MC4R variants modulate α-MSH and setmelanotide induced cellular signaling at multiple levels.

CONTEXT: Melanocortin-4 receptor (MC4R) plays an important role in body weight regulation. Pathogenic MC4R variants are the most common cause of monogenic obesity. OBJECTIVE: We have identified 17 MC4R variants in adult and pediatric patients with obesity. Here, we aimed to functionally characterize these variants by analyzing four different aspects of MC4R signaling. In addition, we aimed to analyze the effect of setmelanotide, a potent MC4R agonist, on these MC4R variants. MATERIALS AND METHODS: Cell surface expression and α-MSH- or setmelanotide-induced cAMP response, ß-arrestin-2 recruitment, and ERK activation were measured in cells expressing either wild type (WT) or variant MC4R. RESULTS: We found a large heterogeneity in the function of these variants. We identified variants with a loss of response for all studied MC4R signaling, variants with no cAMP accumulation or ERK activation but normal ß-arrestin-2 recruitment, and variants with normal cAMP accumulation and ERK activation but decreased ß-arrestin-2 recruitment, indicating disrupted desensitization and signaling mechanisms. Setmelanotide displayed a greater potency and similar efficacy as α-MSH, and induced significantly increased maximal cAMP responses of several variants compared to α-MSH. Despite the heterogeneity in functional response, there was no apparent difference in the obesity phenotype in our patients. DISCUSSION: We show that these obesity-associated MC4R variants affect MC4R signaling differently, yet leading to a comparable clinical phenotype. Our results demonstrate the clinical importance of assessing the effect of MC4R variants on a range of molecular signaling mechanisms to determine their association with obesity, which may aid in improving personalized treatment.

GNB1 and obesity: Evidence for a correlation between haploinsufficiency and syndromic obesity.

Most patients with GNB1 encephalopathy have developmental delay and/or intellectual disability, brain anomalies and seizures. Recently, two cases with GNB1 encephalopathy caused by haploinsufficiency have been reported that also show a Prader-Willi-like phenotype of childhood hypotonia and severe obesity. Here we present three new cases from our expert centre for genetic obesity in which GNB1 truncating and splice variants, probably leading to haploinsufficiency, were identified. They all have obesity, hyperphagia and intellectual deficit. The clinical cases and their weight courses are presented, together with a review of all 68 published cases with GNB1 encephalopathy. Information on weight was not mentioned in most of these articles, so we contacted authors for additional clinical information on weight status and hyperphagia. Of the 42 patients whose weight status we could determine, obesity was present in 8 patients (19%). Obesity is significantly over-represented in the group with truncating and splicing variants. In this group, we see an obesity prevalence of 75%. Since GNB1 has been linked to several key genes in the hypothalamic leptin-melanocortin pathway, which regulates satiety and energy expenditure, our data support the potential association between GNB1 haploinsufficiency and genetic obesity. We also suggest GNB1 is a candidate gene for the known obesity phenotype of the 1p36 microdeletion syndrome given this chromosomal region includes the GNB1 gene. Knowledge of an additional obesity phenotype is important for prognosis, early interventions against obesity and awareness when prescribing weight-inducing medication.

DNA methylation episignature and comparative epigenomic profiling for Pitt-Hopkins syndrome caused by TCF4 variants.

