Chromosome 14q32.2 Imprinted Region Disruption as an Alternative Molecular Diagnosis of Silver-Russell Syndrome

The Journal of Clinical Endocrinology & Metabolism, Jul 2018

Silver-Russell syndrome (SRS) (mainly secondary to 11p15 molecular disruption) and Temple syndrome (TS) (secondary to 14q32.2 molecular disruption) are imprinting disorders with phenotypic (prenatal and postnatal growth retardation, early feeding difficulties) and molecular overlap.

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Chromosome 14q32.2 Imprinted Region Disruption as an Alternative Molecular Diagnosis of Silver-Russell Syndrome

J Clin Endocrinol Metab, July Chromosome 14q32.2 Imprinted Region Disruption as an Alternative Molecular Diagnosis of Silver-Russell Syndrome Sophie Geoffron 14 Walid Abi Habib 14 Sandra Chantot-Bastaraud 13 Be´ atrice Dubern 11 Virginie Steunou 14 Salah Azzi 14 Alexandra Afenjar 19 Tiffanny Busa 18 Ana Pinheiro Canton 14 17 Christel Chalouhi 16 Marie-Noe¨ lle Dufourg 14 Blandine Esteva 14 M e´lanie Fradin 21 David Genevie` ve 15 20 Solveig Heide 13 Bertrand Isidor 8 Agn e`s Linglart 6 7 9 Fanny Morice Picard 4 Catherine Naud-Saudreau 5 Isabelle Oliver Petit 2 Nicole Philip 18 Catherine Pienkowski 2 Marl e`ne Rio 0 3 Sylvie Rossignol 10 12 Maith e´ Tauber 1 2 Julien Thevenon Thuy-Ai Vu-Hong 14 Madeleine D Harbison Jennifer Salem Fr e´d e´ric Brioude 14 Ir e`ne Netchine 14 Eloı¨se Giabicani 14 0 INSERM UMR 1163, Paris Descartes- Sorbonne Paris Cit e ́ Universite ́ , Institut Imagine , F-75015 Paris , France 1 INSERM U1043, Centre de Physiopathologie de Toulouse Purpan , Universit e ́ 2 CHU de Toulouse, Hoˆ pital des Enfants, Unit e ́ d'Endocrinologie, Ob e ́sit e ́, Maladies Osseuses, G e ́n e ́tique et Gyn e ́cologie M e ́dicale , 31059 Toulouse cedex 9 , France 3 APHP, Hoˆ pital Necker- Enfants-Malades, Service de G e ́ne ́ tique , F-75015 Paris , France 4 CHU de Bordeaux, Hoˆ pital Pellegrin-Enfants, Department of Pediatric Dermatology, National Centre for Rare Skin Disorders , 33076 Bordeaux cedex , France 5 Bretagne Sud Hospital Center , Pediatric Endocrinology and Diabetology, 56322 Lorient cedex , France 6 APHP, Bic eˆtre Paris Sud Hospital, Department of Pediatric Endocrinology and Diabetology , 94270 Le Kremlin Bic eˆtre , France 7 INSERM U1169, Bic eˆtre Paris Sud Hospital , 94270 Le Kremlin Bic eˆtre, Universit e ́ Paris-Saclay , France 8 CHU Nantes, Service de G e ́n e ́tique M e ́dicale , 44093 Nantes cedex 1 , France 9 APHP, Bic eˆtre Paris Sud Hospital, Reference Center for Rare Mineral Metabolism Disorders (Fili e`re OSCAR) and the Plateforme d'Expertise Paris Sud Maladies Rares , 94270 Le Kremlin Bic eˆtre , France 10 INSERM U1112, Laboratoire de G e ́n e ́tique M e ́dicale, Institut de G e ́n e ́tique M e ́dicale d'Alsace, Facult e ́ de M e ́decine de Strasbourg , 67091 Strasbourg cedex , France 11 Sorbonne Universit e ́, INSERM, UMRS U1166 (Eq 6) Nutriomics, Institut de Cardiom e ́tabolisme et Nutrition, APHP, Hoˆ pital Armand Trousseau, Service de Nutrition et de Gastroent e ́rologie P e ́diatriques , F-75012 Paris , France 12 Hoˆ pitaux Universitaires de Strasbourg, Service de P e ́diatrie , 67091 Strasbourg cedex , France 13 APHP, Hoˆ pital Armand Trousseau, D e ́partement de G e ́n e ́tique, UF de G e ́n e ́tique Chromosomique , F-75012 Paris , France 14 Sorbonne Universit e ́, INSERM, UMR_S 938 Centre de Recherche Saint Antoine, Assistance Publique - Hoˆ pitaux de Paris (APHP), Hoˆ pital Armand Trousseau, Explorations Fonctionnelles Endocriniennes , F-75012 Paris , France 15 INSERM U1183, Institute of Regenerative Medicine and Biotherapie, Montpellier University, CHU Montpellier , 34295 Montpellier cedex 5 , France 16 APHP, ~ Hoˆ pital Necker-Enfants-Malades, Service de P e ́diatrie G e ́n e ́rale , F-75015 Paris , France 17 Unidade de Endocrinologia Gen e ́tica, Laborato ́ rio de Endocrinologia Celular e Molecular LIM25, Disciplina de Endocrinologia, Faculdade de Medicina da Universidade de S 18 Assistance Publique des Hoˆ pitaux de Marseille, Hoˆ pital Timone Enfants, Centre de R e ́f e ́rence Anomalies du D e ́veloppement et Syndromes Malformatifs Provence Alpes Coˆ te d'Azur, D e ́partement de Ge ́ n e ́tique M e ́dicale et G e ́nomique Fonctionnelle, Aix Marseille Universit e ́ , 13284 Marseille cedex 7 , France 19 Sorbonne Universit e ́, APHP, Hoˆ pital Armand Trousseau, D e ́partement de G e ́n e ́tique Clinique et Centre de R e ́f e ́rence Anomalies du D e ́veloppement et Syndromes Malformatifs et D