NetActivity enhances transcriptional signals by combining gene expression into robust gene set activity scores through interpretable autoencoders.

Grouping gene expression into gene set activity scores (GSAS) provides better biological insights than studying individual genes. However, existing gene set projection methods cannot return representative, robust, and interpretable GSAS. We developed NetActivity, a machine learning framework that generates GSAS based on a sparsely-connected autoencoder, where each neuron in the inner layer represents a gene set. We proposed a three-tier training that yielded representative, robust, and interpretable GSAS. NetActivity model was trained with 1518 GO biological processes terms and KEGG pathways and all GTEx samples. NetActivity generates GSAS robust to the initialization parameters and representative of the original transcriptome, and assigned higher importance to more biologically relevant genes. Moreover, NetActivity returns GSAS with a more consistent definition and higher interpretability than GSVA and hipathia, state-of-the-art gene set projection methods. Finally, NetActivity enables combining bulk RNA-seq and microarray datasets in a meta-analysis of prostate cancer progression, highlighting gene sets related to cell division, key for disease progression. When applied to metastatic prostate cancer, gene sets associated with cancer progression were also altered due to drug resistance, while a classical enrichment analysis identified gene sets irrelevant to the phenotype. NetActivity is publicly available in Bioconductor and GitHub.

Narrowing the diagnostic gap: Genomes, episignatures, long-read sequencing, and health economic analyses in an exome-negative intellectual disability cohort.

PURPOSE: Genome sequencing (GS)-specific diagnostic rates in prospective tightly ascertained exome sequencing (ES)-negative intellectual disability (ID) cohorts have not been reported extensively. METHODS: ES, GS, epigenetic signatures, and long-read sequencing diagnoses were assessed in 74 trios with at least moderate ID. RESULTS: The ES diagnostic yield was 42 of 74 (57%). GS diagnoses were made in 9 of 32 (28%) ES-unresolved families. Repeated ES with a contemporary pipeline on the GS-diagnosed families identified 8 of 9 single-nucleotide variations/copy-number variations undetected in older ES, confirming a GS-unique diagnostic rate of 1 in 32 (3%). Episignatures contributed diagnostic information in 9% with GS corroboration in 1 of 32 (3%) and diagnostic clues in 2 of 32 (6%). A genetic etiology for ID was detected in 51 of 74 (69%) families. Twelve candidate disease genes were identified. Contemporary ES followed by GS cost US$4976 (95% CI: $3704; $6969) per diagnosis and first-line GS at a cost of $7062 (95% CI: $6210; $8475) per diagnosis. CONCLUSION: Performing GS only in ID trios would be cost equivalent to ES if GS were available at $2435, about a 60% reduction from current prices. This study demonstrates that first-line GS achieves higher diagnostic rate than contemporary ES but at a higher cost.

Polymorphic Inversions Underlie the Shared Genetic Susceptibility of Obesity-Related Diseases.

The burden of several common diseases including obesity, diabetes, hypertension, asthma, and depression is increasing in most world populations. However, the mechanisms underlying the numerous epidemiological and genetic correlations among these disorders remain largely unknown. We investigated whether common polymorphic inversions underlie the shared genetic influence of these disorders. We performed an inversion association analysis including 21 inversions and 25 obesity-related traits on a total of 408,898 Europeans and validated the results in 67,299 independent individuals. Seven inversions were associated with multiple diseases while inversions at 8p23.1, 16p11.2, and 11q13.2 were strongly associated with the co-occurrence of obesity with other common diseases. Transcriptome analysis across numerous tissues revealed strong candidate genes for obesity-related traits. Analyses in human pancreatic islets indicated the potential mechanism of inversions in the susceptibility of diabetes by disrupting the cis-regulatory effect of SNPs from their target genes. Our data underscore the role of inversions as major genetic contributors to the joint susceptibility to common complex diseases.

The Combined Treatment of Curcumin with Verapamil Ameliorates the Cardiovascular Pathology in a Williams-Beuren Syndrome Mouse Model.