BACKGROUND: Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder caused by pathogenic variants in TCF4, leading to intellectual disability, specific morphological features, and autonomic nervous system dysfunction. Epigenetic dysregulation has been implicated in PTHS, prompting the investigation of a DNA methylation (DNAm) "episignature" specific to PTHS, for diagnostic purposes and variant reclassification, and further functional insights into the molecular pathophysiology of this disorder. METHODS: A cohort of 67 individuals with genetically confirmed PTHS and three individuals with intellectual disability and a variant of uncertain significance (VUS) in TCF4 were studied. The DNAm episignature was developed with an Infinium Methylation EPIC BeadChip array analysis, using peripheral blood cells. Support vector machine (SVM) modeling and clustering methods were employed to generate a DNAm classifier for PTHS. Validation was extended to an additional cohort of 11 individuals with PTHS. The episignature was further assessed in relation to other neurodevelopmental disorders and its specificity was examined. RESULTS: A specific DNAm episignature for PTHS was established. The classifier exhibited high sensitivity for TCF4 haploinsufficiency and missense variants in the basic helix-loop-helix domain. Notably, seven individuals with TCF4 variants exhibited negative episignatures, suggesting complexities related to mosaicism, genetic factors, and environmental influences. The episignature displayed degrees of overlap with other related disorders and biological pathways. CONCLUSIONS: This study defines a DNAm episignature for TCF4-related PTHS, enabling improved diagnostic accuracy and VUS reclassification. The finding that some cases scored negative underscores the potential for multiple or nested episignatures and emphasizes the need for continued investigation to enhance specificity and coverage across PTHS-related variants.

Evaluation of 100 Dutch cases with 16p11.2 deletion and duplication syndromes; from clinical manifestations towards personalized treatment options.

The 16p11.2 deletion syndrome is a clinically heterogeneous disorder, characterized by developmental delay, intellectual disability, hyperphagia, obesity, macrocephaly and psychiatric problems. Cases with 16p11.2 duplication syndrome have similar neurodevelopmental problems, but typically show a partial 'mirror phenotype' with underweight and microcephaly. Various copy number variants (CNVs) of the chromosomal 16p11.2 region have been described. Most is known about the 'typical' 16p11.2 BP4-BP5 (29.6-30.2 Mb; ~600 kb) deletions and duplications, but there are also several published cohorts with more distal 16p11.2 BP2-BP3 CNVs (28.8-29.0 Mb; ~220 kb), who exhibit clinical overlap. We assessed 100 cases with various pathogenic 16p11.2 CNVs and compared their clinical characteristics to provide more clear genotype-phenotype correlations and raise awareness of the different 16p11.2 CNVs. Neurodevelopmental and weight issues were reported in the majority of cases. Cases with distal 16p11.2 BP2-BP3 deletion showed the most severe obesity phenotype (73.7% obesity, mean BMI SDS 3.2). In addition to the more well defined typical 16p11.2 BP4-BP5 and distal 16p11.2 BP2-BP3 CNVs, we describe the clinical features of five cases with other, overlapping, 16p11.2 CNVs in more detail. Interestingly, four cases had a second genetic diagnosis and 18 cases an additional gene variant of uncertain significance, that could potentially help explain the cases' phenotypes. In conclusion, we provide an overview of our Dutch cohort of cases with various pathogenic 16p11.2 CNVs and relevant second genetic findings, that can aid in adequately recognizing, diagnosing and counseling of individuals with 16p11.2 CNVs, and describe the personalized medicine for cases with these conditions.

Novel PUF60 variant suggesting an interaction between Verheij and Cornelia de Lange syndrome: phenotype description and review of the literature.

Verheij syndrome [VRJS; OMIM 615583] is a rare autosomal dominant neurodevelopmental disorder characterized by distinct clinical features, including growth retardation, intellectual disability, cardiac, and renal anomalies. VRJS is caused by deletions of chromosome 8q24.3 or pathogenic variants in the PUF60 gene. Recently, pathogenic PUF60 variants have been reported in some individuals with VRJS, contributing to the variability in the clinical presentation and severity of the condition. PUF60 encodes a protein involved in regulating gene expression and cellular growth. In this report, we describe a new case of VRJS with developmental delay, cardiac-, and renal abnormalities, caused by a heterozygous pathogenic PUF60 variant. Surprisingly, DNA methylation analysis revealed a pattern resembling the Cornelia de Lange syndrome (CdLS) episignature, suggesting a potential connection between PUF60 and CdLS-related genes. This case report further delineates the clinical and molecular spectrum of VRJS and supports further research to validate the interaction between VRJS and CdLS.

Novel loss-of-function variants expand ABCC9-related intellectual disability and myopathy syndrome.