e ́ficiences Intellectuelles de Causes Rares , F-75012 Paris , France 20 H oˆpital Arnaud de Villeneuve, Unit e ́ de G e ́n e ́tique Clinique, D e ́partement de G e ́n e ́tique M e ́dicale, Maladies Rares et M e ́decine Personnalis e ́e , 34090 Montpellier , France 21 Centre Hospitalier Universitaire (CHU) Hoˆ pital Sud, Service de G e ́n e ́tique Clinique, Centre de R e ́f e ́rence Maladies Rares Centre Lab e ́llis e ́ 'Anomalies du D e ́veloppement'-Ouest , 35203 Rennes cedex 2 , France - *These authors are co-first authors. Abbreviations: aGNRH, gonadotrophin-releasing hormone analog; BMI, body mass index; GH, growth hormone; IG-DMR, intergenic differentially methylated region; LOM, loss of methylation; MLMD, multilocus methylation defect; NH-CSS, Netchine-Harbison clinical scoring system; SDS, standard deviation score; SGA, small for gestational age; SRS, SilverRussell syndrome; TS, Temple syndrome. Paul-Sabatier, 31330 Toulouse, France; 23Centre de R e´f e´rence du Syndrome de Prader Willi, 31059 Toulouse cedex 9, France; 24CHU Dijon, Hoˆ pital d’Enfants, Centre de G e´n e´tique et Centre de R e´f e´rence “Anomalies du D e´veloppement et Syndromes Malformatifs,” 21079 Dijon cedex, France; 25CHU Grenoble-Alpes, Hoˆ pital Couple-Enfants, Centre de G e´n e´tique, Centre de R e´f e´rence “Anomalies du D e´veloppement et Syndromes Malformatifs,” 38700 La Tronche, France; 26Icahn School of Medicine at Mount Sinai, Department of Pediatrics, New York, New York 10029; and 27The MAGIC Foundation, Russell-Silver Syndrome/Small for Gestational Age Research & Education Fund, Warrenville, Illinois 60555 Context: Silver-Russell syndrome (SRS) (mainly secondary to 11p15 molecular disruption) and Temple syndrome (TS) (secondary to 14q32.2 molecular disruption) are imprinting disorders with phenotypic (prenatal and postnatal growth retardation, early feeding difficulties) and molecular overlap. Objective: To describe the clinical overlap between SRS and TS and extensively study the molecular aspects of TS. Patients: We retrospectively collected data on 28 patients with disruption of the 14q32.2 imprinted region, identified in our center, and performed extensive molecular analysis. Results: Seventeen (60.7%) patients showed loss of methylation of the MEG3/DLK1 intergenic differentially methylated region by epimutation. Eight (28.6%) patients had maternal uniparental disomy of chromosome 14 and three (10.7%) had a paternal deletion in 14q32.2. Most patients (72.7%) had a Netchine-Harbison SRS clinical scoring system $4/6, and consistent with a clinical diagnosis of SRS. The mean age at puberty onset was 7.2 years in girls and 9.6 years in boys; 37.5% had premature pubarche. The body mass index of all patients increased before pubarche and/or the onset of puberty. Multilocus analysis identified multiple methylation defects in 58.8% of patients. We identified four potentially damaging genetic variants in genes encoding proteins involved in the establishment or maintenance of DNA methylation. Conclusions: Most patients with 14q32.2 disruption fulfill the criteria for a clinical diagnosis of SRS. These clinical data suggest similar management of patients with TS and SRS, with special attention to their young age at the onset of puberty and early increase of body mass index. (J Clin Endocrinol Metab 103: 2436–2446, 2018) Irisk of perinatal morbidity and mortality and metabolic mpaired fetal growth is associated with an increased problems later in life, according to the Developmental Origins of Health and Disease theory ( 1 ). Imprinted regions are known to play an important role in fetal growth ( 2 ). Paternally expressed genes are mostly involved in growth promotion, whereas maternally expressed genes repress it. Most imprinted regions are methylated on the maternal allele. In humans, only two regions are methylated in the male germ line ( 3, 4 ), the 11p15 H19/IGF2 intergenic differentially methylated region (IG-DMR) and the 14q32.2 MEG3/DLK1:IG-DMR, involved in Silver-Russell syndrome (SRS)/Beckwith-Wiedemann syndrome, and Temple syndrome (TS)/Kagami-Ogata syndrome, respectively. SRS is characterized by fetal and postnatal growth retardation and feeding difficulties ( 5–9 ). Epimutation, resulting in the loss of methylation (LOM) of H19/IGF2: IG-DMR on the paternal allele, is identified in 50% of SRS cases ( 10–12 ). In this region, the imprinting center H19/IGF2:IG-DMR is methylated on the paternal allele, resulting in IGF2 expression. When unmethylated, as on