CRISPR/Cas9-based functional genomics strategy to decipher the pathogenicity of genetic variants in inherited metabolic disorders.

The determination of the functional impact of variants of uncertain significance (VUS) is one of the major bottlenecks in the diagnostic workflow of inherited genetic diseases. To face this problem, we set up a CRISPR/Cas9-based strategy for knock-in cellular model generation, focusing on inherited metabolic disorders (IMDs). We selected variants in seven IMD-associated genes, including seven reported disease-causing variants and four benign/likely benign variants. Overall, 11 knock-in cell models were generated via homology-directed repair in HAP1 haploid cells using CRISPR/Cas9. The functional impact of the variants was determined by analyzing the characteristic biochemical alterations of each disorder. Functional studies performed in knock-in cell models showed that our approach accurately distinguished the functional effect of pathogenic from non-pathogenic variants in a reliable manner in a wide range of IMDs. Our study provides a generic approach to assess the functional impact of genetic variants to improve IMD diagnosis and this tool could emerge as a promising alternative to invasive tests, such as muscular or skin biopsies. Although the study has been performed only in IMDs, this strategy is generic and could be applied to other genetic disorders.

Bilirubin increases viability and decreases osteoclast apoptosis contributing to osteoporosis in advanced liver diseases.

Bilirubin and bile acids have deleterious effects on osteoblasts, which may explain the low bone formation of liver diseases with cholestasis. Although there is some clinical evidence of increased bone resorption in this condition, the effects of these substances on osteoclasts are unknown. The objective was to analyze the effects of bilirubin and bile acids -lithocholic acid (LCA) and ursodeoxycholic acid (UDCA)- on osteoclast viability and apoptosis, and on the expression of osteoclast-related microRNAs (miRNAs). RAW 264.7 cells and human PBMCs were differentiated into osteoclasts. Success in differentiation was assessed by TRAP stain and osteoclast-specific gene expression; osteoclast activity was detected by the resorption pits in Corning® Osteo Assay Surface Plates. Cells were treated with camptothecin (CAM) or with bilirubin, LCA or UDCA, at several concentrations and combinations, including non-treated cells as control. Cell viability was measured using WST-1 assay and apoptosis assessing Caspase-3 by Western blot. Expression of miR-21a, miR-29b, miR-31, miR-148a, miR-155 and miR-223 were analyzed by Real Time. Viability increased gradually in osteoclasts differentiated from RAW 264.7 cells, as the concentration of bilirubin increased, being particularly high with bilirubin 100 µM (61 %) as compared to the untreated control (p < 0.007). Viability decreased significantly with CAM, LCA and UDCA (80 %, 62 % and 27 %, respectively), effects which were abolished by bilirubin. Moreover, bilirubin increased viability in osteoclasts derived from human PBMCs (p < 0.03). Caspase-3 decreased by 46 % with bilirubin 50 µM and increased 10-fold with LCA 100 µM and CAM (p < 0.01). Bilirubin increased miR-21 and miR-148a expression as compared to controls (115 % and 59 %, respectively; p < 0.007). In conclusion, bilirubin increases viability and decreases apoptosis of osteoclasts, and overexpresses the osteoclastogenic miR-21 and miR-148a. The effects of bilirubin counteract the actions of LCA and UDCA. Therefore, bilirubin may contribute to the increased bone resorption and to the development of osteoporosis in advanced liver diseases.

Bilirubin and bile acids in osteocytes and bone tissue. Potential role in the cholestatic-induced osteoporosis.

