. By analysing 731,213 autosomal or X chromosome SNPs and an additional ∼1.9 million imputed variants in 4,694 individuals, we identified several SNPs associated with the selected quantitative trait loci (QTLs) and replicated all the top signals in an independent sample of 1,392 individuals from the same population. Next, to increase power to detect and resolve associations, we further genotyped the whole cohort (6,145 individuals) for 293,875 variants included on the ImmunoChip and MetaboChip custom arrays. Overall, our combined approach led to the identification of 9 genome-wide significant novel independent signals-5 of which were identified only with the custom arrays-and provided confirmatory evidence for an additional 7. Novel signals include: for IL-6, in the ABO gene (rs657152, p = 2.13×10(-29)); for ESR, at the HBB (rs4910472, p = 2.31×10(-11)) and UCN119B/SPPL3 (rs11829037, p = 8.91×10(-10)) loci; for MCP-1, near its receptor CCR2 (rs17141006, p = 7.53×10(-13)) and in CADM3 (rs3026968, p = 7.63×10(-13)); for hsCRP, within the CRP gene (rs3093077, p = 5.73×10(-21)), near DARC (rs3845624, p = 1.43×10(-10)), UNC119B/SPPL3 (rs11829037, p = 1.50×10(-14)), and ICOSLG/AIRE (rs113459440, p = 1.54×10(-08)) loci. Confirmatory evidence was found for IL-6 in the IL-6R gene (rs4129267); for ESR at CR1 (rs12567990) and TMEM57 (rs10903129); for MCP-1 at DARC (rs12075); and for hsCRP at CRP (rs1205), HNF1A (rs225918), and APOC-I (rs4420638). Our results improve the current knowledge of genetic variants underlying inflammation and provide novel clues for the understanding of the molecular mechanisms regulating this complex process.

DNA methylation may partly explain psychotropic drug-induced metabolic side effects: results from a prospective 1-month observational study.

BACKGROUND: Metabolic side effects of psychotropic medications are a major drawback to patients' successful treatment. Using an epigenome-wide approach, we aimed to investigate DNA methylation changes occurring secondary to psychotropic treatment and evaluate associations between 1-month metabolic changes and both baseline and 1-month changes in DNA methylation levels. Seventy-nine patients starting a weight gain inducing psychotropic treatment were selected from the PsyMetab study cohort. Epigenome-wide DNA methylation was measured at baseline and after 1 month of treatment, using the Illumina Methylation EPIC BeadChip. RESULTS: A global methylation increase was noted after the first month of treatment, which was more pronounced (p < 2.2 × 10-16) in patients whose weight remained stable (< 2.5% weight increase). Epigenome-wide significant methylation changes (p < 9 × 10-8) were observed at 52 loci in the whole cohort. When restricting the analysis to patients who underwent important early weight gain (≥ 5% weight increase), one locus (cg12209987) showed a significant increase in methylation levels (p = 3.8 × 10-8), which was also associated with increased weight gain in the whole cohort (p = 0.004). Epigenome-wide association analyses failed to identify a significant link between metabolic changes and methylation data. Nevertheless, among the strongest associations, a potential causal effect of the baseline methylation level of cg11622362 on glycemia was revealed by a two-sample Mendelian randomization analysis (n = 3841 for instrument-exposure association; n = 314,916 for instrument-outcome association). CONCLUSION: These findings provide new insights into the mechanisms of psychotropic drug-induced weight gain, revealing important epigenetic alterations upon treatment, some of which may play a mediatory role.