the maternal allele, it allows H19 expression, a long noncoding RNA (Fig. 1). The key role of IGF2 in prenatal growth is well-established. Maternal uniparental disomy for chromosome 7 [upd( 7 )mat] is seen in ;5% to 10% of patients with SRS ( 13 ). However, for 35% to 40% of patients with SRS, the molecular etiology remains unknown. TS, first clinically and molecularly described in 1991, associates fetal and postnatal growth retardation, hypotonia, obesity, and early puberty ( 14 ). TS is caused by disruption of the 14q32.2 imprinted region, where MEG3/DLK1:IG-DMR is methylated on the paternal allele. MEG3/DLK1:IG-DMR methylation results in DLK1, RTL1, and DIO3 expression, whereas long noncoding RNAs (MEG3 and MEG8), microRNAs, and small nucleolar RNAs are expressed when it is unmethylated (as on the maternal allele) (Fig. 1). In a metaanalysis of 51 patients with TS, the molecular anomalies identified consisted mostly of maternal uniparental disomy of chromosome 14 [upd( 14 )mat] (78.4%), epimutation of MEG3/DLK1:IG-DMR on the paternal allele (11.8%), and paternal deletion of the MEG3/DLK1 domain (9.8%) ( 15 ). A cohort of 32 patients with Clinical assessment and definitions The Netchine-Harbison clinical scoring system (NH-CSS), recently adopted as the clinical definition of SRS by the first international consensus on this syndrome ( 9, 11 ), was applied to each of the 28 patients. This scoring system defines a suspicion of SRS if at least four of the six following criteria are met: ( 1 ) being born small for gestational age (SGA) [birth weight and/or birth length #22 standard deviation score (SDS) for gestational age], ( 2 ) postnatal growth failure (height at 24 6 1 months #22 SDS or height #22 SDS from midparental target height), ( 3 ) relative macrocephaly at birth (head circumference at birth $1.5 SDS above birth weight and/or length SDS), ( 4 ) protruding forehead (forehead projecting beyond the facial plane on a side view as a toddler), ( 5 ) body asymmetry [leg length discrepancy $0.5 cm or arm asymmetry or leg length discrepancy ,0.5 cm with at least two other asymmetrical body parts (one nonface)], and ( 6 ) low body mass index (BMI) (BMI #22 SDS at 24 months) and/or feeding difficulties defined by the use of a feeding tube and/or cyproheptadine for appetite stimulation. See the Supplemental Materials and Methods for auxologic methods. Premature pubarche was defined by the appearance of pubic or axillary hair occurring before eight years in girls and nine years in boys ( 20 ). Precocious puberty was defined by breast development (thelarche) before age 8 years in girls and testicular enlargement before age 9 years in boys ( 21 ). Exaggerated adrenarche was defined by high levels of serum dehydroepiandrosterone sulfate for age (after other diseases were excluded) ( 22 ). Molecular analysis Methylation studies at both 11p15 H19/IGF2:IG-DMR and MEG3/DLK1:IG-DMR loci are described in the Supplemental Data. All patients had hypomethylation at MEG3/DLK1:IGDMR. We distinguished three different mechanisms: upd( 14 )mat, deletion, and LOM by epimutation. Single nucleotide polymorphism microarray analysis We analyzed the DNA samples using Illumina CytoSNP-12 arrays (Illumina, San Diego, CA) to distinguish between MEG3/ DLK1:IG-DMR epimutation, upd( 14 )mat, and large copy number variations. See the Supplemental Materials and Methods for details. IG-DMR and exome variant sequencing Whole-exome sequencing Library preparation, exome capture, sequencing, and data analysis were performed by IntegraGen SA (Evry, France). The sequencing methods and bioinformatics analysis are detailed in the Supplemental Materials and Methods. Statistical analysis The characteristics of the population are described as percentages for qualitative variables or as SDS and mean (range) for 14q32.2 anomalies has recently been reported and, again, most had upd( 14 )mat (71.9%), whereas only 18.8% had epimutations ( 16 ). Clinical overlap between SRS and TS has been previously highlighted in reports of patients presenting with a clinical diagnosis of SRS with no 11p15 disruption or upd( 7 )mat, but for whom chromosome 14q32.2 anomalies were identified ( 17–19 ). Thus, these syndromes overlap in terms of phenotype and may be caused by anomalies of imprinted regions sharing similar molecular organization, both methylated in the male germ line. We identified 28 patients with chromosome 14q32.2 disruption in our molecular diagnostic laboratory. The purpose of this study was to