Williams-Beuren syndrome (WBS) is a rare disorder caused by a recurrent microdeletion with hallmarks of cardiovascular manifestations, mainly supra-valvular aortic stenosis (SVAS). Unfortunately, there is currently no efficient treatment. We investigated the effect of chronic oral treatment with curcumin and verapamil on the cardiovascular phenotype of a murine model of WBS harbouring a similar deletion, CD (complete deletion) mice. We analysed systolic blood pressure in vivo and the histopathology of the ascending aorta and the left ventricular myocardium to determine the effects of treatments and their underlying mechanism. Molecular analysis showed significantly upregulated xanthine oxidoreductase (XOR) expression in the aorta and left ventricular myocardium of CD mice. This overexpression is concomitant with increased levels of nitrated proteins as a result of byproduct-mediated oxidative stress damage, indicating that XOR-generated oxidative stress impacts the pathophysiology of cardiovascular manifestations in WBS. Only the combined therapy of curcumin and verapamil resulted in a significant improvement of cardiovascular parameters via activation of the nuclear factor erythroid 2 (NRF2) and reduction of XOR and nitrated protein levels. Our data suggested that the inhibition of XOR and oxidative stress damage could help prevent the severe cardiovascular injuries of this disorder.

The RD-Connect Genome-Phenome Analysis Platform: Accelerating diagnosis, research, and gene discovery for rare diseases.

Rare disease patients are more likely to receive a rapid molecular diagnosis nowadays thanks to the wide adoption of next-generation sequencing. However, many cases remain undiagnosed even after exome or genome analysis, because the methods used missed the molecular cause in a known gene, or a novel causative gene could not be identified and/or confirmed. To address these challenges, the RD-Connect Genome-Phenome Analysis Platform (GPAP) facilitates the collation, discovery, sharing, and analysis of standardized genome-phenome data within a collaborative environment. Authorized clinicians and researchers submit pseudonymised phenotypic profiles encoded using the Human Phenotype Ontology, and raw genomic data which is processed through a standardized pipeline. After an optional embargo period, the data are shared with other platform users, with the objective that similar cases in the system and queries from peers may help diagnose the case. Additionally, the platform enables bidirectional discovery of similar cases in other databases from the Matchmaker Exchange network. To facilitate genome-phenome analysis and interpretation by clinical researchers, the RD-Connect GPAP provides a powerful user-friendly interface and leverages tens of information sources. As a result, the resource has already helped diagnose hundreds of rare disease patients and discover new disease causing genes.

FOSL2 truncating variants in the last exon cause a neurodevelopmental disorder with scalp and enamel defects.

PURPOSE: We aimed to investigate the molecular basis of a novel recognizable neurodevelopmental syndrome with scalp and enamel anomalies caused by truncating variants in the last exon of the gene FOSL2, encoding a subunit of the AP-1 complex. METHODS: Exome sequencing was used to identify genetic variants in all cases, recruited through Matchmaker exchange. Gene expression in blood was analyzed using reverse transcription polymerase chain reaction. In vitro coimmunoprecipitation and proteasome inhibition assays in transfected HEK293 cells were performed to explore protein and AP-1 complex stability. RESULTS: We identified 11 individuals from 10 families with mostly de novo truncating FOSL2 variants sharing a strikingly similar phenotype characterized by prenatal growth retardation, localized cutis scalp aplasia with or without skull defects, neurodevelopmental delay with autism spectrum disorder, enamel hypoplasia, and congenital cataracts. Mutant FOSL2 messenger RNAs escaped nonsense-mediated messenger RNA decay. Truncated FOSL2 interacts with c-JUN, thus mutated AP-1 complexes could be formed. CONCLUSION: Truncating variants in the last exon of FOSL2 associate a distinct clinical phenotype by altering the regulatory degradation of the AP-1 complex. These findings reveal a new role for FOSL2 in human pathology.

Pathogenic variants in RNPC3 are associated with hypopituitarism and primary ovarian insufficiency.

