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.).

Bi-allelic Mutations in KLHL7 Cause a Crisponi/CISS1-like Phenotype Associated with Early-Onset Retinitis Pigmentosa.

Crisponi syndrome (CS)/cold-induced sweating syndrome type 1 (CISS1) is a very rare autosomal-recessive disorder characterized by a complex phenotype with high neonatal lethality, associated with the following main clinical features: hyperthermia and feeding difficulties in the neonatal period, scoliosis, and paradoxical sweating induced by cold since early childhood. CS/CISS1 can be caused by mutations in cytokine receptor-like factor 1 (CRLF1). However, the physiopathological role of CRLF1 is still poorly understood. A subset of CS/CISS1 cases remain yet genetically unexplained after CRLF1 sequencing. In five of them, exome sequencing and targeted Sanger sequencing identified four homozygous disease-causing mutations in kelch-like family member 7 (KLHL7), affecting the Kelch domains of the protein. KLHL7 encodes a BTB-Kelch-related protein involved in the ubiquitination of target proteins for proteasome-mediated degradation. Mono-allelic substitutions in other domains of KLHL7 have been reported in three families affected by a late-onset form of autosomal-dominant retinitis pigmentosa. Retinitis pigmentosa was also present in two surviving children reported here carrying bi-allelic KLHL7 mutations. KLHL7 mutations are thus associated with a more severe phenotype in recessive than in dominant cases. Although these data further support the pathogenic role of KLHL7 mutations in a CS/CISS1-like phenotype, they do not explain all their clinical manifestations and highlight the high phenotypic heterogeneity associated with mutations in KLHL7.

Novel action of FOXL2 as mediator of Col1a2 gene autoregulation.

FOXL2 belongs to the evolutionarily conserved forkhead box (FOX) superfamily and is a master transcription factor in a spectrum of developmental pathways, including ovarian and eyelid development and bone, cartilage and uterine maturation. To analyse its action, we searched for proteins that interact with FOXL2. We found that FOXL2 interacts with specific C-terminal propeptides of several fibrillary collagens. Because these propeptides can participate in feedback regulation of collagen biosynthesis, we inferred that FOXL2 could thereby affect the transcription of the cognate collagen genes. Focusing on COL1A2, we found that FOXL2 indeed affects collagen synthesis, by binding to a DNA response element located about 65Kb upstream of this gene. According to our hypothesis we found that in Foxl2(-/-) mouse ovaries, Col1a2 was elevated from birth to adulthood. The extracellular matrix (ECM) compartmentalizes the ovary during folliculogenesis, (with type I, type III and type IV collagens as primary components), and ECM composition changes during the reproductive lifespan. In Foxl2(-/-) mouse ovaries, in addition to up-regulation of Col1a2, Col3a1, Col4a1 and fibronectin were also upregulated, while laminin expression was reduced. Thus, by regulating levels of extracellular matrix components, FOXL2 may contribute to both ovarian histogenesis and the fibrosis attendant on depletion of the follicle reserve during reproductive aging and menopause.

FOXL2 modulates cartilage, skeletal development and IGF1-dependent growth in mice.

BACKGROUND: Haploinsufficiency of the FOXL2 transcription factor in humans causes Blepharophimosis/Ptosis/Epicanthus Inversus syndrome (BPES), characterized by eyelid anomalies and premature ovarian failure. Mice lacking Foxl2 recapitulate human eyelid/forehead defects and undergo female gonadal dysgenesis. We report here that mice lacking Foxl2 also show defects in postnatal growth and embryonic bone and cartilage formation. METHODS: Foxl2 (-/-) male mice at different stages of development have been characterized and compared to wild type. Body length and weight were measured and growth curves were created. Skeletons were stained with alcian blue and/or alizarin red. Bone and cartilage formation was analyzed by Von Kossa staining and immunofluorescence using anti-FOXL2 and anti-SOX9 antibodies followed by confocal microscopy. Genes differentially expressed in skull vaults were evaluated by microarray analysis. Analysis of the GH/IGF1 pathway was done evaluating the expression of several hypothalamic-pituitary-bone axis markers by RT-qPCR. RESULTS: Compared to wild-type, Foxl2 null mice are smaller and show skeletal abnormalities and defects in cartilage and bone mineralization, with down-regulation of the GH/IGF1 axis. Consistent with these effects, we find FOXL2 expressed in embryos at 9.5 dpc in neural tube epithelium, in head mesenchyme near the neural tube, and within the first branchial arch; then, starting at 12.5 dpc, expressed in cartilaginous tissue; and at PO and P7, in hypothalamus. CONCLUSIONS: Our results support FOXL2 as a master transcription factor in a spectrum of developmental processes, including growth, cartilage and bone formation. Its action overlaps that of SOX9, though they are antagonistic in female vs male gonadal sex determination but conjoint in cartilage and skeletal development.