clinically and molecularly characterize these patients to determine the clinical overlap with patients with SRS. Furthermore, we sought to identify the mechanism involved in the onset of 14q32.2 epimutation. Patients and Methods Study population The study population consisted of 28 patients with chromosome 14q32.2 disruption. The molecular diagnosis of 25 patients was performed in our laboratory and three upd( 14 ) mat were identified without methylation analysis in other diagnostic laboratories. All patients were either followed in our clinic or were referred by other clinical centers for molecular analysis. A clinical file, including extensive clinical data, growth charts, a detailed phenotypic description, and pictures was completed for all patients. Each patient had been examined by a geneticist and/or a pediatric endocrinologist. Written informed consent for participation was received either from the patients themselves or their parents, in accordance with French national ethics rules for patients recruited in France (Assistance Publique – Hoˆ pitaux de Paris authorization no. 681) and with the institutional review board I00000204 of the Mount Sinai School of Medicine, New York, for patients recruited in the United States. continuous variables. For subgroup comparisons, we used the Wilcoxon Mann-Whitney test and the Fischer test. Results Patients Our cohort was composed of 28 patients (17 girls). Three patients (10.7%) were conceived with the aid of medically assisted procreation: two in vitro fecundations and one intrauterine insemination. The median maternal age was 29.1 (19.8–41.5) years and paternal age was 30.7 (25.8-44.8) years. Molecular diagnosis Classical molecular anomalies found in SRS [i.e., 11p15 epimutation and upd( 7 )mat] were ruled out for all but three patients with upd( 14 )mat not identified in our laboratory. All patients presented with chromosome 14q32.2 hypomethylation at the MEG3/DLK1:IG-DMR, which was secondary to upd( 14 )mat in eight (28.6%) patients or to a paternal deletion of DLK1/MEG3 region in three (10.7%), whereas 17 (60.7%) had MEG3/DLK1:IG-DMR LOM caused by epimutation on the paternal allele. This was ascertained after ruling out a upd( 14 )mat or deletion of the DLK1/MEG3 region by single nucleotide polymorphism microarray. Clinical features The median age at the end of the study was 7.5 (1.3 to 21.6) years. Birth parameters, postnatal growth, dysmorphic anomalies, psychomotor development, and associated malformations are summarized in Table 1. Dysmorphic features such as protruding forehead, prominent heel, tented appearance of the mouth, and acromicria are presented in Supplemental Fig. 1. Eight of the 23 patients, for whom data were available, were treated with recombinant growth hormone (GH) therapy from a mean age of 4.7 (1.1 to 11.3) years according to the SGA indication and posology ( 23 ). NH-CSS Among patients for whom all items of the NH-CSS were available, 72.7% (16/22) had a score $4/6, and consistent with a clinical diagnosis of SRS. One item was missing in six patients, of whom two had an NH-CSS score of 4/5 (compatible with a diagnosis of SRS), one had a score of 3/5 and three had scores of 2/5, which does not fulfill the criteria for a clinical diagnosis of SRS (Supplemental Fig. 2). Among the six patients who did not fulfill the NH-CSS criteria, five (83.37%) had an epimutation and one a deletion (case 26). Puberty and pubarche We collected data on puberty and pubarche for all patients but one (one girl for whom data were not available). At the end of the study, 11 patients had gone into puberty, eight girls and three boys; the oldest among the other nonpubertal patients was an 8.7-year-old girl. Of these 11 patients, six (54.5%) had precocious puberty, including five (62.5%) girls and one (33.3%) boy. Four (66.7%) had epimutations and two (33.3%) had upd( 14 )mat. Puberty occurred early for the other three girls, before age 9 years, and was rapidly progressive, with menarche ,1 year after breast development for two of them. Puberty also started early for the other two boys, at 10.0 and 10.2 years (Table 2). Six (54.5%) patients were treated with gonadotrophin-releasing hormone analogs (aGnRHs) to suppress puberty at a mean age of 7.9 (5.0 to 10.3) years. Six (54.5%) patients had exaggerated adrenarche; four among them were treated with cyproterone acetate. Epiphyseal fusion occurred early in four patients, at 12.7 (11.2 to 13.8) years in girls (n = 3) and 13.8 years in one boy, without