PURPOSE: We aimed to investigate the molecular basis underlying a novel phenotype including hypopituitarism associated with primary ovarian insufficiency. METHODS: We used next-generation sequencing to identify variants in all pedigrees. Expression of Rnpc3/RNPC3 was analyzed by in situ hybridization on murine/human embryonic sections. CRISPR/Cas9 was used to generate mice carrying the p.Leu483Phe pathogenic variant in the conserved murine Rnpc3 RRM2 domain. RESULTS: We described 15 patients from 9 pedigrees with biallelic pathogenic variants in RNPC3, encoding a specific protein component of the minor spliceosome, which is associated with a hypopituitary phenotype, including severe growth hormone (GH) deficiency, hypoprolactinemia, variable thyrotropin (also known as thyroid-stimulating hormone) deficiency, and anterior pituitary hypoplasia. Primary ovarian insufficiency was diagnosed in 8 of 9 affected females, whereas males had normal gonadal function. In addition, 2 affected males displayed normal growth when off GH treatment despite severe biochemical GH deficiency. In both mouse and human embryos, Rnpc3/RNPC3 was expressed in the developing forebrain, including the hypothalamus and Rathke's pouch. Female Rnpc3 mutant mice displayed a reduction in pituitary GH content but with no reproductive impairment in young mice. Male mice exhibited no obvious phenotype. CONCLUSION: Our findings suggest novel insights into the role of RNPC3 in female-specific gonadal function and emphasize a critical role for the minor spliceosome in pituitary and ovarian development and function.

Genomic and phenotypic characterization of 404 individuals with neurodevelopmental disorders caused by CTNNB1 variants.

PURPOSE: Germline loss-of-function variants in CTNNB1 cause neurodevelopmental disorder with spastic diplegia and visual defects (NEDSDV; OMIM 615075) and are the most frequent, recurrent monogenic cause of cerebral palsy (CP). We investigated the range of clinical phenotypes owing to disruptions of CTNNB1 to determine the association between NEDSDV and CP. METHODS: Genetic information from 404 individuals with collectively 392 pathogenic CTNNB1 variants were ascertained for the study. From these, detailed phenotypes for 52 previously unpublished individuals were collected and combined with 68 previously published individuals with comparable clinical information. The functional effects of selected CTNNB1 missense variants were assessed using TOPFlash assay. RESULTS: The phenotypes associated with pathogenic CTNNB1 variants were similar. A diagnosis of CP was not significantly associated with any set of traits that defined a specific phenotypic subgroup, indicating that CP is not additional to NEDSDV. Two CTNNB1 missense variants were dominant negative regulators of WNT signaling, highlighting the utility of the TOPFlash assay to functionally assess variants. CONCLUSION: NEDSDV is a clinically homogeneous disorder irrespective of initial clinical diagnoses, including CP, or entry points for genetic testing.

CIBERER: Spanish national network for research on rare diseases: A highly productive collaborative initiative.

CIBER (Center for Biomedical Network Research; Centro de Investigación Biomédica En Red) is a public national consortium created in 2006 under the umbrella of the Spanish National Institute of Health Carlos III (ISCIII). This innovative research structure comprises 11 different specific areas dedicated to the main public health priorities in the National Health System. CIBERER, the thematic area of CIBER focused on rare diseases (RDs) currently consists of 75 research groups belonging to universities, research centers, and hospitals of the entire country. CIBERER's mission is to be a center prioritizing and favoring collaboration and cooperation between biomedical and clinical research groups, with special emphasis on the aspects of genetic, molecular, biochemical, and cellular research of RDs. This research is the basis for providing new tools for the diagnosis and therapy of low-prevalence diseases, in line with the International Rare Diseases Research Consortium (IRDiRC) objectives, thus favoring translational research between the scientific environment of the laboratory and the clinical setting of health centers. In this article, we intend to review CIBERER's 15-year journey and summarize the main results obtained in terms of internationalization, scientific production, contributions toward the discovery of new therapies and novel genes associated to diseases, cooperation with patients' associations and many other topics related to RD research.

Short stature with low insulin-like growth factor 1 availability due to pregnancy-associated plasma protein A2 deficiency in a Saudi family.

In 2016 a new syndrome with postnatal short stature and low IGF1 bioavailability caused by biallelic loss-of-function mutations in the gene encoding the metalloproteinase pregnancy-associated plasma protein A2 (PAPP-A2) was described in two families. Here we report two siblings of a third family from Saudi Arabia with postnatal growth retardation and decreased IGF1 availability due to a new homozygous nonsense mutation (p.Glu886* in exon 7) in PAPPA2. The two affected males showed progressively severe short stature starting around 8 years of age, moderate microcephaly, decreased bone mineral density, and high circulating levels of total IGF1, IGFBP3, and the IGF acid-labile subunit (IGFALS), with decreased free IGF1 concentrations. Interestingly, circulating IGF2 and IGFBP5 were not increased. An increase in growth velocity and height was seen in the prepuberal patient in response to rhIGF1. These patients contribute to the confirmation of the clinical picture associated with PAPP-A2 deficiency and that the PAPPA2 gene should be studied in all patients with short stature with this characteristic phenotype. Hence, pediatric endocrinologists should measure circulating PAPP-A2 levels in the study of short stature as very low or undetectable levels of this protein can help to focus the diagnosis and treatment.