BACKGROUND AND AIMS: Osteoporosis is a common complication in patients with primary biliary cholangitis. Both bilirubin and lithocholic acid (LCA) result in detrimental effects on osteoblastic cells, and ursodeoxycholic acid (UDCA) counteracts these outcomes. However, there is no information on the consequences of these retained substances of cholestasis and sera from cholestatic patients in osteocytes. METHODS: The impact of bilirubin, LCA, UDCA and serum from jaundiced patients on viability, differentiation, mineralization and apoptosis has been assessed in MLO-Y4 and MLO-A5 osteocyte cell lines. Effects on gene expression were assessed in these cells and in human bone fragments. RESULTS: Lithocholic acid 10 µmol/L and bilirubin 50 µmol/L decreased viability in MLO-Y4 and MLO-A5 cells (11% and 53% respectively; P ≤ .01). UDCA alone or combined with LCA or bilirubin increased cell viability. Jaundiced sera decreased cell viability (56%), an effect which was reverted by UDCA. Bilirubin decreased differentiation by 47% in MLO-Y4 (P ≤ .01) and mineralization (87%) after 21 days in MLO-A5 (P ≤ .03). Both bilirubin and LCA increased apoptosis in MLO-Y4, and UDCA diminished the apoptotic effect. Moreover, bilirubin down-regulated RUNX2 and up-regulated RANKL gene expression in bone tissue, MLO-Y4 and MLO-A5 cells, and LCA up-regulated RANKL expression in bone tissue. UDCA 100 µmol/L increased the gene expression of all these genes in bone tissue and MLO-Y4 cells and neutralized the decreased RUNX2 expression induced by bilirubin. CONCLUSION: Bilirubin and LCA have damaging consequences in osteocytes by decreasing viability, differentiation and mineralization, increasing apoptosis and modifying gene expression, effects that are neutralized by UDCA.

Bile acids and bilirubin effects on osteoblastic gene profile. Implications in the pathogenesis of osteoporosis in liver diseases.

Osteoporosis in advanced cholestatic and end-stage liver disease is related to low bone formation. Previous studies have demonstrated the deleterious consequences of lithocholic acid (LCA) and bilirubin on osteoblastic cells. These effects are partially or completely neutralized by ursodeoxycholic acid (UDCA). We have assessed the differential gene expression of osteoblastic cells under different culture conditions. The experiments were performed in human osteosarcoma cells (Saos-2) cultured with LCA (10 µM), bilirubin (50 µM) or UDCA (10 and 100 µM) at 2 and 24 h. Expression of 87 genes related to bone metabolism and other signalling pathways were assessed by TaqMan micro fluidic cards. Several genes were up-regulated by LCA, most of them pro-apoptotic (BAX, BCL10, BCL2L13, BCL2L14), but also MGP (matrix Gla protein), BGLAP (osteocalcin), SPP1 (osteopontin) and CYP24A1, and down-regulated bone morphogenic protein genes (BMP3 and BMP4) and DKK1 (Dickkopf-related protein 1). Parallel effects were observed with bilirubin, which up-regulated apoptotic genes and CSF2 (colony-stimulating factor 2) and down-regulated antiapoptotic genes (BCL2 and BCL2L1), BMP3, BMP4 and RUNX2. UDCA 100 µM had specific consequences since differential expression was observed, up-regulating BMP2, BMP4, BMP7, CALCR (calcitonin receptor), SPOCK3 (osteonectin), BGLAP (osteocalcin) and SPP1 (osteopontin), and down-regulating pro-apoptotic genes. Furthermore, most of the differential expression changes induced by both LCA and bilirubin were partially or completely neutralized by UDCA. Conclusion: Our observations reveal novel target genes, whose regulation by retained substances of cholestasis may provide additional insights into the pathogenesis of osteoporosis in cholestatic and end-stage liver diseases.

Functional relevance of the BMD-associated polymorphism rs312009: novel involvement of RUNX2 in LRP5 transcriptional regulation.

LRP5 is an osteoporosis susceptibility gene. Association analyses reveal that individual single-nucleotide polymorphisms (SNPs) determine variation in bone mineral density (BMD) among individuals as well as fracture risk. In a previous study, we identified a lumbar spine BMD-associated SNP, rs312009, located in the LRP5 5' region. A RUNX2 binding site was identified in this region by gel-shift experiments. Here we test the functionality of this SNP and examine whether RUNX2 is indeed a regulator of LRP5 expression. Gene reporter assays were used to test rs312009 functionality. Bioinformatic predictive tools and gel-shift and gene reporter assays were used to identify and characterize additional RUNX2 binding elements in the 3.3-kb region upstream of LRP5. Allelic differences in the transcriptional activity of rs312009 were observed in two osteoblastic cell lines, the T allele being a better transcriber than the C allele. RUNX2 cotransfection in HeLa cells revealed that the LRP5 5' region responded to RUNX2 in a dose-dependent manner and that the previously identified RUNX2 binding site participated in this response. Also, RUNX2 inhibition by RNAi led to nearly 60% reduction of endogenous LRP5 mRNA in U-2 OS cells. Four other RUNX2 binding sites were identified in the 5' region of LRP5. Luciferase experiments revealed the involvement of each of them in the RUNX2 response. The allelic differences observed point to the involvement of rs312009 as a functional SNP in the observed association. To our knowledge, this is the first time that the direct action of RUNX2 on LRP5 has been described. This adds evidence to previously described links between two important bone-regulating systems: the RUNX2 transcription-factor cascade and the Wnt signaling pathway.