aGnRH treatment. The mean final height of the girls (n = 4) was 143.5 (141.0 to 145.0) cm, corresponding to 23.6 (24.0 to 23.3) SDS, according to Sempe´ ( 24 ), with a mean pubertal growth spurt of 12.8 (10.0 to 17.0) cm without aGnRH and 25.5 cm for the girl who was treated. One boy had a final height of 150.0 cm (23.9 SDS), with a pubertal growth spurt of 19.3 cm; the second had a final height of 169 cm (20.8 SDS), far from his target height (+2.7 SDS). None of these patients received recombinant GH treatment. All clinical data concerning puberty and pubarche of these 11 patients are summarized in Supplemental Table 1. Metabolic outcomes The age of adiposity rebound was precocious for 93.8% (15/16 for whom data were available) of the patients, with a mean at 2.1 (1.0 to 6.5) years. Twelve patients (75.0%) had adiposity rebound by the age of 2 years. Among patients with precocious adiposity rebound, only one needed enteral feeding. For this patient, nutrition intake is on the decrease but she experienced complete anorexia. For all other patients, BMI had spontaneously grown precociously. The BMI of all patients for whom puberty had started increased markedly (.1 SDS) before the onset of pubarche and/or central puberty. Extensive molecular analysis Methylation analysis of 18 imprinted loci We studied the methylation levels at 18 imprinted loci, using TaqMan allele-specific methylated multiplex real-time quantitative polymerase chain reaction, as previously described ( 19 ), and methylation specific multiplex ligation-dependent probe amplification (for chromosomes 6, 7, and 14) for 23 patients (Fig. 2). We found that 58.8% of patients with epimutation of the MEG3/DLK1:IG-DMR had methylation defects within at least one of the other studied loci. The upd( 14 )mat group, as well as one patient with a paternal deletion (case 25), had normal methylation levels at all other studied loci, as expected for patients with a cytogenetic defect of the 14q32.2 region. We did not perform these analysis for two patients with deletions and the three with upd( 14 )mat not identified in our laboratory. Screening for regulatory cis-element defects We searched for large deletions or duplications of the 14q32.2 region, using Illumina CytoSNP-12 arrays and multiplex ligation-dependent probe amplification to identify a potential cis-element defect within the MEG3/DLK1: IG-DMR and the MEG3:TSS-DMR that could lead to epimutations. No genetic defects were identified. We then looked for point mutations or small insertions/deletions by sequencing the entire MEG3/DLK1:IG-DMR and MEG3: TSS-DMR. We identified a homozygous variation (C.A) within the IG-DMR at NC_000014.8:g.101274313C.A in one patient. All other copy number variations that we found were already reported in public single nucleotide polymorphism databases, making it highly unlikely that they disturb any regulatory cis-elements of either DMR. Screening for regulatory trans-acting factors We carried out extensive mutation screening by whole exome-sequencing to look for trans-acting factor defects (Continued ) that could be involved in the establishment, maintenance, or reading of DNA methylation marks and/or related to imprinting disorders for all patients with epimutations. We first looked for heterozygous or homozygous missense/nonsense mutations or frameshifts insertions/ deletions in a common defective gene or different genes from the same family. Several genes were mutated in at least four patients, but their function or cellular localization made them very unlikely to be involved in the hypomethylation of the MEG3/DLK1:IG-DMR. We then examined genes encoding proteins known to be involved in the establishment/maintenance of DNA methylation marks related to imprinting, along with some of their binding proteins. The variations found within genes that could be involved in the hypomethylation defect are presented in Table 3. We validated these variations by Sanger sequencing and determined their parental transmission. Discussion Here we report clinical and molecular data on a large cohort of 28 patients with 14q32.2 imprinted region disruptions. The underlying mechanism for most of the patients (60.7%) is epigenetic, with a hypomethylation at n (%) 8 8 8 8/8 (100) 7 5/7 (71.4) 8 7 6/8 (75.0) 5/5 (100.0) 6/8 (75.0) 1/6 (16.7) 6/7 (85.7) 5/7 (71.4) 2/7 (28.6) 5/8 (62.5) 6/7 (85.7) 5/7 (71.4) 2/5 (40.0) 3/5 (60.0) 1/5 (20.0) 1/8 (12.5) 1/5 (20.0%) 1/6 (16.7%) aBicornurate uterus (n = 1), renal agenesis (n = 1), bilateral