Genome-wide association study shows BCL11A associated with persistent fetal hemoglobin and amelioration of the phenotype of beta-thalassemia.

beta-Thalassemia and sickle cell disease both display a great deal of phenotypic heterogeneity, despite being generally thought of as simple Mendelian diseases. The reasons for this are not well understood, although the level of fetal hemoglobin (HbF) is one well characterized ameliorating factor in both of these conditions. To better understand the genetic basis of this heterogeneity, we carried out genome-wide scans with 362,129 common SNPs on 4,305 Sardinians to look for genetic linkage and association with HbF levels, as well as other red blood cell-related traits. Among major variants affecting HbF levels, SNP rs11886868 in the BCL11A gene was strongly associated with this trait (P < 10(-35)). The C allele frequency was significantly higher in Sardinian individuals with elevated HbF levels, detected by screening for beta-thalassemia, and patients with attenuated forms of beta-thalassemia vs. those with thalassemia major. We also show that the same BCL11A variant is strongly associated with HbF levels in a large cohort of sickle cell patients. These results indicate that BCL11A variants, by modulating HbF levels, act as an important ameliorating factor of the beta-thalassemia phenotype, and it is likely they could help ameliorate other hemoglobin disorders. We expect our findings will help to characterize the molecular mechanisms of fetal globin regulation and could eventually contribute to the development of new therapeutic approaches for beta-thalassemia and sickle cell anemia.

Heritability of cardiovascular and personality traits in 6,148 Sardinians.

In family studies, phenotypic similarities between relatives yield information on the overall contribution of genes to trait variation. Large samples are important for these family studies, especially when comparing heritability between subgroups such as young and old, or males and females. We recruited a cohort of 6,148 participants, aged 14-102 y, from four clustered towns in Sardinia. The cohort includes 34,469 relative pairs. To extract genetic information, we implemented software for variance components heritability analysis, designed to handle large pedigrees, analyze multiple traits simultaneously, and model heterogeneity. Here, we report heritability analyses for 98 quantitative traits, focusing on facets of personality and cardiovascular function. We also summarize results of bivariate analyses for all pairs of traits and of heterogeneity analyses for each trait. We found a significant genetic component for every trait. On average, genetic effects explained 40% of the variance for 38 blood tests, 51% for five anthropometric measures, 25% for 20 measures of cardiovascular function, and 19% for 35 personality traits. Four traits showed significant evidence for an X-linked component. Bivariate analyses suggested overlapping genetic determinants for many traits, including multiple personality facets and several traits related to the metabolic syndrome; but we found no evidence for shared genetic determinants that might underlie the reported association of some personality traits and cardiovascular risk factors. Models allowing for heterogeneity suggested that, in this cohort, the genetic variance was typically larger in females and in younger individuals, but interesting exceptions were observed. For example, narrow heritability of blood pressure was approximately 26% in individuals more than 42 y old, but only approximately 8% in younger individuals. Despite the heterogeneity in effect sizes, the same loci appear to contribute to variance in young and old, and in males and females. In summary, we find significant evidence for heritability of many medically important traits, including cardiovascular function and personality. Evidence for heterogeneity by age and sex suggests that models allowing for these differences will be important in mapping quantitative traits.

Differential secretion of the mutated protein is a major component affecting phenotypic severity in CRLF1-associated disorders.

Crisponi syndrome (CS) and cold-induced sweating syndrome type 1 (CISS1) are disorders caused by mutations in CRLF1. The two syndromes share clinical characteristics, such as dysmorphic features, muscle contractions, scoliosis and cold-induced sweating, with CS patients showing a severe clinical course in infancy involving hyperthermia, associated with death in most cases in the first years of life. To evaluate a potential genotype/phenotype correlation and whether CS and CISS1 represent two allelic diseases or manifestations at different ages of the same disorder, we carried out a detailed clinical analysis of 19 patients carrying mutations in CRLF1. We studied the functional significance of the mutations found in CRLF1, providing evidence that phenotypic severity of the two disorders mainly depends on altered kinetics of secretion of the mutated CRLF1 protein. On the basis of these findings, we believe that the two syndromes, CS and CISS1, represent manifestations of the same disorder, with different degrees of severity. We suggest renaming the two genetic entities CS and CISS1 with the broader term of Sohar-Crisponi syndrome.

