Best Practice & Research Clinical Endocrinology & Metabolism 26 (2012) 133143

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Best Practice & Research Clinical Endocrinology & Metabolism

journal homepage: www.elsevier.com/locate/beem

Genetic testing in patients with obesity

F. Phan-Hug, MD, Consultant Pediatric Endocrinologist a, J.S. Beckmann, PhD, Professor, Head of Department b, c, S. Jacquemont, MD, Associate physician b, *
a

EndocrinologyDiabetology Unit, Department of Pediatrics, CHUV, University Hospital, 16 Chemin de Monttan, 1007 Lausanne, Switzerland Service of Medical Genetics, CHUV, 2 av. Pierre Decker, Lausanne University Hospital, 1011 Lausanne, Switzerland c Department of Medical Genetics, University of Lausanne, Lausanne, Switzerland
b

Keywords: early-onset obesity heritability leptin melanocortin pathway genomic structural variants 16p11.2

The obesity epidemic is associated with the recent availability of highly palatable and inexpensive caloric food as well as important changes in lifestyle. Genetic factors, however, play a key role in regulating energy balance and numerous twin studies have estimated the BMI heritability between 40 and 70%. While common variants, identied through genome-wide association studies (GWAS) point toward new pathways, their effect size are too low to be of any use in the clinic. This review therefore concentrates on genes and genomic regions associated with very high risks of human obesity. Although there are no consensus guidelines, we review how the knowledge on these causal factors can be translated into the clinic for diagnostic purposes. We propose genetic workups guided by clinical manifestations in patients with severe early-onset obesity. While etiological diagnoses are unequivocal in a minority of patients, new genomic tools such as Comparative Genomic Hybridization (CGH) array, have allowed the identication of novel causal loci and next-generation sequencing brings the promise of accelerated pace for discoveries relevant to clinical practice. 2011 Elsevier Ltd. All rights reserved.

Introduction The obesity epidemic is clearly associated with the recent availability of highly palatable and inexpensive caloric food. In this new obesogenic environment, however, there remains a wide
* Corresponding author. Tel.: 41 21 314 55 93. E-mail address: sebastien.jacquemont@chuv.ch (S. Jacquemont). 1521-690X/$ see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.beem.2011.11.010

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distribution of adiposity and Body Mass Index (BMI) levels among individuals. The common interpretation of such distribution is variability in lifestyle which allows some individuals to remain lean and drives other to develop severe/morbid obesity.1 Lifestyle refers to a composite set of behaviors, such as physical exercise, quantity and quality of food intake, which are thought to be determined by socioeconomic factors.2,3 There is however compelling evidence that genetic factors, by controlling food intake, play a key role in regulating BMI and heritability studies provide evidence for a substantial genetic contribution with heritability estimates (h2) of w40%70%.46 Recent Twin studies7 show very high concordance rate in BMI between identical twins (66%) (who share the same genome except for somatic mutations and epigenetic factors) when compared to fraternal twins (28%) (equivalent to brothers or sisters who share 50% of their genome). Studies of twins reared apart represent an even more powerful research paradigm. In the latter case, BMI of the adopted twins is associated with the BMI of the biological parents but not with that of the adoptive ones.8 Obesity is often considered to have a complex multifactorial etiology, with the contribution of numerous genetic and non-genetic factors. Hence, estimates of heritability for complex disorders are shaped by a composite set of genetic factors, inherited or occurring de novo (accidently), which are strongly inuenced by environmental factors: e.g. in the context of food restriction, an individual may not develop obesity regardless of strong predisposing genetic factors.4 One should however stress that Mendelian entities also exist, wherein mutations in a single gene can represent alone, a very strong, highly-penetrant risk factor. In this review we will describe different genes and syndromes that have been described as causal factors of human obesity (or strongly predisposing). Although there are no consensus guidelines, we will then review how this knowledge has been translated into the clinic for diagnostic purposes mainly. Since studying and establishing associations or causal relationships is by design more accessible to extreme phenotypes, most of the syndromes described here involve severe earlyonset obesity. Non-syndromic monogenic forms of obesity Most of the identied monogenic forms of obesity disrupt hypothalamic functions. The hypothalamus receives input from numerous hormones several of which (glucocorticoids, estrogen, leptin, ghrelin) are key players regulating food intake and metabolism. The hypothalamus then responds by motor outputs to key regulatory sites including the anterior and posterior pituitary glands, the cerebral cortex, premotor and motor neurons in the brainstem and spinal cord, and autonomic (parasympathetic and sympathetic) preganglionic neurons. Their action results in endocrine, behavioral, and autonomic responses inuencing energy balance.9 Leptin and leptin receptor Serum leptin is produced by adipose tissue in levels proportional to its mass. The vast majority of obese patients are leptin-resistant rather than decient. Measuring very low leptin levels in the serum will help orient towards the diagnosis of leptin-deciency, which responds well to replacement therapy.10 Although extremely rare, autosomal recessive loss-of-function mutations in the Leptin (LEP, OMIM 164160) or Leptin receptor (LEPR, OMIM 601007) genes 11,12 have been shown to be central in the regulation of energy balance, neuroendocrine function and immunity.13 Patients with congenital leptin or leptin receptor-deciency present with normal birth weight, severe early-onset hyperphagia from the rst months of life, impaired satiety and, when food is denied, aggressive behavior. Motor and cognitive development is normal. Pubertal delay, signs of hypothyroidism (with slightly elevated thyroid stimulating hormone (TSH) levels) and reduced adult height point to neuroendocrine dysfunction (hypothalamic hypogonadism and hypothyroidism and reduced growth hormone secretion).14,15 Growth during childhood can be normal or slightly delayed and adult height is reduced due to absent pubertal growth spurt. Patients also have a susceptibility to infections with abnormal T-cell function and number.