Analysis of three functional polymorphisms in relation to osteoporosis phenotypes: replication in a Spanish cohort.

Osteoporosis is a complex disease involving many putative genetic factors. Association analysis of functional SNPs in candidate genes is an important tool for their identification. However, this approach is affected by limited power, population stratification, and other drawbacks that lead to discordant results. Replication in independent cohorts is essential. We performed association analyses of three functional polymorphisms previously associated with bone phenotypes--namely, Ala222Val in MTHFR, Ile1062Val in LRP6, and -13910C>T in LCT--in a cohort of 944 postmenopausal Spanish women, all of them with lumbar spine (LS) bone mineral density (BMD) data and most with femoral neck (FN) BMD and fracture data. We found significant differences between genotypes only for the MTHFR polymorphism and vertebral factures, with an OR of 2.27 (95% CI 1.17-4.38) for the TT vs. CC/CT genotypes, P = 0.018. We present genotype and allele frequency data for LCT -13910C>T for a Spanish population, where the T allele (conferring lactase persistence) has a frequency of 38.6%. Genotype frequencies were consistent with observed clines in Europe and with the prevalence of lactase nonpersistence. The LCT -13910C>T polymorphism was significantly associated with height and weight, such that T allele carriers were 0.88 cm taller (95% CI 0.08-1.59 cm, P = 0.032, adjusted by age) than CC individuals and TT homozygotes were 1.91 kg heavier than CC/CT individuals (95% CI 0.11-3.71 kg, P = 0.038, adjusted by age). In conclusion, no significant association was observed between the studied polymorphisms and LS BMD or FN BMD in postmenopausal Spanish women, and only MTHFR Ala222Val was associated with vertebral fractures.

Effect of IL-1beta, PGE(2), and TGF-beta1 on the expression of OPG and RANKL in normal and osteoporotic primary human osteoblasts.

The RANKL/RANK/OPG pathway is essential for bone remodeling regulation. Many hormones and cytokines are involved in regulating gene expression in most of the pathway components. Moreover, any deregulation of this pathway can alter bone metabolism, resulting in loss or gain of bone mass. Whether osteoblasts from osteoporotic and nonosteoporotic patients respond differently to cytokines is unknown. The aim of this study was to compare the effect of interleukin (IL)-1beta, proftaglandin E(2) (PGE(2)), and transforming growth factor-beta1 (TGF-beta1) treatments on OPG and RANKL gene expression in normal (n = 11) and osteoporotic (n = 8) primary osteoblasts. OPG and RANKL mRNA levels of primary human osteoblastic (hOB) cell cultures were assessed by real-time PCR. In all cultures, OPG mRNA increased significantly in response to IL-1beta treatment and decreased in response to TGF-beta1 whereas PGE(2) treatment had no effect. RANKL mRNA levels were significantly increased by all treatments. Differences in OPG and RANKL responses were observed between osteoporotic and nonosteoporotic hOB: in osteoporotic hOB, the OPG response to IL-1beta treatment was up to three times lower (P = 0.009), whereas that of RANKL response to TGF-beta1 was five times higher (P = 0.002) after 8 h of treatment, as compared with those in nonosteoporotic hOBs. In conclusion, osteoporotic hOB cells showed an anomalous response under cytokine stimulation, consistent with an enhanced osteoclastogenesis resulting in high levels of bone resorption.

A haplotype-based analysis of the LRP5 gene in relation to osteoporosis phenotypes in Spanish postmenopausal women.