The forkhead transcription factor Foxl2 is sumoylated in both human and mouse: sumoylation affects its stability, localization, and activity.

The FOXL2 forkhead transcription factor is expressed in ovarian granulosa cells, and mutated FOXL2 causes the blepharophimosis, ptosis and epicanthus inversus syndrome (BPES) and predisposes to premature ovarian failure. Inactivation of Foxl2 in mice demonstrated its indispensability for female gonadal sex determination and ovary development and revealed its antagonism of Sox9, the effector of male testis development. To help to define the regulatory activities of FOXL2, we looked for interacting proteins. Based on yeast two-hybrid screening, we found that FOXL2 interacts with PIAS1 and UBC9, both parts of the sumoylation machinery. We showed that human FOXL2 is sumoylated in transfected cell lines, and that endogenous mouse Foxl2 is comparably sumoylated. This modification changes its cellular localization, stability and transcriptional activity. It is intriguing that similar sumoylation and regulatory consequences have also been reported for SOX9, the male counterpart of FOXL2 in somatic gonadal tissues.

FOXL2 inactivation by a translocation 171 kb away: analysis of 500 kb of chromosome 3 for candidate long-range regulatory sequences.

A translocation breakpoint 171 kb 5' of the transcription start of FOXL2 causes blepharophimosis/ptosis/epicanthus inversus syndrome (BPES) and associated premature ovarian failure. The breakpoint falls within another gene, MRPS22, that has been sequenced in 500 kb of continuous DNA. MRPS22 encodes 20 exons and a number of alternative transcripts. Three CpG islands (>91% identical) are followed by noncoding exons 4-12 and coding exons 13-20. The 3'UTR extends into the 3'UTR of COPB2. Based on the sequence, three reported translocations that cause BPES all fall within intron 6 of MRPS22. Comparisons reveal conserved segments in introns 6, 11, and 12 of human and mouse. Notably intron 11 sequence is also deleted in goat PIS syndrome (which combines craniofacial defects, female infertility, and XX sex reversal). The conserved sequences are candidates for models in which they are distant enhancers or otherwise affect higher order chromatin structure to impose long-range cis regulation of FOXL2 expression.

Overgrowth of a mouse model of the Simpson-Golabi-Behmel syndrome is independent of IGF signaling.

The type 1 Simpson-Golabi-Behmel overgrowth syndrome (SGBS1) is caused by loss-of-function mutations of the X-linked GPC3 gene encoding glypican-3, a cell-surface heparan sulfate proteoglycan that apparently plays a negative role in growth control by an unknown mechanism. Mice carrying a Gpc3 gene knockout exhibited several phenotypic features that resemble clinical hallmarks of SGBS1, including somatic overgrowth, renal dysplasia, accessory spleens, polydactyly, and placentomegaly. In Gpc3/DeltaH19 double mutants (lacking GPC3 and also carrying a deletion around the H19 gene region that causes bialellic expression of the closely linked Igf2 gene by imprint relaxation), the Gpc3-null phenotype was exacerbated, while additional SGBS1 features (omphalocele and skeletal defects) were manifested. However, results from a detailed comparative analysis of growth patterns in double mutants lacking GPC3 and also IGF2, IGF1, or the type 1 IGF receptor (IGF1R) provided conclusive genetic evidence inconsistent with the hypothesis that GPC3 acts as a growth suppressor by sequestering or downregulating an IGF ligand. Nevertheless, our data are compatible with a model positing that there is downstream convergence of the independent signaling pathways in which either IGFs or (indirectly) GPC3 participate.

Foxl2 disruption causes mouse ovarian failure by pervasive blockage of follicle development.

FOXL2 mutations cause gonadal dysgenesis or premature ovarian failure (POF) in women, as well as eyelid/forehead dysmorphology in both sexes (the 'blepharophimosis-ptosis-epicanthus inversus syndrome', BPES). Here we report that mice lacking Foxl2 recapitulate relevant features of human BPES: males and females are small and show distinctive craniofacial morphology with upper eyelids absent. Furthermore, in mice as in humans, sterility is confined to females. Features of Foxl2 null animals point toward a new mechanism of POF, with all major somatic cell lineages failing to develop around growing oocytes from the time of primordial follicle formation. Foxl2 disruption thus provides a model for histogenesis and reproductive competence of the ovary.