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Melanocortin pathway POMC (pro-opiomelanocortin, OMIM #609734) encodes a precursor peptide whose downstream products include adrenocorticotrophin (ACTH) and a-, b-, g-melanocyte stimulating hormones (MSH). a-MSH is involved in satiety mechanisms in the hypothalamus, as well as the production of melanin. Autosomal recessive loss-of-function mutations in POMC, which are extremely rare, manifest as the association of early-onset obesity, ACTH-deciency and subsequent hypocortisolaemia. Other symptoms may include hypopigmentation of the skin (typically red hair) and hyperinsulinaemia, but patients show no other hypothalamic dysfunction. Heterozygous carriers of loss-of-function mutations in POMC have an elevated risk of developing obesity as shown in a large pedigree of a Turkish family were 11 of 12 heterozygotes were obese or overweight compared to only 1of 7 intrafamilial controls.16 In addition to null mutations in POMC, a heterozygous mutation in the region encoding b-MSH, leading to isolated deciency of b-MSH, a MC4R ligand derived from POMC, has also been described in association with increased risk of obesity.17,18 This deciency causes a phenotype similar to that observed in MC4R-deciency (childhood obesity, hyperphagia and increased linear growth without the other symptoms of POMC deciency). Pro-hormone convertase 1 (PCSK1, OMIM #600955) cleaves respectively, POMC into ACTH, proinsulin into insulin and gonadotropin releasing hormone (GnRH) precursor into GnRH and induces processing of various propeptides in the gut. Autosomal recessive null mutations in this gene are responsible of severe early-onset obesity, ACTH-deciency inducing hypocorticism, postprandial hypoglycemia (due to elevated proinsulin levels), and GnRH-deciency inducing hypogonadotropic hypogonadism. Severe absorptive dysfunction of the small intestine in neonatal life has been reported and small intestine dysfunction later in life is a particular sign that may help diagnose these patients.19 Serum leptin levels are elevated and appropriate to body mass.20 Carriers of heterozygous mutations are reported as clinically normal.21 Melanocortin 4 Receptor (MC4R, OMIM,*155541) mutations represent the most frequent known monogenic etiology of severe obesity: they are found in 16% of children with this condition22 and in the European population, the prevalence of deleterious MC4R mutations was estimated to be w1/1000. These mutations are characterized by either dominant or recessive mode of inheritance. The later being less frequent and more severe due to the lower residual protein activity.23 MC4R patients present with hyperphagia and severe hyperinsulinaemia. While there are no signs of either hypothalamic hypogonadism, GH- or ACTH-deciency, children manifest typically accelerated linear growth that is more pronounced than in BMI-matched controls. Height is increased up to 2.5 SD in childhood and 0.5 SD in adults. This may be related to the role of MC4R in the regulation of growth hormone resulting in increased GnRH levels.24 As a result, the clinical examination of these children reveals a big child with high fat and lean mass. While no specic complication is associated with MC4R-deciency, children may have a high risk of sleep apnea and adults an increased risk of glucose intolerance. Lower prevalence of hypertension induced by reduced sympathetic nervous system activity has also been reported.25,26 SIM1 (OMIM *603128) encodes a transcription factor involved in the hypothalamic development (paraventricular and supraoptic nuclei). SIM1 expressing neurons are responsible for the production of multiple neuropeptides including oxytocine, vasopressin, cortico-releasing hormone (CRH), (TRH), and somatostatin. While deleterious point mutations have not been identied in patients,27 deletions of SIM1 were reported by several groups to be associated with early-onset obesity, developmental delay and increased linear growth.2830 While complete knock-out of the gene in mice is lethal, haploinsufcient animals demonstrate hyperphagia and obesity correlated with oxytocine reduction.31 Developmental delay has been observed in all patients 2830 and reects the widespread involvement of SIM in neurogenesis. Syndromic forms of obesity The rst recognized syndromic forms of obesity have been described in association with other symptoms or malformations, which, in combination with their rare occurrence, allowed one to delineate them clinically in the absence of any knowledge on causal genes. In these syndromes, obesity and intellectual disabilities are often associated. The phenotype and functional studies have shown that many of these syndromes also involve hypothalamic dysfunction.32

