Technologies, strategies, and cautions when deconvoluting genome-wide association signals: FTO in focus.

Genome-wide association studies have revealed a plethora of genetic variants that correlate with polygenic conditions. However, causal molecular mechanisms have proven challenging to fully define. Without such information, the associations are not physiologically useful or clinically actionable. By reviewing studies of the FTO locus in the genetic etiology of obesity, we wish to highlight advances in the field fueled by the evolution of technical and analytic strategies in assessing the molecular bases for genetic associations. Particular attention is drawn to extrapolating experimental findings from animal models and cell types to humans, as well as technical aspects used to identify long-range DNA interactions and their biological relevance with regard to the associated trait. A unifying model is proposed by which independent obesogenic pathways regulated by multiple FTO variants and genes are integrated at the primary cilium, a cellular antenna where signaling molecules that control energy balance convene.

Endocrine and behavioural features of Lowe syndrome and their potential molecular mechanisms.

BACKGROUND: Lowe syndrome (LS) is an X linked disease caused by pathogenic variants in the OCRL gene that impacts approximately 1 in 500 000 children. Classic features include congenital cataract, cognitive/behavioural impairment and renal tubulopathy. METHODS: This study is a retrospective review of clinical features reported by family based survey conducted by Lowe Syndrome Association. Frequency of non-ocular clinical feature(s) of LS and their age of onset was summarised. An LS-specific therapy effectiveness scale was used to assess the response to the administered treatment. Expression of OCRL and relevant neuropeptides was measured in postmortem human brain by qPCR. Gene expression in the mouse brain was determined by reanalysis of publicly available bulk and single cell RNA sequencing. RESULTS: A total of 137 individuals (1 female, 89.1% white, median age 14 years (range 0.8-56)) were included in the study. Short stature (height <3rd percentile) was noted in 81% (n=111) individuals, and 15% (n=20) received growth hormone therapy. Undescended testis was reported in 47% (n=64), and median age of onset of puberty was 15 years. Additional features were dental problems (n=77, 56%), bone fractures (n=63, 46%), hypophosphataemia (n=60, 44%), developmental delay and behavioural issues. OCRL is expressed in human and mouse hypothalami, and in hypothalamic cell clusters expressing Ghrh, Sst, Oxt, Pomc and pituitary cells expressing Gh and Prl. CONCLUSIONS: There is a wide spectrum of the clinical phenotype of LS. Some of the features may be partly driven by the loss of function of OCRL in the hypothalamus and the pituitary.

Gene expression atlas of energy balance brain regions.

Energy balance is controlled by interconnected brain regions in the hypothalamus, brainstem, cortex, and limbic system. Gene expression signatures of these regions can help elucidate the pathophysiology underlying obesity. RNA sequencing was conducted on P56 C57BL/6NTac male mice and E14.5 C57BL/6NTac embryo punch biopsies in 16 obesity-relevant brain regions. The expression of 190 known obesity-associated genes (monogenic, rare, and low-frequency coding variants; GWAS; syndromic) was analyzed in each anatomical region. Genes associated with these genetic categories of obesity had localized expression patterns across brain regions. Known monogenic obesity causal genes were highly enriched in the arcuate nucleus of the hypothalamus and developing hypothalamus. The obesity-associated genes clustered into distinct "modules" of similar expression profile, and these were distinct from expression modules formed by similar analysis with genes known to be associated with other disease phenotypes (type 1 and type 2 diabetes, autism, breast cancer) in the same energy balance-relevant brain regions.

Potential Issues in the Freezing of Breast Milk.

Frost-free freezers affect the storage of breast milk.

Bardet-Biedl syndrome proteins regulate intracellular signaling and neuronal function in patient-specific iPSC-derived neurons.