Gene-specific facial dysmorphism in Axenfeld-Rieger syndrome caused by FOXC1 and PITX2 variants.

Axenfeld-Rieger syndrome is a genetic condition characterized by ocular and systemic features and is most commonly caused by variants in the FOXC1 or PITX2 genes. Facial dysmorphism is part of the syndrome but the differences between both genes have never been systematically assessed. Here, 11 facial traits commonly reported in Axenfeld-Rieger syndrome were assessed by five clinical geneticists blinded to the molecular diagnosis. Individuals were drawn from the Australian and New Zealand Registry of Advanced Glaucoma in Australia or recruited through the Genetic and Ophthalmology Unit of l'Azienda Socio-Sanitaria Territoriale Grande Ospedale Metropolitano Niguarda in Italy. Thirty-four individuals from 18 families were included. FOXC1 variants were present in 64.7% of individuals and PITX2 variants in 35.3% of individuals. A thin upper lip (55.9%) and a prominent forehead (41.2%) were common facial features shared between both genes. Hypertelorism/telecanthus (81.8% vs 25.0%, p = 0.002) and low-set ears (31.8% vs 0.0%, p = 0.036) were significantly more prevalent in individuals with FOXC1 variants compared with PITX2 variants. These findings may assist clinicians in reaching correct clinical and molecular diagnoses, and providing appropriate genetic counseling.

MADloy: robust detection of mosaic loss of chromosome Y from genotype-array-intensity data.

BACKGROUND: Accurate protocols and methods to robustly detect the mosaic loss of chromosome Y (mLOY) are needed given its reported role in cancer, several age-related disorders and overall male mortality. Intensity SNP-array data have been used to infer mLOY status and to determine its prominent role in male disease. However, discrepancies of reported findings can be due to the uncertainty and variability of the methods used for mLOY detection and to the differences in the tissue-matrix used. RESULTS: We created a publicly available software tool called MADloy (Mosaic Alteration Detection for LOY) that incorporates existing methods and includes a new robust approach, allowing efficient calling in large studies and comparisons between methods. MADloy optimizes mLOY calling by correctly modeling the underlying reference population with no-mLOY status and incorporating B-deviation information. We observed improvements in the calling accuracy to previous methods, using experimentally validated samples, and an increment in the statistical power to detect associations with disease and mortality, using simulation studies and real dataset analyses. To understand discrepancies in mLOY detection across different tissues, we applied MADloy to detect the increment of mLOY cellularity in blood on 18 individuals after 3 years and to confirm that its detection in saliva was sub-optimal (41%). We additionally applied MADloy to detect the down-regulation genes in the chromosome Y in kidney and bladder tumors with mLOY, and to perform pathway analyses for the detection of mLOY in blood. CONCLUSIONS: MADloy is a new software tool implemented in R for the easy and robust calling of mLOY status across different tissues aimed to facilitate its study in large epidemiological studies.

Constraint and conservation of paired-type homeodomains predicts the clinical outcome of missense variants of uncertain significance.

The need to interpret the pathogenicity of novel missense variants of unknown significance identified in the homeodomain of X-chromosome aristaless-related homeobox (ARX) gene prompted us to assess the utility of conservation and constraint across these domains in multiple genes compared to conventional in vitro functional analysis. Pathogenic missense variants clustered in the homeodomain of ARX contribute to intellectual disability (ID) and epilepsy, with and without brain malformation in affected males. Here we report novel c.1112G>A, p.Arg371Gln and c.1150C>T, p.Arg384Cys variants in male patients with ID and severe seizures. The third case of a male patient with a c.1109C>T, p.Ala370Val variant is perhaps the first example of ID and autism spectrum disorder (ASD), without seizures or brain malformation. We compiled data sets of pathogenic variants from ClinVar and presumed benign variation from gnomAD and demonstrated that the high levels of sequence conservation and constraint of benign variation within the homeodomain impacts upon the ability of publicly available in silico prediction tools to accurately discern likely benign from likely pathogenic variants in these data sets. Despite this, considering the inheritance patterns of the genes and disease variants with the conservation and constraint of disease variants affecting the homeodomain in conjunction with current clinical assessments may assist in predicting the pathogenicity of missense variants, particularly for genes with autosomal recessive and X-linked patterns of disease inheritance, such as ARX. In vitro functional analysis demonstrates that the transcriptional activity of all three variants was diminished compared to ARX-Wt. We review the associated phenotypes of the published cases of patients with ARX homeodomain variants and propose expansion of the ARX-related phenotype to include severe ID and ASD without brain malformations or seizures. We propose that the use of the constraint and conservation data in conjunction with consideration of the patient phenotype and inheritance pattern may negate the need for the experimental functional validation currently required to achieve a diagnosis.

