Camille Gosseaume

Thierry Fournier

Isabelle Jéru

Marie-Léone Vignaud

Isabelle Missotte

Françoise Archambeaud

Xavier Debussche

Céline Droumaguet

Bruno Fève

Sophie Grillot

Bruno Guerci

Sylvie Hieronimus

Yves Horsmans

Estelle Nobecourt

Catherine Pienkowski

Christine Poitou

Jean-Paul Thissen

Olivier Lascols

Séverine Degrelle

Vassilis Tsatsaris

Corinne Vigouroux
email: corinne.vigouroux@inserm.fr

Camille Vatier
email: camille.vatier@aphp.fr

Perinatal, Metabolic and Reproductive Features in PPARG-related Lipodystrophy

Keywords: diabetes mellitus, lipodystrophy, placenta, PPARG, pregnancy complications

PPARG-related lipodystrophy is a rare disease due to variants in PPARG encoding a major regulator of adipocyte differentiation, also involved in placenta formation.

We show, in the largest cohort of patients with PPARG-related lipodystrophy reported to date (n=26), that the disease is associated with early and severe metabolic complications and decreased gestational age at birth. In affected women, metabolic complications during pregnancy lead to increased newborns' birth weight. However, 85.7% of patients not exposed to fetal dysmetabolic environment (with a very likely paternally-inherited disease), were born small-for-gestational age. Placental PPARG variants originating from the fetus may thus impair fetal growth and parturition. Close metabolic follow-up of patients and monitoring of fetal growth in their descendants, are needed in PPARG-related lipodystrophy.

Abstract

Objective : The adipogenic PPARG-encoded PPARγ nuclear receptor also displays essential placental functions. We evaluated metabolic, reproductive and perinatal features of patients with PPARG-related lipodystrophy.

Methods: Current and retrospective data were collected in patients referred to a National Rare Diseases Reference Centre.

Results: 26 patients from 15 unrelated families were studied (18 women, median age 43 years). They carried monoallelic PPARG variants except a homozygous patient with congenital generalized lipodystrophy. Among heterozygous patients aged 16 or more (n=24), 92% had diabetes, 96% partial lipodystrophy (median age at diagnosis 24 and 37 years, respectively), 78% hypertriglyceridemia, 71% liver steatosis and 58% hypertension. Mean BMI was 26 ± 5.0 kg/m 2 . Women (n=16) were frequently affected by acute pancreatitis (n=6) and/or polycystic ovary syndrome (n=12). Eleven women obtained one or several pregnancies, all complicated by diabetes (n=8), hypertension (n=4) and/or hypertriglyceridemia (n=10). We analysed perinatal data of patients according to the presence (n=8) or absence (n=9) of maternal dysmetabolic environment. The median gestational age at birth was low in both groups (37 and 36 weeks of amenorrhea, respectively). As expected, birth weight was higher in patients exposed to a fetal dysmetabolic environment of maternal origin. In contrast, 85.7% of non-exposed patients, in whom the variant is, or is very likely to be, paternally-inherited, were small for gestational age.

Conclusions: Lipodystrophy-related PPARG variants induce early metabolic complications.

Our results suggest that placental expression of PPARG pathogenic variants carried by affected fetuses could impair prenatal growth and parturition. This justifies careful pregnancy monitoring in affected families.

INTRODUCTION

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor that controls the expression of genes involved in numerous physiological pathways. PPARγ modulates adipocyte differentiation and function, energy homeostasis and insulin sensitivity, and is the target of the antidiabetic drugs such as thiazolidinediones [START_REF] Tontonoz | Fat and beyond: the diverse biology of PPARgamma[END_REF]. Pathogenic variants of PPARG are responsible for familial partial lipodystrophy type 3 (FPLD3), an autosomal dominant rare disease characterized by a limited capacity of peripheral adipose tissue to store excess energy as triglycerides, leading to the progressive development of severe insulin resistance, hypertriglyceridemia, non-alcoholic fatty liver disease, and frequent hypertension [START_REF] Barroso | Dominant negative mutations in human PPARγ associated with severe insulin resistance, diabetes mellitus and hypertension[END_REF][START_REF] Semple | PPAR gamma and human metabolic disease[END_REF].