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder caused by mutations in genes encoding components of the primary cilium and is characterized by hyperphagic obesity. To investigate the molecular basis of obesity in human BBS, we developed a cellular model of BBS using induced pluripotent stem cell-derived (iPSC-derived) hypothalamic arcuate-like neurons. BBS mutations BBS1M390R and BBS10C91fsX95 did not affect neuronal differentiation efficiency but caused morphological defects, including impaired neurite outgrowth and longer primary cilia. Single-cell RNA sequencing of BBS1M390R hypothalamic neurons identified several downregulated pathways, including insulin and cAMP signaling and axon guidance. Additional studies demonstrated that BBS1M390R and BBS10C91fsX95 mutations impaired insulin signaling in both human fibroblasts and iPSC-derived neurons. Overexpression of intact BBS10 fully restored insulin signaling by restoring insulin receptor tyrosine phosphorylation in BBS10C91fsX95 neurons. Moreover, mutations in BBS1 and BBS10 impaired leptin-mediated p-STAT3 activation in iPSC-derived hypothalamic neurons. Correction of the BBS mutation by CRISPR rescued leptin signaling. POMC expression and neuropeptide production were decreased in BBS1M390R and BBS10C91fsX95 iPSC-derived hypothalamic neurons. In the aggregate, these data provide insights into the anatomic and functional mechanisms by which components of the BBSome in CNS primary cilia mediate effects on energy homeostasis.

Functional genomic characterization of the FTO locus in African Americans.

BACKGROUND: SNPs in the first intron of the fat mass and obesity-associated (FTO) gene represent the strongest genome-wide associations with adiposity [body mass index (BMI)]; the molecular basis for these associations is under intense investigation. In European populations, the focus of most genome-wide association studies conducted to date, the single nucleotide polymorphisms (SNPs) have indistinguishable associations due to the high level of linkage disequilibrium (LD). However, in African American (AA) individuals, reduced LD and increased haplotype diversity permit finer distinctions among obesity-associated SNPs. Such distinctions are important to mechanistic inferences and for selection of disease SNPs relevant to specific populations. METHODS: To identify specific FTO SNP(s) directly related to adiposity, we performed: 1) haplotype analyses of individual-level data in 3,335 AAs from the Atherosclerosis Risk in Communities Cohort (ARIC) study; as well as 2) statistical fine-mapping using summary statistics from a study of FTO in over 20 000 AAs and over 1000 functional genomic annotations. RESULTS: Our haplotype analyses suggest that in AAs at least two distinct signals underlie the intron 1 FTO-adiposity signal. Fine mapping showed that two SNPs have the highest posterior probability of association (PPA) with BMI: rs9927317 (PPA = 0.94) and rs62033405 (PPA = 0.99). These variants overlap possible enhancer sites and the 5'-regions of transcribed genes in the substantia nigra, chondrocytes, and white adipocytes. CONCLUSIONS: We found two SNPs in FTO with the highest probability of direct association with BMI in AAs, as well as tissue-specific mechanisms by which these variants may contribute to the pathogenesis of obesity.

Ciliary gene RPGRIP1L is required for hypothalamic arcuate neuron development.

Intronic polymorphisms in the α-ketoglutarate-dependent dioxygenase gene (FTO) that are highly associated with increased body weight have been implicated in the transcriptional control of a nearby ciliary gene, retinitis pigmentosa GTPase regulator-interacting protein-1 like (RPGRIP1L). Previous studies have shown that congenital Rpgrip1l hypomorphism in murine proopiomelanocortin (Pomc) neurons causes obesity by increasing food intake. Here, we show by congenital and adult-onset Rpgrip1l deletion in Pomc-expressing neurons that the hyperphagia and obesity are likely due to neurodevelopmental effects that are characterized by a reduction in the Pomc/Neuropeptide Y (Npy) neuronal number ratio and marked increases in arcuate hypothalamic-paraventricular hypothalamic (ARH-PVH) axonal projections. Biallelic RPGRIP1L mutations result in fewer cilia-positive human induced pluripotent stem cell-derived (iPSC-derived) neurons and blunted responses to Sonic Hedgehog (SHH). Isogenic human ARH-like embryonic stem cell-derived (ESc-derived) neurons homozygous for the obesity-risk alleles at rs8050136 or rs1421085 have decreased RPGRIP1L expression and have lower numbers of POMC neurons. RPGRIP1L overexpression increases POMC cell number. These findings suggest that apparently functional intronic polymorphisms affect hypothalamic RPGRIP1L expression and impact development of POMC neurons and their derivatives, leading to hyperphagia and increased adiposity.

FTO mediates cell-autonomous effects on adipogenesis and adipocyte lipid content by regulating gene expression via 6mA DNA modifications.