Circular DNA intermediates in the generation of large human segmental duplications.

BACKGROUND: Duplications of large genomic segments provide genetic diversity in genome evolution. Despite their importance, how these duplications are generated remains uncertain, particularly for distant duplicated genomic segments. RESULTS: Here we provide evidence of the participation of circular DNA intermediates in the single generation of some large human segmental duplications. A specific reversion of sequence order from A-B/C-D to B-A/D-C between duplicated segments and the presence of only microhomologies and short indels at the evolutionary breakpoints suggest a circularization of the donor ancestral locus and an accidental replicative interaction with the acceptor locus. CONCLUSIONS: This novel mechanism of random genomic mutation could explain several distant genomic duplications including some of the ones that took place during recent human evolution.

Nested Inversion Polymorphisms Predispose Chromosome 22q11.2 to Meiotic Rearrangements.

Inversion polymorphisms between low-copy repeats (LCRs) might predispose chromosomes to meiotic non-allelic homologous recombination (NAHR) events and thus lead to genomic disorders. However, for the 22q11.2 deletion syndrome (22q11.2DS), the most common genomic disorder, no such inversions have been uncovered as of yet. Using fiber-FISH, we demonstrate that parents transmitting the de novo 3 Mb LCR22A-D 22q11.2 deletion, the reciprocal duplication, and the smaller 1.5 Mb LCR22A-B 22q11.2 deletion carry inversions of LCR22B-D or LCR22C-D. Hence, the inversions predispose chromosome 22q11.2 to meiotic rearrangements and increase the individual risk for transmitting rearrangements. Interestingly, the inversions are nested or flanking rather than coinciding with the deletion or duplication sizes. This finding raises the possibility that inversions are a prerequisite not only for 22q11.2 rearrangements but also for all NAHR-mediated genomic disorders.

Heterozygous rare genetic variants in non-syndromic early-onset obesity.

BACKGROUND: Obesity is a very heterogeneous disorder at both the clinical and molecular levels and with high heritability. Several monogenic forms and genes with strong effects have been identified for non-syndromic severe obesity. Novel therapeutic interventions are in development for some genetic forms, emphasizing the importance of determining genetic contributions. OBJECTIVE: We aimed to define the contribution of rare single-nucleotide genetic variants (RSVs) in candidate genes to non-syndromic severe early-onset obesity (EOO; body mass index (BMI) >+3 standard deviation score, <3 years). METHODS: Using a pooled DNA-sequencing approach, we screened for RSVs in 15 obesity candidate genes in a series of 463 EOO patients and 480 controls. We also analysed exome data from 293 EOO patients from the "Viva la Familia" (VLF) study as a replication dataset. RESULTS: Likely or known pathogenic RSVs were identified in 23 patients (5.0%), with 7 of the 15 genes (BDNF, FTO, MC3R, MC4R, NEGR1, PPARG and SIM1) harbouring RSVs only in cases (3.67%) and none in controls. All were heterozygous changes, either de novo (one in BDNF) or inherited from obese parents (seven maternal, three paternal), and no individual carried more than one variant. Results were replicated in the VLF study, where 4.10% of probands carried RSVs in the overrepresented genes. RSVs in five genes were either absent (LEP) or more common in controls than in cases (ADRB3, LEPR, PCSK1 and PCSK2) in both obese datasets. CONCLUSIONS: Heterozygous RSVs in several candidate genes of the melanocortin pathway are found in ~5.0% patients with EOO. These results support the clinical utility of genetic testing to identify patients who might benefit from targeted therapeutic intervention.