Loss-of-function mutation of ABCC9, the gene encoding the SUR2 subunit of ATP sensitive-potassium (KATP) channels, was recently associated with autosomal recessive ABCC9-related intellectual disability and myopathy syndrome (AIMS). Here we identify nine additional subjects, from seven unrelated families, harboring different homozygous LoF variants in ABCC9 and presenting with a conserved range of clinical features. All variants are predicted to result in severe truncations or in-frame deletions within SUR2, leading to the generation of non-functional SUR2-dependent KATP channels. Affected individuals show psychomotor delay and intellectual disability of variable severity, microcephaly, corpus callosum and white matter abnormalities, seizures, spasticity, short stature, muscle fatigability, and weakness. Heterozygous parents do not show any conserved clinical pathology but report multiple incidences of intrauterine fetal death, which were also observed in an eighth family included in this study. In vivo studies of abcc9 LoF in zebrafish revealed an exacerbated motor response to pentylenetetrazole, a pro-convulsive drug, consistent with impaired neurodevelopment associated with an increased seizure susceptibility. Our findings define an ABCC9 LoF related phenotype, expanding the genotypic and phenotypic spectrum of AIMS and reveal novel human pathologies arising from KATP channel dysfunction.

Mirror Hand Movements Caused by a Deletion of the DCC Gene.

This case report describes the evaluation of 16-year-old twins with chromosomal microarray and mirror movements.

DNA methylation episignature, extension of the clinical features, and comparative epigenomic profiling of Hao-Fountain syndrome caused by variants in USP7.

PURPOSE: Hao-Fountain syndrome (HAFOUS) is a neurodevelopmental disorder caused by pathogenic variants in USP7. HAFOUS is characterized by developmental delay, intellectual disability, speech delay, behavioral abnormalities, autism spectrum disorder, seizures, hypogonadism, and mild dysmorphic features. We investigated the phenotype of 18 participants with HAFOUS and performed DNA methylation (DNAm) analysis, aiming to generate a diagnostic biomarker. Furthermore, we performed comparative analysis with known episignatures to gain more insight into the molecular pathophysiology of HAFOUS. METHODS: We assessed genomic DNAm profiles of 18 individuals with pathogenic variants and variants of uncertain significance (VUS) in USP7 to map and validate a specific episignature. The comparison between the USP7 cohort and 56 rare genetic disorders with earlier reported DNAm episignatures was performed with statistical and functional correlation. RESULTS: We mapped a sensitive and specific DNAm episignature for pathogenic variants in USP7 and utilized this to reclassify the VUS. Comparative epigenomic analysis showed evidence of HAFOUS similarity to a number of other rare genetic episignature disorders. CONCLUSION: We discovered a sensitive and specific DNAm episignature as a robust diagnostic biomarker for HAFOUS that enables VUS reclassification in USP7. We also expand the phenotypic spectrum of 9 new and 5 previously reported individuals with HAFOUS.

Delineation of the adult phenotype of Coffin-Siris syndrome in 35 individuals.

Coffin-Siris syndrome (CSS) is a rare multisystemic autosomal dominant disorder. Since 2012, alterations in genes of the SWI/SNF complex were identified as the molecular basis of CSS, studying largely pediatric cohorts. Therefore, there is a lack of information on the phenotype in adulthood, particularly on the clinical outcome in adulthood and associated risks. In an international collaborative effort, data from 35 individuals ≥ 18 years with a molecularly ascertained CSS diagnosis (variants in ARID1B, ARID2, SMARCA4, SMARCB1, SMARCC2, SMARCE1, SOX11, BICRA) using a comprehensive questionnaire was collected. Our results indicate that overweight and obesity are frequent in adults with CSS. Visual impairment, scoliosis, and behavioral anomalies are more prevalent than in published pediatric or mixed cohorts. Cognitive outcomes range from profound intellectual disability (ID) to low normal IQ, with most individuals having moderate ID. The present study describes the first exclusively adult cohort of CSS individuals. We were able to delineate some features of CSS that develop over time and have therefore been underrepresented in previously reported largely pediatric cohorts, and provide recommendations for follow-up.