SNPs in the first intron of α-ketoglutarate-dependent dioxygenase (FTO) convey effects on adiposity by mechanisms that remain unclear, but appear to include modulation of expression of FTO itself, as well as other genes in cisFTO expression is lower in fibroblasts and iPSC-derived neurons of individuals segregating for FTO obesity risk alleles. We employed in vitro adipogenesis models to investigate the molecular mechanisms by which Fto affects adipocyte development and function. Fto expression was upregulated during adipogenesis, and was required for the maintenance of CEBPB and Cebpd/CEBPD expression in murine and human adipocytes in vitro. Fto knockdown decreased the number of 3T3-L1 cells that differentiated into adipocytes as well as the amount of lipid per mature adipocyte. This effect on adipocyte programming was conveyed, in part, by modulation of CCAAT enhancer binding protein (C/ebp)ß-regulated transcription. We found that Fto also affected Cebpd transcription by demethylating DNA N6-methyldeoxyadenosine in the Cebpd promoter. Fto is permissive for adipogenesis and promotes maintenance of lipid content in mature adipocytes by enabling C/ebpß-driven transcription and expression of Cebpd These findings are consistent with the loss of fat mass in mice segregating for a dominant-negative Fto allele.

DMSO increases efficiency of genome editing at two non-coding loci.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) has become the tool of choice for genome editing. Despite the fact that it has evolved as a highly efficient means to edit/replace coding sequence, CRISPR/Cas9 efficiency for "clean" editing of non-coding DNA remains low. We set out to introduce a single base-pair substitution in two intronic SNPs at the FTO locus without altering nearby non-coding sequence. Substitution efficiency increased up to 10-fold by treatment of human embryonic stem cells (ESC) with non-toxic levels of DMSO (1%) before CRISPR/Cas9 delivery. Treatment with DMSO did not result in CRISPR/Cas9 off-target effects or compromise the chromosomal stability of the ESC. Twenty-four hour treatment of human ESC with DMSO before CRISPR/Cas9 delivery may prove a simple means to increase editing efficiency of non-coding DNA without incorporation of undesirable mutations.

The role of Rpgrip1l, a component of the primary cilium, in adipocyte development and function.

Genetic variants within the FTO (α-ketoglutarate-dependent dioxygenase) gene have been strongly associated with a modest increase in adiposity as a result of increased food intake. These risk alleles are associated with decreased expression of both FTO and neighboring RPGRIP1L (retinitis pigmentosa GTPase regulator-interacting protein 1 like). RPGRIP1L encodes a protein that is critical to the function of the primary cilium, which conveys extracellular information to the cell. Rpgrip1l+/- mice exhibit increased adiposity, in part, as a result of hyperphagia. Here, we describe the effects of Rpgrip1l in adipocytes that may contribute to the adiposity phenotype observed in these animals and possibly in humans who segregate for FTO risk alleles. Loss of Rpgrip1l in 3T3-L1 preadipocytes increased the number of cells that are capable of differentiating into mature adipocytes. Knockout of Rpgrip1l in mature adipocytes using Adipoq-Cre did not increase adiposity in mice that were fed chow or a high-fat diet. We also did not observe any effects of Rpgrip1l knockdown in mature 3T3-L1 adipocytes. Thus, to the extent that Rpgrip1l affects cell-autonomous adipose tissue function, it may do so as a result of the effects conveyed in preadipocytes in which the primary cilium is functionally important. We propose that decreased RPGRIP1L expression in preadipocytes in humans who segregate for FTO obesity risk alleles may increase the storage capacity of adipose tissue.-Martin Carli, J. F., LeDuc, C. A., Zhang, Y., Stratigopoulos, G., Leibel, R. L. The role of Rpgrip1l, a component of the primary cilium, in adipocyte development and function.

Hypomorphism of Fto and Rpgrip1l causes obesity in mice.