Nevertheless, the natural history of the disease, regarding metabolic complications and reproductive consequences of insulin resistance, remains largely unknown, since large cohorts of patients have not been described.

Besides its well-known role in adipogenesis, PPARγ has been shown to play an essential role in the formation of the placenta, a transitional organ essential for embryo implantation, pregnancy maintenance and outcome, and fetal development and growth. The placenta is a post-zygotic organ, and is therefore genetically identical to the fetus. PPARG, the human PPAR-encoding gene, is expressed by both the villous and extravillous cytotrophoblasts of the chorionic villi, i.e. the structural and functional unit of the placenta (4). PPARis activated during the differentiation of villous cytotrophoblasts into syncytiotrophoblasts [START_REF] Ruebner | Regulation of the human endogenous retroviral Syncytin-1 and cell-cell fusion by the nuclear hormone receptors PPARγ/RXRα in placentogenesis[END_REF][START_REF] Tarrade | PPARγ/RXRα Heterodimers Are Involved in Human CGβ Synthesis and Human Trophoblast Differentiation[END_REF], which ensure placental endocrine functions. PPARalso controls the migration and invasion of extravillous cytotrophoblasts and is therefore essential for the anchorage of chorionic villi into the uterine wall (7) -a major pre-requisite for embryo implantation and growth -, remodeling of uterine spiral arteries, and immune tolerance during pregnancy.

However, only a few and conflicting results are available regarding the pathophysiological role of PPARG in pregnancy outcomes in humans [START_REF] Lane | Pharmacological activation of peroxisome proliferatoractivated receptor γ (PPAR-γ) protects against hypoxia-associated fetal growth restriction[END_REF][START_REF] Holdsworth-Carson | Peroxisome proliferator-activated receptors are altered in pathologies of the human placenta: gestational diabetes mellitus, intrauterine growth restriction and preeclampsia[END_REF][START_REF] Díaz | Placental Expression of Peroxisome Proliferator-Activated Receptor γ (PPARγ): Relation to Placental and Fetal Growth[END_REF].

Two case reports of pregnancies complicated by premature birth and placental anomalies were reported in women with FPLD3 [START_REF] Madhra | A complicated pregnancy in a patient with lipodystrophic diabetes attributable to a peroxisome proliferator-activated receptor gamma (PPARG) mutation[END_REF][START_REF] Castell | Vascular placental abnormalities and newborn death in a pregnant diabetic woman with familial partial lipodystrophy type 3: a possible role for peroxisome proliferator-activated receptor γ[END_REF], but whether this could be due to severe maternal lipodystrophy-linked metabolic complications and/or to the effects of placental expression of PPARG variants carried by the fetus remains unknown.

The cohort of patients and families referred to our reference centre provides a unique opportunity to study the consequences of FPLD3-related PPARG variants on metabolic characteristics, reproductive phenotype of affected women, and perinatal features. In addition to the characterisation of the FPLD3 phenotype in an important cohort of patients, our results bring data in favor of a deleterious role of placental expression of lipodystrophy-associated PPARG variants on the course of pregnancy and on fetal growth.

METHODS

This study includes 26 patients from 15 unrelated families referred in our French Reference Centre for Rare Diseases of Insulin Secretion and Insulin Sensitivity (PRISIS) for lipodystrophy and/or insulin resistance, and diagnosed with PPARG-related lipodystrophy.

PPARG variants were identified using a lipodystrophy-dedicated diagnostic gene panel.

Capture (SeqCapEZ enrichment protocol, Roche NimbleGen) of exons and flanking intronic sequences of 23 genes was followed by massively parallel sequencing on a MiSeq platform (Illumina Inc, San Diego, CA, USA). Data were analysed using the Sophia Genetics DDM pipeline®. PPARG variants were confirmed by Sanger sequencing and described based on the isoform NM_015869.4. Written informed consents were collected according to legal procedures for molecular investigations. This study was approved by the Comité de Protection des Personnes Ile-de-France 5 (Paris, France) and conducted in accordance with the Declaration of Helsinki and the Guideline for Good Clinical Practice.