cryptorchidism (n = 2), nephrocalcinosis (n = 1). bMultiple agenesis (n = 3), crowded teeth (n = 3), delayed tooth eruption (n = 2). cAneurysm of the interatrial septum (n = 2), interatrial communication (n = 1). the paternal MEG3/DLK1:IG-DMR, whereas it has been reported to be between 11.8% and 18.8% in recent publications, consisting mostly of upd( 14 )mat ( 15, 16 ). We have described as many 14q32.2 epimutations as those already published (n = 17; Table 4). Epimutations at 14q32.2 have been rarely reported, and the mechanisms responsible for their occurrence are unknown. We therefore extensively studied the molecular pattern of the 17 patients with epimutations. We first looked for genetic disruption of MEG3/DLK1: Characteristics of Pubarche and Puberty for 11 Patients >9 Years of Age Pubarche Premature pubarche Exaggerated adrenarche Age at pubarche onset, y Bone age advancement during puberty Central puberty Age at thelarche or testicle enlargement, y Age at menarche, ya Delay between thelarche and menarche, ya BMI At pubarche onset (SDS) At central puberty onset (SDS) aWithout GnRH analogs. Total 3/8 (37.5%) 6/10 (60.0%) 9/9 (100.0%) — — — 2.1 (0.2–6.1) 1.9 (20.9 to 6.1) Girls (n = 8) 2/6 (33.3%) 4/7 (57.1%) 8.6 (6.3–12.0) 8/8 (100.0%) 7.2 (4.0–8.5) 10.2 (8.9–12.0) 1.9 (0.5–3.5) 2.1 (0.2-6.1) 1.8 (20.9 to 6.1) Boys (n = 3) 1/2 (50.0%) 2/3 (66.7%) 9.0 (8.0–10.0) 1/1 (100.0%) 9.6 (8.6–10.2) — — 2.3 (1.0–3.6) 2.4 (1.1–3.6) IG-DMR; however, we found no relevant sequence anomaly within this DMR. A large proportion of these patients (58.8%) had multilocus methylation defects (MLMDs). MLMDs have already been reported in patients with SRS with epimutations of H19/IGF2:IG-DMR and Beckwith-Wiedemann syndrome (another imprinted disorder leading to overgrowth secondary to abnormal methylation of the 11p15 region), although in much lower proportions, at 10% and 25%, respectively ( 25 ). The high proportion of our patients with MLMDs prompted us to look for genetic anomalies in trans-acting factors involved in the regulation of specific parental methylation at imprinted loci. However, we did not find any commonly disrupted genes in these patients, and only four patients had variations within genes known to be involved in the establishment or maintenance of methylation. Two of these patients, for whom we identified two paternally inherited variants in the KAP1 (rare variant) and UHRF1BP1 (unreported variation) genes, showed no evidence of MLMD, making it unlikely that these candidate genes are involved in the hypomethylation process of MEG3/ DLK1:IG-DMR. Another patient (case 13), with MLMD affecting six loci aside from 14q32.2, inherited an unreported maternal variation in the ARID4A gene, which has been shown to be involved in the maintenance of methylation at the Snrpn locus in mice. Finally, the fourth patient inherited a rare compound heterozygous variant of the SETDB1 gene, which is also a key player in the methylation process of imprinted genes. This patient showed hypomethylation only at 14q32.2 and no evidence of MLMD. These variations, as well as the ;60% of MLMDs identified among these patients, and the absence of cis-regulatory element defects within either MEG3/DLK1:IG-DMR or MEG3:TSS-DMRs, strongly Upd( 14 )mat: maternal uniparental disomy of chromosome 14. In our cohort, short stature was defined as length ,22 SDS at 24 mo; no definition for other cohorts. References 5–14 can be found in the Supplemental Data. suggest that these imprinting disturbances may be secondary to the dysfunction of one or several trans-acting factors. However, the potential involvement of the identified genetic variants in the LOM mechanism will require further investigation and/or identification of variants of these genes in additional patients. Molecular analysis allowed us to distinguish among three types of anomalies in the 14q32.2 region: epimutations, upd( 14 )mat, and deletions. Identification of deletions in these patients is important because of the risk of recurrence if inherited from the father. Aside from the need for genetic counseling, the identification of deletions would also modify the therapeutic strategy. Indeed, some reports have highlighted an increased risk of thyroid cancer [papillar carcinoma ( 26 )] in patients with a large deletion in the 14q32.2 region and/or mental disability in those with a YY1 deletion, a gene within the same 14q32 region ( 27 ). Among the three patients with deletions in this study, two had an already identified