Functional Insight into and Refinement of the Genomic Boundaries of the JARID2-Neurodevelopmental Disorder Episignature.

JARID2 (Jumonji, AT-rich interactive domain 2) haploinsufficiency is associated with a clinically distinct neurodevelopmental syndrome. It is characterized by intellectual disability, developmental delay, autistic features, behavior abnormalities, cognitive impairment, hypotonia, and dysmorphic features. JARID2 acts as a transcriptional repressor protein that is involved in the regulation of histone methyltransferase complexes. JARID2 plays a role in the epigenetic machinery, and the associated syndrome has an identified DNA methylation episignature derived from sequence variants and intragenic deletions involving JARID2. For this study, our aim was to determine whether patients with larger deletions spanning beyond JARID2 present a similar DNA methylation episignature and to define the critical region involved in aberrant DNA methylation in 6p22-p24 microdeletions. We examined the DNA methylation profiles of peripheral blood from 56 control subjects, 13 patients with (likely) pathogenic JARID2 variants or patients carrying copy number variants, and three patients with JARID2 VUS variants. The analysis showed a distinct and strong differentiation between patients with (likely) pathogenic variants, both sequence and copy number, and controls. Using the identified episignature, we developed a binary model to classify patients with the JARID2-neurodevelopmental syndrome. DNA methylation analysis indicated that JARID2 is the driver gene for aberrant DNA methylation observed in 6p22-p24 microdeletions. In addition, we performed analysis of functional correlation of the JARID2 genome-wide methylation profile with the DNA methylation profiles of 56 additional neurodevelopmental disorders. To conclude, we refined the critical region for the presence of the JARID2 episignature in 6p22-p24 microdeletions and provide insight into the functional changes in the epigenome observed when regulation by JARID2 is lost.

The Narrative of a Patient with Leptin Receptor Deficiency: Personalized Medicine for a Rare Genetic Obesity Disorder.

Leptin receptor (LEPR) deficiency is a rare genetic disorder that affects the body's ability to regulate appetite and weight. For patients and their families, the disorder seriously disrupts daily life; however, little is published about this impact. We here report the experiences of a 10.5-year-old girl with leptin receptor deficiency and her family. The diagnosis of this rare genetic obesity had a deep impact on the life of the child and her family. It led to a better understanding of the cause of the impaired appetite regulation and early-onset obesity with subsequently less judgement by others and improved cooperation of their social network and school on maintaining a healthy lifestyle for this girl. A strict eating regimen and lifestyle measures resulted in the first year after diagnosis in a significantly decreased body mass index (BMI), followed by BMI stabilization, still categorized as obesity class three. However, the troublesome challenge of how to manage the disruptive behaviour due to hyperphagia remained. Eventually, due to treatment with targeted pharmacotherapy, i.e., melanocortin-4 receptor agonists, her BMI continued to decrease due to resolving hyperphagia. The daily routine of the family and the atmosphere at home positively changed as they were no longer dominated by the food-focused behaviour of the child and the adherence to the strict eating regimen. This case report demonstrates the importance and impact of a rare genetic obesity disorder diagnosis in a family. Additionally, it highlights the value of genetic testing in patients with a high suspicion of a genetic obesity disorder as it can eventually lead to personalized treatment, such as guidance by specialized healthcare professionals and educated caregivers or targeted pharmacotherapy.

Genetic Obesity Disorders: Body Mass Index Trajectories and Age of Onset of Obesity Compared with Children with Obesity from the General Population.