LRP5 encodes the low-density lipoprotein receptor-related protein 5, a transmembrane protein involved in Wnt signaling. LRP5 is an important regulator of osteoblast growth and differentiation, affecting bone mass in vertebrates. Whether common variations in LRP5 are associated with normal BMD variation or osteoporotic phenotypes is of great relevance. We used a haplotype-based approach to search for common disease-associated variants in LRP5 in a cohort of 964 Spanish postmenopausal women. Twenty-four SNPs were selected, covering the LRP5 region, including the missense changes p.V667M and p.A1330V. The SNPs were genotyped and evaluated for association with BMD at the lumbar spine (LS) or femoral neck (FN) and with osteoporotic fracture, at single SNP and haplotype levels, by regression methods. Association with LS BMD was found for SNP 1, rs312009, located in the 5'-flanking region (p = 0.011, recessive model). SNP 6, rs2508836, in intron 1, was also associated with BMD, both at LS (p = 0.025, additive model) and FN (p = 0.031, recessive model). Two polymorphisms were associated with fracture: SNP 11, rs729635, in intron 1, and SNP 15, rs643892, in intron 5 (p = 0.007 additive model and p = 0.019 recessive model, respectively). Haplotype analyses did not provide additional information, except for haplotype "GC" of the block located at the 3'end of the gene. This haplotype spans intron 22 and the 3' untranslated region and was associated with FN BMD (p = 0.029, one copy of the haplotype versus none). In silico analyses showed that SNP 1 (rs312009) lies in a putative RUNX2 binding site. Electro-mobility shift assays confirmed RUNX2 binding to this site.

Promoter 2 -1025 T/C polymorphism in the RUNX2 gene is associated with femoral neck bmd in Spanish postmenopausal women.

Stimulation of bone formation is a key therapeutic target in osteoporosis. Runx2 is a runt domain transcription factor essential to osteoblast differentiation, bone remodeling, and fracture healing. Runx2 knockout mice exhibit a complete lack of ossification, while overexpression of this gene in transgenic mice results in an osteoporotic phenotype. Thus, RUNX2 is a good candidate for the genetic determination of osteoporosis. In this association study, the effects of the -330 G/T polymorphism in promoter 1 and the -1025 T/C polymorphism (rs7771980) in promoter 2 of RUNX2 were tested in relation to lumbar spine (LS) and femoral neck (FN) bone mineral density (BMD) in a cohort of 821 Spanish postmenopausal women. The minor allele frequencies for the two polymorphisms were 0.15 and 0.07, respectively. The two polymorphisms, located more than 90 kb apart, were not in linkage disequilibrium (D' = 0.27, r (2) = 0.028). In an ANCOVA test adjusting by weight, height, age, and years since menopause, the -330 G/T polymorphism was not associated with any of the phenotypes analyzed, while we found the -1025 T/C polymorphism to be associated with FN BMD (p = 0.001). In particular, individuals carrying the TC genotype had higher mean adjusted FN BMD values than those bearing the TT genotype. Our results highlight the importance of this RUNX2 promoter 2 polymorphism in FN BMD determination.

Relationship between pituitary and adipose tissue after hypothalamic denervation in the female rat. A morphometric immunohistochemical study.

Neonatal administration of monosodium glutamate (MSG) to rats produces severe lesions in certain hypothalamic nuclei, with repercussions in different neuroendocrine axes, and serves as a model for their study. In addition, adipose tissue, as a target organ, is known to be directly related to several neurondocrine axes. We used 21-day-old female Sprague-Dawley rats that had received a neonatal treatment with MSG (4 mg/g body weight, i.p., from day 2 up to day 10 of age) in addition to control rats (injected with 10% NaCl solution, on a similar schedule). We performed a specific immunohistochemical study on each anterior-pituitary cell population, along with the morphometry of these cells and of the parietal and visceral adipose tissue, and measured the levels of serum leptin and triglycerides. The MSG animals evinced significant changes in volume density (VD), cell density (CD), and cell size (CS) in the corticotropes, thyrotropes, and LH gonadotropes, but not in the somatotropes, lactotropes, and FSH gonadotropes. The modification common to the three cell types was a hyperplasia, but with different results depending on cell size. Furthermore, in the MSG rats significant changes were also observed in the VD, CD, and CS of the adipose tissue, consisting of adipogenesis and decrease of adipocyte size in visceral fat, together with probable lipogenesis as judged by an increase in adipocyte size in the parietal fat. The serum levels of leptin and triglycerides appeared significantly higher in MSG animals. For the first time in this animal model, and at the level of three neuroendocrine axes, our results suggest changes that correlate hypothalamic damage, cellular pituitary alterations, and the response of the adipose tissue as a target organ for MSG insult.

Morphological and biochemical changes of pituitary gonadotropes in male golden hamsters submitted to short and long photoperiods.