Current metabolic data, as well as obstetrical and neonatal retrospective data, were collected through analysis of medical records and patient interviews after written informed consent.

Collected data included gender and age, age at clinical and molecular diagnosis of lipodystrophy and at diagnosis of complications, type and parental origin of the variant when available, physical characteristics (clinical lipodystrophy and body fat distribution, presence of muscle hypertrophy, acanthosis nigricans and hyperandrogenism in women). Weight, height, triglyceride, aspartate transaminase, alanine transaminase, and leptin serum levels, HbA1c, presence of diabetes and/or hypertension, liver steatosis assessed by echography, history of acute pancreatitis and use of insulin therapy were recorded, as well as, in women, age at menarche, presence of polycystic ovary syndrome (PCOS) as defined by the Rotterdam criteria [START_REF] Teede | Recommendations from the international evidencebased guideline for the assessment and management of polycystic ovary syndrome † ‡[END_REF], antecedent of treatment for impaired fertility, number and outcomes of pregnancies. Patients' perinatal data were collected, including prenatal metabolic environment (maternal hypertriglyceridemia before pregnancy, diagnosis of hypertension or diabetes during pregnancy, or pre-eclampsia), exposure to maternal smoking during pregnancy, diagnosis of oligo/polyhydramnios, and neonatal data (gestational age at birth, birth weight and height, neonatal hypoglycemia).

Data are expressed as mean/SD and/or median/25 th quartile (Q25). Differences between groups were analyzed with non parametric Mann-Whitney-Wilcoxon test or Fisher exact tests.

Statistical analyses were performed with using GraphPad Prism version 8 (GraphPad softwear, San Diego, CA, USA). Values of p<0.05 were considered significant.

RESULTS

Patients, molecular diagnosis and parental inheritance of PPARG variants

PPARG variants associated with a typical familial partial lipodystrophy (FPLD) phenotype were observed in 19 independent families (31 patients), referred to our Reference Center. Five patients were excluded from the analysis: three patients died before the inclusion, a patient did not wish to participate and a patient was lost to follow-up. We therefore studied 26 patients [START_REF] Martine | Peroxisome Proliferator-Activated Receptor-γ Mutations Responsible for Lipodystrophy With Severe Hypertension Activate the Cellular Renin-Angiotensin System[END_REF] The parental origin of the PPARG variant was confirmed by molecular analyses in ten among 25 heterozygous patients : 6 of them carried a maternally-inherited, and 4 a paternallyinherited PPARG variant (Table 1).

Lipodystrophy and metabolic phenotype of patients carrying heterozygous PPARG variants

Table 2 presents the main metabolic features of the patients carrying heterozygous PPARG variants, with the exception of Patient 15, a premature newborn who died 24 hours after birth [START_REF] Castell | Vascular placental abnormalities and newborn death in a pregnant diabetic woman with familial partial lipodystrophy type 3: a possible role for peroxisome proliferator-activated receptor γ[END_REF]. Their median age at investigation was 45 years. Patient 8, a 83 years old man, was diagnosed after familial screening. He presented with high blood pressure, diabetes and hypertriglyceridemia diagnosed in the elderly, but did not present with obvious clinical

Reproductive phenotype of women with FPLD3

Seventeen adult women with FPLD3, due to heterozygous PPARG variants, were studied, with a median age at investigation of 48 years old (Table 2). Their median age at first menstruation was 12 years. After clinical, biological and ultrasound examination, PCOS was diagnosed in 12 among 16 patients (75%) using the Rotterdam criteria [START_REF] Ikram | The Rotterdam Study: 2018 update on objectives, design and main results[END_REF]. Eleven among 12 women trying to conceive obtained pregnancy, two of them through medically-assisted procreation (ovarian stimulation in Patient 7, in vitro fertilization in Patient 6). Six pregnancies among 25 did not lead to a live birth : one was interrupted by abortion, four by early miscarriages (two in the context of diabetes), and one by fetal death (occuring in a patient with diabetes). Among the 11 women who had at least one pregnancy, all had metabolic alterations, i.e. either diabetes (n=5), hypertension (n=4), and/or hypertriglyceridemia (n=10) diagnosed before pregnancy, and/or developed gestational diabetes (n=3). Preeclampsia was diagnosed in three patients.