OBJECTIVE: We sought to assess body mass index trajectories of children with genetic obesity to identify optimal early age of onset of obesity (AoO) cut-offs for genetic screening. STUDY DESIGN: This longitudinal, observational study included growth measurements from birth onward of children with nonsyndromic and syndromic genetic obesity and control children with obesity from a population-based cohort. Diagnostic performance of AoO was evaluated. RESULTS: We describe the body mass index trajectories of 62 children with genetic obesity (29 nonsyndromic, 33 syndromic) and 298 controls. Median AoO was 1.2 years in nonsyndromic genetic obesity (0.4 and 0.6 years in biallelic LEPR and MC4R; 1.7 in heterozygous MC4R); 2.0 years in syndromic genetic obesity (0.9, 2.3, 4.3, and 6.8 years in pseudohypoparathyroidism, Bardet-Biedl syndrome, 16p11.2del syndrome, and Temple syndrome, respectively); and 3.8 years in controls. The optimal AoO cut-off was ≤3.9 years (sensitivity, 0.83; specificity, 0.49; area under the curve, 0.79; P < .001) for nonsyndromic and ≤4.7 years (sensitivity, 0.82; specificity, 0.37; area under the curve, 0.68; P = .001) for syndromic genetic obesity. CONCLUSIONS: Optimal AoO cut-off as single parameter to determine which children should undergo genetic testing was ≤3.9 years. In case of older AoO, additional features indicative of genetic obesity should be present to warrant genetic testing. Optimal cut-offs might differ across different races and ethnicities.

DNA methylation episignature and comparative epigenomic profiling of HNRNPU-related neurodevelopmental disorder.

PURPOSE: HNRNPU haploinsufficiency is associated with developmental and epileptic encephalopathy 54. This neurodevelopmental disorder is characterized by developmental delay, intellectual disability, speech impairment, and early-onset epilepsy. We performed genome-wide DNA methylation (DNAm) analysis in a cohort of individuals to develop a diagnostic biomarker and gain functional insights into the molecular pathophysiology of HNRNPU-related disorder. METHODS: DNAm profiles of individuals carrying pathogenic HNRNPU variants, identified through an international multicenter collaboration, were assessed using Infinium Methylation EPIC arrays. Statistical and functional correlation analyses were performed comparing the HNRNPU cohort with 56 previously reported DNAm episignatures. RESULTS: A robust and reproducible DNAm episignature and global DNAm profile were identified. Correlation analysis identified partial overlap and similarity of the global HNRNPU DNAm profile to several other rare disorders. CONCLUSION: This study demonstrates new evidence of a specific and sensitive DNAm episignature associated with pathogenic heterozygous HNRNPU variants, establishing its utility as a clinical biomarker for the expansion of the EpiSign diagnostic test.

Successful naltrexone-bupropion treatment after several treatment failures in a patient with severe monogenic obesity.

We describe the therapeutic journey of a 33-year-old patient with early-onset obesity (BMI 56.7 kg/m2) and hyperphagia due to a likely pathogenic heterozygous melanocortin-4 receptor (MC4R) gene variant. She was unsuccessfully treated with several intensive lifestyle interventions, gastric bypass surgery (-40 kg weight loss, followed by +39.8 kg weight regain), liraglutide 3 mg (-3.8% weight loss with sustained hyperphagia), and metformin treatment. However, naltrexone-bupropion treatment led to -48.9 kg (-26.7%) weight loss, of which -39.9 kg (-38.3%) was fat mass, in 17 months of treatment. Importantly, she reported improved hyperphagia and quality of life. We describe the potential beneficial effects of naltrexone-bupropion on weight, hyperphagia, and quality of life in a patient with genetic obesity. This extensive journey shows that various anti-obesity agents can be initiated, subsequently terminated when ineffective and substituted with other anti-obesity agents to identify the most efficient anti-obesity treatment.

Prevalence of congenital anomalies in the Dutch Caribbean islands of Aruba, Bonaire, and Curaçao.