Noncoding polymorphisms in the fat mass and obesity-associated (FTO) gene represent common alleles that are strongly associated with effects on food intake and adiposity in humans. Previous studies have suggested that the obesity-risk allele rs8050136 in the first intron of FTO alters a regulatory element recognized by the transcription factor CUX1, thereby leading to decreased expression of FTO and retinitis pigmentosa GTPase regulator-interacting protein-1 like (RPGRIP1L). Here, we evaluated the effects of rs8050136 and another potential CUX1 element in rs1421085 on expression of nearby genes in human induced pluripotent stem cell-derived (iPSC-derived) neurons. There were allele-dosage effects on FTO, RPGRIP1L, and AKT-interacting protein (AKTIP) expression, but expression of other vicinal genes, including IRX3, IRX5, and RBL2, which have been implicated in mediating functional effects, was not altered. In vivo manipulation of CUX1, Fto, and/or Rpgrip1l expression in mice affected adiposity in a manner that was consistent with CUX1 influence on adiposity via remote effects on Fto and Rpgrip1l expression. In support of a mechanism, mice hypomorphic for Rpgrip1l exhibited hyperphagic obesity, as the result of diminished leptin sensitivity in Leprb-expressing neurons. Together, the results of this study indicate that the effects of FTO-associated SNPs on energy homeostasis are due in part to the effects of these genetic variations on hypothalamic FTO, RPGRIP1L, and possibly other genes.

Hypomorphism for RPGRIP1L, a ciliary gene vicinal to the FTO locus, causes increased adiposity in mice.

Common polymorphisms in the first intron of FTO are associated with increased body weight in adults. Previous studies have suggested that a CUX1-regulatory element within the implicated FTO region controls expression of FTO and the nearby ciliary gene, RPGRIP1L. Given the role of ciliary genes in energy homeostasis, we hypothesized that mice hypomorphic for Rpgrip1l would display increased adiposity. We find that Rpgrip1l⁺/⁻ mice are hyperphagic and fatter, and display diminished suppression of food intake in response to leptin administration. In the hypothalamus of Rpgrip1l⁺/⁻ mice, and in human fibroblasts with hypomorphic mutations in RPGRIP1L, the number of AcIII-positive cilia is diminished, accompanied by impaired convening of the leptin receptor to the vicinity of the cilium, and diminished pStat3 in response to leptin. These findings suggest that RPGRIP1L may be partly or exclusively responsible for the obesity susceptibility signal at the FTO locus.

Cut-like homeobox 1 (CUX1) regulates expression of the fat mass and obesity-associated and retinitis pigmentosa GTPase regulator-interacting protein-1-like (RPGRIP1L) genes and coordinates leptin receptor signaling.

The first intron of FTO contains common single nucleotide polymorphisms associated with body weight and adiposity in humans. In an effort to identify the molecular basis for this association, we discovered that FTO and RPGRIP1L (a ciliary gene located in close proximity to the transcriptional start site of FTO) are regulated by isoforms P200 and P110 of the transcription factor, CUX1. This regulation occurs via a single AATAAATA regulatory site (conserved in the mouse) within the FTO intronic region associated with adiposity in humans. Single nucleotide polymorphism rs8050136 (located in this regulatory site) affects binding affinities of P200 and P110. Promoter-probe analysis revealed that binding of P200 to this site represses FTO, whereas binding of P110 increases transcriptional activity from the FTO as well as RPGRIP1L minimal promoters. Reduced expression of Fto or Rpgrip1l affects leptin receptor isoform b trafficking and leptin signaling in N41 mouse hypothalamic or N2a neuroblastoma cells in vitro. Leptin receptor clusters in the vicinity of the cilium of arcuate hypothalamic neurons in C57BL/6J mice treated with leptin, but not in fasted mice, suggesting a potentially important role of the cilium in leptin signaling that is, in part, regulated by FTO and RPGRIP1L. Decreased Fto/Rpgrip1l expression in the arcuate hypothalamus coincides with decreased nuclear enzymatic activity of a protease (cathepsin L) that has been shown to cleave full-length CUX1 (P200) to P110. P200 disrupts (whereas P110 promotes) leptin receptor isoform b clustering in the vicinity of the cilium in vitro. Clustering of the receptor coincides with increased leptin signaling as reflected in protein levels of phosphorylated Stat3 (p-Stat3). Association of the FTO locus with adiposity in humans may reflect functional consequences of A/C alleles at rs8050136. The obesity-risk (A) allele shows reduced affinity for the FTO and RPGRIP1L transcriptional activator P110, leading to the following: 1) decreased FTO and RPGRIP1L mRNA levels; 2) reduced LEPR trafficking to the cilium; and, as a consequence, 3) a diminished cellular response to leptin.