recurrent deletion, leading to cognitive delay and thyroid cancer (Supplemental Figure 3). This cancer risk must be considered and carefully evaluated for patients who are candidates for recombinant GH treatment because of short stature. We compared the clinical data from all published cases with our cohort (Table 4). We noticed the relatively high frequencies of NH-CSS items among the previously published patients (being born SGA, relative macrocephaly, feeding difficulties, short stature, and protruding forehead). Asymmetry was rare, as in our patients. In patients with SRS, asymmetry is also the less frequent sign when looking at the overall SRS group (57.3%) ( 14 ). As in our study, neonatal hypotonia, acromicria, obesity, and early puberty were frequent signs among patients with 14q32.2 anomalies. In our cohort, we found no substantial differences between the clinical features associated with epimutations and those associated with upd ( 14 )mat. The overlap between TS and SRS phenotypes has already been established in reported cases ( 17–19 ). Most of the patients in our cohort with a 14q32.2 disruption meet the criteria for a clinical diagnosis of SRS, as do other recently reported patients ( 16 ). Features classically reported in TS, such as neonatal hypotonia, acromicria, clinodactyly precocious puberty, obesity, and psychomotor delay, can also be identified in patients with SRS with 11p15 epimutations or upd( 7 )mat. Indeed, although certain classic characteristics are shared across the different molecular causes of SRS, others may be more common to one molecular cause than another ( 7, 9, 11 ). As such, patients with SRS can have a large spectrum of symptoms that are not included in the NH-CSS. The NHCSS was developed to provide easy, rapid, and sensitive diagnosis of SRS but is not meant to be exhaustive. As recently published in the first SRS international consensus ( 9 ), SRS is a clinical diagnosis, and molecular testing is useful for confirmation and stratification of the diagnosis. In our cohort, almost 3/4 of patients with 14q32.2 disruption fulfill the clinical criteria of the NHCSS ($4/6) and therefore may be considered as falling within the SRS phenotype. As also proposed in the SRS consensus for CDKN1C and IGF2 mutation, 14q32.2 disruption may be an alternative molecular diagnosis of SRS. In this retrospective analysis, we focused on pubarche and puberty in the oldest patients. All but three patients of the cohort were born SGA and puberty was precocious or early for all patients. SGA patients are known to develop early and rapidly progressive puberty ( 22, 28 ). These data are concordant with our clinical experience (work under submission) with patients with SRS with 11p15 disruption or upd(7)mat, who frequently present with aggressive early adrenarche and puberty. GnRH analogs have been shown to be beneficial for patients born SGA of small stature at pubertal onset (with or without a clinical diagnosis of SRS), together with recombinant GH treatment ( 29, 30 ). This therapy is now considered to be a possible personalized treatment of patients with SRS (9). Adding aGnRH to recombinant GH treatment may also be considered for patients with 14q32.2 anomalies. Although the role of DLK1 in the regulation of puberty is still unknown, a recent familial report suggests a link between DLK1 anomalies and pubertal onset ( 31 ). All of our patients started pubarche or puberty after a substantial rise in BMI. DLK1 expression has been implicated in the homeostasis of fat metabolism in studies in mice and humans, showing a role in preadipocyte differentiation, regulation of hypothalamic satiety, circulating leptin concentrations, peripheral adipose tissue activity, and muscle mass development ( 32–37 ). As in patients with SRS, the BMI of patients with 14q32.2 anomalies should be carefully followed to avoid an excessive and uncontrollable increase during infancy ( 38–40 ). The SRS international consensus recommends maintaining a ratio of weight/expected weight for height at 75% to 85% and BMI at 12 to 14 kg/m2 until the age of 4 years in those with SRS (9). Likely, it is also very important for patients with 14q32 disruption to try to limit their weight gain in their early life to avoid the onset of obesity and metabolic complications usually observed in this group of patients. When looking at the age at diagnosis, we identified two groups: one with early diagnosis (n = 17 before 3.2 years) and one with late diagnosis (n = 11 .8.5 years). The