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PraderWilli syndrome (PWS; OMIM # 176270) occurs in 1:25,000 births and is a well-known syndromic form of obesity associating neonatal feeding difculties related to hypotonia, childhood-onset obesity, intellectual disabilities and behavioral symptoms including obsessive compulsive disorder and autism. Seventeen % of patients with a 15q11-13 deletion and 64% of those with maternal uniparental disomy are diagnosed with a current or previous psychiatric illness.33 Manifestations of hypothalamic dysfunction include short stature (due to growth hormone-deciency) as well as cryptorchidism/ micropenis and delayed puberty (secondary hypogonadism)34. ACTH-deciency has also been reported. Different nutritional phases have been identied35: while hyperphagia is severe between 5 and 13 years of age, satiety may normalize in some adults.33 Adult life is marked by cardiovascular complications, obstructive and central sleep apnea and morbid obesity-related diabetes.36 In affected PWS patients, the paternal locus is absent due to a deletion (in 75% of the cases) or the loss of the entire paternal chromosome 15, due to the presence of two maternal chromosomes 15 (uniparental maternal disomy in 25% of the cases).36 Since the PWS locus (15q11.2q12) is under parental imprinting, methylation prole is the accepted screening method detecting both mutation mechanisms. Deletions are also visualized by CGH arrays. Growth hormone treatment improves the fat/lean body mass proportion and nal height. Early-onset intervention, including diet, sports and psychotherapy have been proven to be successful.37,38 Loss of oxytocin-producing neurons (working as an anorexic factor) and high levels of ghrelin (orexigen hormone) have been reported.3941 Maternal uniparental disomy of chromosome 14 (OMIM # 608149) has to be considered in a child with a PraderWilli-like phenotype with neonatal hypotonia inducing initial feeding problems, developmental delay, mild intellectual disabilities, early-onset truncal obesity, pre- and post-natal growth retardation with advanced bone age and premature puberty leading to reduced adult height. Syndromes of ciliary dysfunctions Certain ciliopathies are associated with obesity but the mechanisms underlying the dysfunction in energy balance regulation are still unknown. Knock-out models with loss-of-function of the ciliated hypothalamic POMC neurons may play a role in the pathogenesis of the obesity seen in BardetBiedl syndrome (BBS).42 Some studies suggest that cilia-related proteins are involved in leptin receptor signaling.43 Other studies showed ciliar dysfunction in the differentiating preadipocytes that will induce early adipocyte maturation and thus an early ability to accumulate fat and hence promote obesity.44 BardetBiedl syndrome (OMIM #209900) is a primary disorder of the cilium,45 involving the kidney and the eye. Retinitis pigmentosa and polydactyly are typical clinical signs and prognosis is associated to kidney dysfunction. The prevalence is estimated between 1/125,000 and 1/175,000 (orphanet, http://www.orpha.net/orphacom/cahiers/docs/FR/Prevalence_des_maladies_rares_par_prevalence_ decroissante_ou_cas.pdf) and 12 genes (BBS1 to BBS12) coding for cilia function have been implicated. The transmission is autosomal recessive and the severity of clinical manifestations (obesity, hypogenitalism, development delay) is variable and potentially related to the effects of additional mutations in other BBS genes resulting in true oligogenicity.42 Alstrom syndrome (#203800), a closely related ciliopathy is a differential diagnosis to consider. It is caused by recessive mutations in ALMS1, which codes for a putative ciliary protein. The risk of obesity is similar but diabetes is more frequent and cognition may be spared in 50% of the cases. A conerod retinal dystrophy in this pathology usually develops within a few weeks after birth and leads to blindness, usually by the second decade of life. Most patients develop mild-to-moderate slowly progressive bilateral sensoneural hearing loss and polydactyly is absent.46,47 Albright hereditary osteodystrophy, AHO (OMIM #103580) is a monogenic obesity disorder caused by heterozygous loss-of-function mutations in the GNAS1 gene which is under parental imprinting. GNAS1 codes for the G-protein a subunit (Gsa) which mediates receptor-stimulated cAMP generation. AHO patients are characterized by short stature, skeletal and neurocognitive defects. Subcutaneous ossications and shortening of long bones (4th and 5th metacarpals in particular) are characteristic. Obesity is only observed in mutations of the active maternal GNAS1 allele which leads to pseudohypoparathyroidism type 1A, (OMIM #103580) with severe Gsa-deciency and hormone resistance (PTH, TSH). Paternal mutations (on the less active allele) have little effects on Gsa expression or hormone sensitivity