Golden hamsters are seasonal breeders, and a pattern of regression-recrudescence in their hypothalamic-pituitary axis is observed when adult animals are exposed to less than 12.5 h daylight for a prolonged period of time. The purpose of the present work was to investigate the possible morphological and biochemical changes of gonadotrope cell population in male golden hamsters submitted to both short (SP) and long photoperiods (LP). Thus, adult male golden hamsters were exposed to SP (6 h light, 18 h darkness) for 8, 16, 22 and 28 weeks or maintained under LP (14 h light, 10 h darkness). Pituitaries were processed using both light and electron microscopy, and serum levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were assessed by heterologous radioimmunoassay. Volume density [VD = Sigma cell area/reference area (RA)] and cell density (CD = number of cells/RA) of gonadotropes were measured with an image analysis system (Imaging Technology, Software Optimas 5.2). When analyzing the gonadotrope population from animals submitted either to SP or LP, no significant differences were found. At the ultrastructural level, we found a decrease (p < 0.05) in the exocytotic profiles and the individual mean area of secretory granules of both gonadotropes in hamsters maintained under SP for 16 and 22 weeks. Exposure to short days resulted in a decline in serum levels showing a nadir after 16 weeks (p < 0.05). Gonadotropin levels of the hamsters under prolonged exposure to short cycles spontaneously returned to normal values after 22 weeks. In summary, we found a decrease in the ultrastructural and biochemical parameters suggesting an altered release of FSH and LH in male golden hamsters submitted to SP, with a spontaneous recrudescence phase appearing at the end of the study.

Influence of photoinhibition on the morphology and function of pituitary lactotropes in male golden hamsters.

Inhibition of prolactin (PRL) secretion has been previously shown in pituitaries from male and female hamsters exposed to short photoperiods. The purpose of the present study was to analyze the possible quantitative immunohistochemical and ultrastructural changes of PRL cells in male golden hamsters undergoing regression and spontaneous recrudescence, correlating the morphological findings with circulating PRL levels. Thus, adult male golden hamsters were exposed for 8, 16, 22 and 28 weeks to either short photoperiods (SP: 6 h light, 18 h darkness) or long photoperiods (LP: 14 h light, 10 h darkness). Pituitaries were processed for both light and electron microscopy, and serum levels of PRL were assessed by radioimmunoassay (RIA). Volume density (VD = cell area/reference area) and cell density (CD = number of cells/reference area) of lactotropes were measured with an image analysis system (Imaging Technology, Software Optimas 5.2). One hundred lactotropes were recorded for measuring several ultrastructural parameters. When analyzing the lactrotrope cell population from animals submitted to SP, the VD and CD were found to be significantly (p < 0.05) diminished with respect to those of the groups submitted to LP at weeks 8, 16 and 22. However, at week 28 a spontaneous recrudescence appeared. The lactotropes from animals submitted to LP for 8, 16, 22 and 28 weeks exhibited numerous large electrondense secretory granules. The rough endoplasmic reticulum (RER) presented some flat cisternae and numerous free ribosomes. Animals submitted to SP for 8 weeks showed a number of medium and large secretory granules, and the RER exhibited mainly numerous free ribosomes. In those animals submitted to SP for 16 and 22 weeks, lactotropes were found smaller and showed small and medium-sized secretory granules decreased in number. The Golgi complex exhibited some immature granules and dilated cisternae, while the RER did not present differences with respect to the 8-week SP group. The hamsters submitted to SP for 28 weeks presented medium and large secretory granules, and the RER exhibited dilated irregular cisternae. The ultrastructural morphometric parameters showed a decrease (p < 0.05) in the number of secretory granules and exocytotic profiles, as well as a diminution (p < 0.05) in the areas of Golgi complex, RER, secretory granules, and individual mean area of secretory granule during the 16 and 22 weeks of SP. Serum PRL levels were severely reduced under SP. This decrease was greater at 16 weeks than at 8 weeks of exposure. When hamsters were kept for 22 weeks under SP, PRL levels started their recovery. Thus, at the end of the experiment, the PRL values returned to near those of LP group. In summary, we found quantitative immunohistochemical and ultrastructural, as well as biochemical changes that suggest an inhibition of synthesis, storage and release of PRL in male golden hamsters submitted to SP, with a spontaneous recrudescence appearing at the end of the study.