Association of plastin 3 expression with disease severity in spinal muscular atrophy only in postpubertal females.

OBJECTIVE: To investigate the potential association of plastin 3 (PLS3) expression levels in the blood with disease severity in spinal muscular atrophy (SMA). DESIGN: Measurement of PLS3 messenger RNA levels in the blood of patients with types I, II, and III SMA. SETTING: Pediatric Neuromuscular Clinical Research Network SMA Natural History study. PARTICIPANTS: A cohort of 88 patients of both sexes who had SMA. MAIN OUTCOME MEASURES: Levels of PLS3 messenger RNA in relation to SMA type and SMN2 copy number. RESULTS: Prepubertal female and younger male (<11 years) patients had approximately 2-fold-higher levels of PLS3 expression than did postpubertal female and older male (≥11 years) patients, respectively (P ≤ .001). Expression of PLS3 in male patients did not correlate with SMA clinical type or SMN2 copy number in either age group (P > .10). In postpubertal female patients, PLS3 expression was greatest in patients with type III SMA, was intermediate in patients with type II SMA, and was lowest in patients with type I SMA. Expression of PLS3 correlated with SMA type, SMN2 copy number, and the gross motor function measure only in postpubertal female patients. CONCLUSION: The PLS3 gene may be an age- and/or puberty-specific and sex-specific modifier of SMA.

Functional consequences of the human leptin receptor (LEPR) Q223R transversion.

Perturbations in the functional integrity of the leptin axis are obvious candidates for mediation of altered adiposity. In a large number of genetic association studies in humans, the nonconservative LEPR Q223R allele has been inconsistently associated with adiposity. Subtle, long-term effects of such genetic variants can be obscured by effects of the environment and other confounders that render definitive inferences difficult to reach. We directly assessed the biological effects of this variant in 129P3/J mice segregating for the humanized Lepr allele at codon 223. No effects of this allele were detected on body weight, composition, or energy expenditure in animals fed diets of varying fat content over periods as long as 235 days. In vitro, Q223R did not affect leptin signaling as reflected by activation of STAT3. We conclude that Q223R is unlikely to play a significant role in regulation of human adiposity. This approach to vetting of human allelic variation might be more widely used.

Regulation of Fto/Ftm gene expression in mice and humans.

Two recent, large whole-genome association studies (GWAS) in European populations have associated a approximately 47-kb region that contains part of the FTO gene with high body mass index (BMI). The functions of FTO and adjacent FTM in human biology are not clear. We examined expression of these genes in organs of mice segregating for monogenic obesity mutations, exposed to underfeeding/overfeeding, and to 4 degrees C. Fto/Ftm expression was reduced in mesenteric adipose tissue of mice segregating for the Ay, Lep ob, Lepr db, Cpe fat, or tub mutations, and there was a similar trend in other tissues. These effects were not due to adiposity per se. Hypothalamic Fto and Ftm expression were decreased by fasting in lean and obese animals and by cold exposure in lean mice. The fact that responses of Fto and Ftm expression to these manipulations were almost indistinguishable suggested that the genes might be coregulated. The putative overlapping regulatory region contains at least two canonical CUTL1 binding sites. One of these nominal CUTL1 sites includes rs8050136, a SNP associated with high body mass. The A allele of rs8050136 preferentially bound CUTL1[corrected] in human fibroblast DNA. 70% knockdown of CUTL1 expression in human fibroblasts decreased FTO and FTM expression by 90 and 65%, respectively. Animals and humans with various genetic interruptions of FTO or FTM have phenotypes reminiscent of aspects of the Bardet-Biedl obesity syndrome, a confirmed "ciliopathy." FTM has recently been shown to be a ciliary basal body protein.

Role of a founder c.201_202delCT mutation and new phenotypic features of congenital lipoid adrenal hyperplasia in Palestinians.