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and lead to the AHO phenotype without hormone resistance (Pseudopseudohypoparathyroidism, OMIM #612463).48 Data suggest that CNS-specic disruption of Gsa is sufcient to produce severe obesity with abnormal melanocortin signaling and reduction in energy expenditure due to reduced sympathetic nerve activity.49 Obesity may therefore be related to lower energy expenditure without hyperphagia.50 Other rare syndromic disorders of obesity with associated hypothalamic dysfunction The Rohhadnet syndrome51 associates rapid onset obesity with hypothalamic, autonomic dysregulation and neuroendocrine tumors. The prognosis of this disorder depends on the central hypoventilation which can be lethal. A remarkable feature is the apparent normality of their rst 24 years of life, with sudden onset of rapid weight gain and obesity. The onset and severity of the different symptoms and the hypothalamicpituitary endocrine dysfunction is variable. Although very similar to the congenital hypoventilation syndrome (OMIM #209880), candidate gene (PHOX2B, TRKB, and BDNF) screening yielded no mutations.51,52 The report of monozygotic twins discordant for the diagnosis of Rohhadnet syndrome, suggested an alternative to a strictly genetic etiology (involving autoimmune or epigenetic factors),53 though the role of de novo appearing mutations, a well-known phenomenon in discordant twins, cant be ruled out. The association of intellectual disability, obesity and hypogonadotropic hypogonadism is a sequence found in many syndromes including PWS and BBS previously detailed in this review. This association suggests that many genes involved in neurodevelopment may impact hypothalamic function. Fragile X syndrome (OMIM #300624) is the most common genetic cause of inherited intellectual disabilities and autism. The rate of obesity in adolescence with fragile X syndrome is approximately 30%, a rate signicantly higher than in the general population. In addition, some fragile X patients present with a PraderWilli-like phenotype and associated hyperphagia and hypogonadotropic hypogonadism.54 This association is also encountered in other forms of obesity. Mutations in CUL4B (OMIM *300304), which encodes a ubiquitin E3 ligase subunit, cause an X-linked syndrome associating the aforementioned sequence with aggressive behavior and epilepsy.55 BorjesonForssman syndrome (OMIM # 301900) due to mutations in the X-linked PHF6 gene also associates intellectual disabilities, hypogonadism, gynecomastia and obesity.56 Investigating copy number variants Genes are normally present in two copies (diploid) except for those encoded by the sex chromosomes. Copy number variants (CNVs) (also known as genomic structural variants, GSVs), represent a deviation from the normal number of copies. Such changes are generated by deletions (one copy is left) or duplications (gain resulting in three copies).57 CNVs may be deleterious and associated with clinical manifestations when encompassed genes have tightly regulated expression. CNV-mediated diseases are regrouped under the denomination of genomic disorders. The recent introduction of array CGH (comparative genomic hybridization) in routine clinical practice has led to the identication of a urry of new genomic disorders including several obesityassociated syndromes, often associated with developmental delay and intellectual disabilities (DD/ ID). It is hard at this stage to exclude that the frequency of this particular association does not reect an ascertainment bias due to the fact that CGH array is primarily used in clinics to explore the etiologies of DD/ID.58 The 16p11.2 region Deletion of a 600 kb segment (chromosome 16: 29.530.1 Mb) at the 16p11.2 locus has been recently associated with highly-penetrant form of severe obesity (OMIM #611913).59 Carriers of this deletion are also at risk of neurodevelopmental disorders such as autism and language impairment.60 The cumulative risk for an adult carrier to develop obesity is approximately 70 percent (our own unpublished data) and the deletion is found in z0.6% of cases with severe obesity and in up to z3% of the patients associating obesity and DD/ID.59 The onset of BMI increase is variable and may appear as