A six-attribute classification of genetic mosaicism.

Mosaicism denotes an individual who has at least two populations of cells with distinct genotypes that are derived from a single fertilized egg. Genetic variation among the cell lines can involve whole chromosomes, structural or copy-number variants, small or single-nucleotide variants, or epigenetic variants. The mutational events that underlie mosaic variants occur during mitotic cell divisions after fertilization and zygote formation. The initiating mutational event can occur in any types of cell at any time in development, leading to enormous variation in the distribution and phenotypic effect of mosaicism. A number of classification proposals have been put forward to classify genetic mosaicism into categories based on the location, pattern, and mechanisms of the disease. We here propose a new classification of genetic mosaicism that considers the affected tissue, the pattern and distribution of the mosaicism, the pathogenicity of the variant, the direction of the change (benign to pathogenic vs. pathogenic to benign), and the postzygotic mutational mechanism. The accurate and comprehensive categorization and subtyping of mosaicisms is important and has potential clinical utility to define the natural history of these disorders, tailor follow-up frequency and interventions, estimate recurrence risks, and guide therapeutic decisions.

Further definition of the proximal 19p13.3 microdeletion/microduplication syndrome and implication of PIAS4 as the major contributor.

The proximal 19p13.3 microdeletion/microduplication (prox19p13.3del/dup) syndrome is a recently described disorder with common clinical features including developmental delay, intellectual disability, speech delay, facial dysmorphic features with ear defects, anomalies of the hands and feet, umbilical hernia and hypotonia. While deletions are associated with macrocephaly, patients with duplications have microcephaly. The smallest region of overlap in multiple patients (113.5 kb) included three genes and one pseudogene, with a suggested major role of PIAS4 in determination of the phenotype and head size in these patients. Here, we refine the prox19p13.3del/dup with four additional patients: two with microdeletions, one with microduplication and one family with single-nucleotide nonsense variant in PIAS4. The patient with the PIAS4 loss of function variant displayed a phenotype quite similar to deletion patients -including the macrocephaly and many other core features of the syndrome. Patient's SNV was inherited from her mother who is similarly affected. Thus, our data indicate that PIAS4 is a major contributor to the proximal 19p13.3del/dup syndrome phenotype. In summary, we report the first patient with a pathogenic variant in PIAS4- and three additional rearrangements at the proximal 19p13.3 locus. These observations add further evidence about the molecular basis of this microdeletion/microduplication syndrome.

Novel genes involved in severe early-onset obesity revealed by rare copy number and sequence variants.

Obesity is a multifactorial disorder with high heritability (50-75%), which is probably higher in early-onset and severe cases. Although rare monogenic forms and several genes and regions of susceptibility, including copy number variants (CNVs), have been described, the genetic causes underlying the disease still remain largely unknown. We searched for rare CNVs (>100kb in size, altering genes and present in <1/2000 population controls) in 157 Spanish children with non-syndromic early-onset obesity (EOO: body mass index >3 standard deviations above the mean at <3 years of age) using SNP array molecular karyotypes. We then performed case control studies (480 EOO cases/480 non-obese controls) with the validated CNVs and rare sequence variants (RSVs) detected by targeted resequencing of selected CNV genes (n = 14), and also studied the inheritance patterns in available first-degree relatives. A higher burden of gain-type CNVs was detected in EOO cases versus controls (OR = 1.71, p-value = 0.0358). In addition to a gain of the NPY gene in a familial case with EOO and attention deficit hyperactivity disorder, likely pathogenic CNVs included gains of glutamate receptors (GRIK1, GRM7) and the X-linked gastrin-peptide receptor (GRPR), all inherited from obese parents. Putatively functional RSVs absent in controls were also identified in EOO cases at NPY, GRIK1 and GRPR. A patient with a heterozygous deletion disrupting two contiguous and related genes, SLCO4C1 and SLCO6A1, also had a missense RSV at SLCO4C1 on the other allele, suggestive of a recessive model. The genes identified showed a clear enrichment of shared co-expression partners with known genes strongly related to obesity, reinforcing their role in the pathophysiology of the disease. Our data reveal a higher burden of rare CNVs and RSVs in several related genes in patients with EOO compared to controls, and implicate NPY, GRPR, two glutamate receptors and SLCO4C1 in highly penetrant forms of familial obesity.