first group was typically diagnosed because of being born SGA and failure to thrive, whereas the second was diagnosed later because of precocious puberty and obesity. Because of the late diagnosis in these latter patients, which correspond to the classical presentation of TS described until now, none benefited from early care including active weight and pubertal management or recombinant GH therapy. Among a series of published cases with 14q32.2 disruption, only half were treated with an aGnRH during their precocious puberty and only about a fourth were treated with concomitant or isolated recombinant GH therapy ( 16 ). Using NH-CSS to screen these patients allows a more precocious diagnosis and the possibility of proposing an earlier specific management of their metabolic, growth, and pubertal issues. Further studies will be necessary to evaluate if patients with 14q32 disruption will benefit from this earlier management as do patients with SRS. Conclusion We provide clinical and molecular data to support that, as raised in the SRS international consensus, 14q32.2 disruption may be considered as an alternative molecular diagnosis of SRS. These patients should be managed with a close follow-up for early onset of obesity, pubarche, precocious puberty, and short stature. 14q32.2 disruption should be investigated in case of suspected SRS without 11p15 LOM or upd( 7 )mat, especially in the presence of more specific characteristics of TS such as neonatal hypotonia, acromicria, hyperphagia, and/or early obesity. In patients with MEG3/DLK1:IG-DMR hypomethylation, an additional molecular analysis must be carried out to identify any paternal deletion within the 14q32.2 region because of the different prognosis and management of these patients. Elucidation of the mechanisms that control epimutation should be a research priority because of the high prevalence of MLMD in these patients. Acknowledgments We thank the patients, their families and physicians, the “Association Française des Familles ayant un enfant atteint du Syndrome Silver-Russell ou ne´ Petit pour l’ Aˆge Gestationnel (AFIF/PAG),” and The MAGIC Foundation for their support. We also thank Mrs. Laurence Perin, Mrs. Nathalie Thibaut, Mrs. Cristina Das Neves, Mrs. Marilyne le Jule, and Mrs. Evelyne Tagodoe, the diagnostic and research technicians of the Pediatric Endocrinology Department, Trousseau Pediatric Hospital, and Dr. Boris Keren for his help in molecular analysis. Financial Support: This work was supported by the Institut National de la Sant e´ et de la Recherche Me´dicale (INSERM) and funding from the Universit e´ Pierre et Marie Curie (UPMC-Paris VI). W.A.H. was supported by the People Programme Marie Curie Actions (MCA) of the European Union’s Seventh Framework Programme FP7/ITN Ingenium 2007–2013 under REA grant agreement no. 290123 and by the Soci e´te´ Française d’Endocrinologie et Diabe´tologie Pe´diatrique. A.P.C. was supported by CNPq under grant no. 233262/20148. F.B. was supported by a Novo Nordisk “Growth Hormone, Growth and Metabolism” grant. F.B. and I.N. are members of the European Congenital Imprinting Disorders Network European Cooperation in Science and Technology (BM1208). Correspondence and Reprint Requests: Ire`ne Netchine, MD, PhD, Explorations Fonctionnelles Endocriniennes, H oˆpital Armand Trousseau, 26 avenue du Dr Arnold Netter, 75571 Paris cedex 12, France. E-mail: . Disclosure Summary: The authors have nothing to disclose. Geoffron et al 1. Barker DJ , Osmond C , Golding J , Kuh D , Wadsworth ME . Growth in utero, blood pressure in childhood and adult life, and mortality from cardiovascular disease . 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Geoffron, Sophie, Abi Habib, Walid, Chantot-Bastaraud, Sandra, Dubern, Béatrice, Steunou, Virginie, Azzi, Salah, Afenjar, Alexandra, Busa, Tiffanny, Pinheiro Canton, Ana, Chalouhi, Christel, Dufourg, Marie-Noëlle, Esteva, Blandine, Fradin, Mélanie, Geneviève, David, Heide, Solveig, Isidor, Bertrand, Linglart, Agnès, Morice Picard, Fanny, Naud-Saudreau, Catherine, Oliver Petit, Isabelle, Philip, Nicole, Pienkowski, Catherine, Rio, Marlène, Rossignol, Sylvie, Tauber, Maithé, Thevenon, Julien, Vu-Hong, Thuy-Ai, Harbison, Madeleine D, Salem, Jennifer, Brioude, Frédéric, Netchine, Irène, Giabicani, Eloïse. Chromosome 14q32.2 Imprinted Region Disruption as an Alternative Molecular Diagnosis of Silver-Russell Syndrome, The Journal of Clinical Endocrinology & Metabolism, 2018, 2436-2446, DOI: 10.1210/jc.2017-02152