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early as 2 years of age, but is most visible post-puberty. Adulthood onset has also been observed (our own unpublished data). At present, there are no data pointing towards particular candidate genes, among the 28 known coding genes encoded by this 600 kb deletion. The presence in almost all carriers of similar breakpoints (16: 29.530.1 Mb) prevented this far the study of smaller overlapping deletions and therefore the identication of a critical region or candidate genes.61 Interestingly, the duplication of the same region (patients carry a total of three copies and have therefore an excess in gene dosage), dened by the same breakpoints, was associated with the mirror BMI phenotype: children who carry this duplication are underweight in z 50% of the cases and adults have an 8-fold increased risk of being clinically underweight (BMI < 18.5).61 Currently, there are no data identifying the gene(s) responsible of the morbid phenotype. At the same 16p11.2 locus, a non-overlapping 220 kb long deletion, 500 kb telomeric of the aforementioned CNV, has independently been associated with childhood-onset obesity and insulin resistance (OMIM #613444), however, it is unclear whether the insulin levels in these patients are signicantly higher than in other age- and BMI-matched children.62 Far less is known on the full spectrum of clinical symptoms associated with this second CNV. While data on the penetrance of obesity among carriers of this CNV are still lacking, a series of recently reported carriers of this deletion suggested of a week association with DD/ID.63 Numerous studies point towards SH2B1, which is known to be involved in leptin and insulin signaling, as the most likely causal gene in this 220 kb deletion.62 CNVs encompassing genes involved in the downstream signaling pathway of MC4R have been reported in rare groups of patients with childhood-onset obesity: deletions of BDNF (Brain derived neurotrophic factor)64,65 were associated with obesity and developmental delay in a group of children with a WAGRO syndrome (OMIM #612469). This is consistent with the fact that BDNF is implicated in the regulation of body weight, with reduced expression during fasting and weight loss in mice triggered by BDNF administration.66 Evidence suggests that BDNF-expressing neurons lie downstream of MC4R neuronal pathways.67,68 Other well-known syndromes that we previously detailed, such as PraderWilli syndrome, can be caused by deletions (in 70% of the case69) visible by CGH arrays. As illustrated by these examples, unbiased whole-genome investigations using CGH array can detect CNVs without any prior information or suspicion on candidate genes or locus. Such procedure is therefore warranted as part of the routine and systematic etiological workup in patients with severe/morbid obesity with DD/ID (Fig. 1). In addition to its diagnostic value, this procedure may lead to the identication of new regions or genes implicated in energy balance. To sum up, the frequent association of DD/ID in patients with early-onset obesity across these different etiologies, suggests that many genes involved in energy balance play an essential role in other central nervous system (CNS) functions. In fact, every mutation identied so far in monogenic or monolocus forms of obesity, affects genes involved in energy balance at the CNS. Conversely, cognitive and behavioral assessments of children with idiopathic early-onset obesity show an array of cognitive decits. In many cases, childhood-onset obesity may be viewed as a neurodevelopmental disorder thus highlighting the fact that obesity can essentially also be considered as a CNS disease. The study of the common, low-penetrance susceptibility loci for obesity, described hereunder, also pinpointed the central role of the CNS in the etiology of this condition.70 The contribution of common variants Genome-wide association studies (GWAS) have identies over 32 common loci70 associated with BMI. Single nucleotide polymorphism (SNPs) tagging these regions do not represent as yet a practical tool for the clinician due to the small effect sizes and poor predictive power of these variants (e.g. the strongest effects recorded are observed in homozygous carriers of FTO risk allele which result in a 3 4 kg increase in weight). However these SNPs point towards many genes or pathways that may harbor rare mutations with large effects (e.g. MC4R, SH2B1 have been repeatedly tagged by SNPs70). The loci identied through GWAS could eventually turn out to be useful in clinical practice with the advent of cost-effective high-throughput sequencing methods, though many problems need rst to be solved (this could be the subject of another review).