BACKGROUND: Congenital anomalies represent an important global health issue. Data on the prevalence and pattern of congenital anomalies in the Caribbean region are scarce and lacking altogether in Aruba, Bonaire and Curaçao (ABC islands). METHODS: We performed a population-based surveillance study to determine the prevalence of structural congenital anomalies in the ABC islands, including all live births and stillbirths between January 1, 2008 and December 31, 2017 with major congenital anomalies according to EUROCAT guide 1.5. Terminations of pregnancy for fetal anomaly were included as well. Cases were identified by active case ascertainment, using multiple sources including pediatric patient files and discharge letters, delivery records, and clinical genetic patient files. Total and subgroup prevalence rates were compared between the three islands and to the French West Indies and Northern Netherlands. RESULTS: Total prevalence of congenital anomalies on the ABC islands was 242.97 per 10,000 births. Total prevalence of congenital anomalies in Bonaire (325.15 per 10,000 births) was higher compared to Aruba (233.29 per 10,000 births) and Curaçao (238.58 per 10,000 births), which was mainly attributable to a higher prevalence of limb anomalies, in particular polydactyly, in Bonaire. Total prevalence of congenital anomalies on the ABC islands was comparable to the French West Indies (248.69 per 10,000 births) but significantly lower compared to the Northern Netherlands (298.98 per 10,000 births). In the subgroup prevalence analysis, the prevalence of polydactyly and atrial septal defect on the ABC islands was significantly higher compared with the French West Indies and the Northern Netherlands, while the prevalence of congenital anomalies of the kidney and urinary tract and genetic disorders was significantly lower. CONCLUSIONS: This is the first study to establish the prevalence and pattern of congenital anomalies on the ABC islands, which is important to inform healthcare managers and policymakers and to provide a basis for continuous surveillance of congenital anomalies.

Lymphedema as first clinical presentation of Cantu Syndrome: reversed phenotyping after identification of gain-of-function variant in ABCC9.

Cantu Syndrome (CS), [OMIM #239850] is characterized by hypertrichosis, osteochondrodysplasia, and cardiomegaly. CS is caused by gain-of-function (GOF) variants in the KCNJ8 or ABCC9 genes that encode pore-forming Kir6.1 and regulatory SUR2 subunits of ATP-sensitive potassium (KATP) channels. Many subjects with CS also present with the complication of lymphedema. A previously uncharacterized, heterozygous ABCC9 variant, p.(Leu1055_Glu1058delinsPro), termed indel1055, was identified in an individual diagnosed with idiopathic lymphedema. The variant was introduced into the equivalent position of rat SUR2A, and inside-out patches were used to characterize the KATP channels formed by Kir6.2 and WT or mutant SUR2A subunits coexpressed in Cosm6 cells. The indel1055 variant causes gain-of-function of the channel, with an increase of the IC50 for ATP inhibition compared to WT. Retrospective consideration of this individual reveals clear features of Cantu Syndrome. An additional heterozygous ABCC9 variant, p.(Ile419Thr), was identified in a second individual diagnosed with lymphedema. In this case, there were no additional features consistent with CS, and the properties of p.(Ile416Thr) (the corresponding mutation in rat SUR2A)--containing channels were not different from WT. This proof-of-principle study shows that idiopathic lymphedema may actually be a first presentation of otherwise unrecognized Cantu Syndrome, but molecular phenotyping of identified variants is necessary to confirm relevance.

Episignature Mapping of TRIP12 Provides Functional Insight into Clark-Baraitser Syndrome.

Clark-Baraitser syndrome is a rare autosomal dominant intellectual disability syndrome caused by pathogenic variants in the TRIP12 (Thyroid Hormone Receptor Interactor 12) gene. TRIP12 encodes an E3 ligase in the ubiquitin pathway. The ubiquitin pathway includes activating E1, conjugating E2 and ligating E3 enzymes which regulate the breakdown and sorting of proteins. This enzymatic pathway is crucial for physiological processes. A significant proportion of TRIP12 variants are currently classified as variants of unknown significance (VUS). Episignatures have been shown to represent a powerful diagnostic tool to resolve inconclusive genetic findings for Mendelian disorders and to re-classify VUSs. Here, we show the results of DNA methylation episignature analysis in 32 individuals with pathogenic, likely pathogenic and VUS variants in TRIP12. We identified a specific and sensitive DNA methylation (DNAm) episignature associated with pathogenic TRIP12 variants, establishing its utility as a clinical biomarker for Clark-Baraitser syndrome. In addition, we performed analysis of differentially methylated regions as well as functional correlation of the TRIP12 genome-wide methylation profile with the profiles of 56 additional neurodevelopmental disorders.