CONTEXT: Congenital lipoid adrenal hyperplasia (CLAH), caused by mutations in steroidogenic acute regulatory protein (StAR), is most frequent in Japanese and Palestinians. We report eight Palestinians from four unrelated families with CLAH. OBJECTIVE: The objective of the study was to identify the mutation(s) in StAR, correlate genotype with phenotype, and determine whether the common mutation represents a founder mutation. PATIENTS AND SETTING: Clinical, histopathological, and molecular genetic characterization was performed in these eight patients. RESULTS: All affected individuals (three XY, five XX) presented neonatally with undetectable adrenocortical hormones and are responding to replacement therapy. Only two sisters had neurodevelopmental deficits. Histopathological findings of excised XY gonads included accumulation of fat in Leydig cells. Significantly, already at 1 yr of age, positive placental alkaline phosphatase and octamer binding transcription factor staining indicated neoplastic potential. Sequence analysis of StAR revealed homozygosity for c.201_202delCT mutation in all eight cases, causing premature termination of the StAR protein. This mutation was confirmed to be a founder mutation using both an intragenic microsatellite and several single nucleotide polymorphism markers. Screening of 100 normal Jerusalem Palestinians detected no carriers of this mutation. CONCLUSION: CLAH is rare in the general Palestinian population. In most Palestinian cases, a founder c.201_202delCT mutation in StAR is the cause. The observed early neonatal presentation may reflect the major StAR protein truncation caused by this mutation. A crucial role for StAR in the central nervous system was not supported with normal neurological examinations in six of eight cases. Finally, we advocate early gonadectomy in XY CLAH cases, given the early onset of neoplastic changes observed histologically.

Positive control of tylosin biosynthesis: pivotal role of TylR.

Control of tylosin production in Streptomyces fradiae features interplay between a repressor, TylQ, and an activator, TylS, during regulation of tylR. The latter encodes a pathway-specific activator that controls most of the tylosin-biosynthetic (tyl) genes that are subject to regulation. This was established by targeted gene disruption applied separately to tylR and tylS together with transcript analysis involving reverse transcription polymerase chain reaction (RT-PCR). TylR controls multiple genes that encode the synthesis or addition of all three tylosin sugars, plus polyketide ring oxidation, and at least one of the polyketide synthase (PKS) megagenes, tylGI. (Expression of a few tyl genes, plus the resistance determinants tlrB and tlrD, together with some ancillary or unassigned genes, is not apparently regulated during fermentation, consistent with constitutive expression.) In contrast, the only gene known for sure to be directly controlled by TylS is tylR, and there are very few additional candidates. These include the mycinose-biosynthetic gene, tylJ, and two previously unassigned genes, ORF12* (tylU) plus ORF11* (tylV). TylS also controls the PKS genes [tylGIII-tylGIV-tylGV] although not in obligatory fashion. These genes can be transcribed (i.e. tylosin can be produced) in a tylS-KO strain by forcing overexpression of tylR using a foreign promoter. We therefore suspect that TylS might control the PKS genes indirectly, although this remains to be established unequivocally. Conceivably, the direct effects of TylS are exerted exclusively on other regulators. Tylosin production levels were elevated when tylS or (especially) tylR was overexpressed in S. fradiae wild-type and yield increments of industrial significance were generated by similar manipulation of an enhanced production strain.

Regulation of tylosin production and morphological differentiation in Streptomyces fradiae by TylP, a deduced gamma-butyrolactone receptor.

During promoter-probe analysis carried out in Streptomyces lividans, the TylP protein powerfully inhibited reporter gene expression from the tylP promoter, raising the likelihood that tylP is autoregulated in its native host, Streptomyces fradiae. Also in S. lividans, TylP negatively controlled the tylQ promoter, even though tylQ could still be switched off in S. fradiae strains specifically disrupted in tylP. Under the latter conditions, tylosin production was brought forward and enhanced, whereas overexpression of tylP resulted in reduced levels of the antibiotic, accompanied by barely detectable transcription from multiple genes of the tylosin biosynthetic cluster. Unexpectedly, overexpression of tylP reduced transcription of tylS, a transcriptional activator essential for tylosin production. This was probably a direct effect, as TylP also reduced expression from the tylS promoter in S. lividans. For these several reasons, we conclude that TylP acts as a repressor during tylosin biosynthesis. In addition, TylP influences morphological differentiation in S. fradiae. On solid media, strains in which tylP was disrupted sporulated significantly earlier than wild type and, in liquid culture, displayed hyperfragmentation.