F. Phan-Hug et al. / Best Practice & Research Clinical Endocrinology & Metabolism 26 (2012) 133143 Fig. 1. Group A: patients with associated ID (Intellectual disabilities). Group B: patients with hypothalamic dysfunction (neuroendocrine abnormalities). Bold & italic: genes, Bold: diagnosis. Boxed items: handles for differential diagnosis orientation. CGH array: This analysis should be performed systematically in the association of early-onset severe obesity and ID. *: conditions which can be classically diagnosed through CGH array (although any of the genes presented can be involved in a CNV). Gray background: diagnoses associated with neuroendocrine abnormalities. 139

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Genetic testing in the clinic There is no international consensus or guidelines regarding genetic testing for patients with obesity. Although unambiguous causal mutations have been published,59,71 the interpretation and prognostic value of mutations with incomplete penetrance and variable expressivity is challenging in all common and complex phenotypes. Therefore, we recommend, at this stage, to limit genetic testing to severe, early-onset and/or hypothalamic-related symptoms (hyperphagia, neuroendocrine dysfunction) and/ or syndromic forms of obesity. Investigating the more severe end of the spectrum will increase the chances of identifying a relevant genetic etiology. Hopefully, in the near future, testing will be extended to less severe forms. Various denitions are used in studies investigating childhood obesity, but to date, none of them is considered ideal and their use is mostly based on current practice or on practical aspects.72 Early-onset obesity is dened as BMI > 2 SDS or >95th percentile for age and sex, appearing before 5 years of age, that is before the normal adiposity rebound. An early adiposity rebound is associated with increased risk of adult obesity. Morbid or severe obesity is clearly dened in adults as a BMI > 40. In children, there is an ongoing debate as to what may be the most appropriate threshold: BMI ! 3 SD or !99 percentile.73 Family history is relevant in all cases but does not represent an indication or exclusion criteria for genetic testing. A dominant mutation or CNV may affect several individuals in the family. Conversely, the absence of family history may suggest a de novo mutation (appearing accidently during conception or fetal development) or an autosomal recessive form. The associated features should help the clinician to prioritize the etiological workup (Fig. 1). In children with rapid onset obesity a pituitary dysfunction (growth hormone-deciency, Cushing disease, Hypothyroidism or CNS-tumors typically cranyopharyngeoma) should be ruled out rst before any genetic testing (Fig. 1), since these etiologies require prompt treatment. Once these treatable etiologies are excluded, there are several clinical elements which may guide the clinician in his diagnostic workup. Obesity with associated intellectual disabilities and or additional features (Fig. 1 group A) Intellectual disabilities or signicant learning difculties are common features in childhood-onset obesity. Conversely, obesity is frequent in patients with intellectual disabilities (ID).74 This suggests that mutations in genes disrupting neurodevelopment are often non-specic and may also affect circuitry involved in food intake regulation. The approach in these cases stems from the large clinical experience accumulated in the genetic investigation of ID. The most sensitive screening analysis, available in clinical practice, is array CGH analysis which allows the identication of CNVs genomewide without prior information on disease etiology. Depending on the platform, this nowadays routine analysis which has a resolution ranging from 10000 to 100000 base pair, will for example identify deletions responsible of PraderWilli syndrome due to a 15q deletion (70% of the cases) as well as any sizeable deletion or duplication encompassing a known candidate gene or region (e.g. deletions of SIM1, BDNF, SH2B1 have been reported in obese patients, and new causal relationships, such as the deletion of the 16p11.2 region, have been established using this method59). CNVs may represent up to 20% of all mutations but the vast majority of smaller mutational events are not detectable by current CGH arrays. In the latter case, clinicians will need to gather anamnestic, clinical and endocrine data to orient their etiological workups. Several well-known syndromic forms associate remarkable features, allowing to establish or suspect a clinical diagnosis which can be conrmed by targeted mutation screening of the corresponding candidate genes (Fig. 1): e.g. ciliopathies, Albright syndrome, PWS, etc. Isolated early-onset or severe obesity (Fig. 1, group B) Impairment in food intake or energy expenditure seems to be an important clinical marker for the indication of genetic testing especially in early-onset obesity (Fig. 1). The yield of conrmed molecular diagnosis in this group of patients is still very low. Screening for MC4R mutations should be prescribed systematically as a rst line of investigation. Mutations in this gene alone explain between 1 and 6% of the etiology of severe early-onset obesity.75,76 There are no specic clinical or endocrine symptoms that

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strongly point to MC4R as the typical accelerated height and hyperinsulinaemia seen in MC4R patients that are also present in other patients with idiopathic or severe obesity. The penetrance of obesity in carriers of MC4R mutations is age-related. In adults it increases from 37% to 60% between 20 and 40 years of age (longitudinal dataset). In addition there is a generational effect with a discrepancy between the penetrance in children <18 years (79%) and older carriers >50 years (40%).75 This may suggest that the phenotypes in these monogenic forms are also very sensitive to the recent obesogenic environment. If MC4R mutations have been excluded, the presence of any neuroendocrine abnormality (hypogonadotropic hypogonadism, hypothyroidism or growth retardation) orients towards a complex group of differential diagnosis (Fig. 1 group B in Fig. 1) related to hypothalamic dysfunction. The type of endocrine abnormality will suggest a non-syndromic (absence of ID or malformative features) hypothalamic form of obesity. The majority of patients with early-onset and severe obesity remain without an etiological diagnosis. Either because the clinical spectrum associated to one of the aforementioned genes is broader that what is currently suspected (and therefore the wrong genes were selected in the list of differential diagnosis) or because the causal gene has not yet been identied or recognized as an etiology of morbid obesity. In both situations, exome sequencing (and full genome sequencing in the long term), will likely identify causal mutations in many currently unsolved cases. The thorough and unbiased nature of these high-throughput approaches have signicantly shaped the eld of Mendelian disease in the past 2 years by identifying numerous new rare de novo or inherited causal mutations. The rapidly evolving cost-effectiveness will allow a systematic implementation of these next-generation DNA sequencing diagnostic techniques in the clinic and it is likely that the diagnostic workup for severe early-onset obesity will be profoundly transformed in the near future.77,78 Clinical practice:  In children with rapid onset obesity a pituitary dysfunction or a CNS-tumor should rst be ruled out.  Genetic testing should be considered for severe, early-onset and/or syndromic forms of obesity and/or hypothalamic-related symptoms.  Neuroendocrine Dysfunction (hypogonadism, hypothyroidism, ACTH deciency, GH deciency) point to a group of differential diagnosis involving genes in the leptin melanocortin signaling pathway and can also be encountered in syndromic forms of obesity  Normal or tall stature seen in the majority of children with early-onset obesity does not rule out, as previously thought, genetic causes.  MC4R mutation should be screened rst in all early-onset obesities without associated medical conditions or malformations.  CGH array has emerged as a powerful tool to explore early-onset obesity especially when associated with intellectual disability and/or medical conditions or malformations. It will detect all genomic rearrangement without any a priori hypothesis. References
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