Perinatal characteristics of patients with FPLD3

Perinatal data were available in 17 patients with heterozygous PPARG variants (Table 3 and Supplemental Table ). A majority of them (9 among 17, 52.9%) was born premature ( 37 weeks of amenorrhea). The median gestational age at birth was 36 weeks of amenorrhea (range 31-41). The median birth weight of patients was 2200g (range 1200-4000g). Six patients (42.9%) were small for gestational age and five patients ( Polyhydramnios was diagnosed during the prenatal life of Patient 15, who was born premature and died in the neonatal period [START_REF] Castell | Vascular placental abnormalities and newborn death in a pregnant diabetic woman with familial partial lipodystrophy type 3: a possible role for peroxisome proliferator-activated receptor γ[END_REF]. Patient 25 was born in a context of maternal diabetes, hypertension, and preeclampsia (Table 3 and Supplemental Table ).

Both the maternal dysmetabolic environment, or the placental expression of a PPARG variant carried by the fetus, could contribute to perinatal deleterious outcomes. Therefore, we compared the perinatal characteristics of patients with heterozygous PPARG variants according to the presence or absence of fetal exposure to a maternal dysmetabolic environment. Data from 17 patients were available (Table 4). Patients whose mothers were diagnosed with diabetes, and/or high blood pressure during pregnancy and/or hypertriglyceridemia before pregnancy were considered as exposed to a dysmetabolic environment during fetal life (n=8). All heterozygous patients carrying a maternally-inherited variant (n=6), but none of those carrying a paternally-inherited variant (n=4), reach the criteria to be classified in this group (Table 4 and Supplemental Table ).

The maternal age at delivery (median 25 years) was similar in patients exposed or not to a dysmetabolic intrauterine environment (Table 4). Importantly, the median gestational age at birth was low, and the prevalence of prematurity was high, both in patients exposed to maternal metabolic alterations (37 weeks of amenorrhea, and 37.5% of prevalence, respectively), and in patients not exposed (36 weeks of amenorrhea, and 66.7% of prevalence), without any statistical difference between the two groups. Newborns were frequently large for gestational age in the group exposed to maternal dysmetabolic environment, as expected (71.4% of them, versus 0% in the non-exposed group, p=0.02). In contrast, the great majority of patients with PPARG variants who developed in a favorable metabolic intrauterine environment was born small for gestational age (n=6, 85.7%, versus 0% in the dysmetabolic group, p=0.005). To note, patients born small for gestational age were preserved from maternal smoking during their fetal life, which is a known cause of low birth weight.

Perinatal and metabolic features of the patient carrying the homozygous PPARG (p.Tyr173Cys) variant

Patient 17, born from parents originating from the same island of the New Caledonia archipelago, carried a homozygous PPARG variant. She was born preterm and small for gestational age (610g, 28 weeks of amenorrhea), after cesarean delivery for fetal distress in the context of retro-placental hematoma. Diabetes, severe acanthosis nigricans and hypertriglyceridemia were diagnosed before pregnancy in her mother. Her father was known to suffer form severe hypertension with renal insufficiency without other information. Patient 17 presented at birth with generalized lipoatrophy, muscular hypertrophy, hepatosplenomegaly, signs of virilization, and major hypertriglyceridemia (Suppl Figure ).

Hyperinsulinemia and hypoleptinemia were reported at the age of 2, and diabetes was diagnosed at the age of 6 years and 10 months. She showed severe developmental delay secondary to perinatal brain damage, with growth retardation and signs of bone dysplasia.

Molecular investigations were performed for congenital generalized lipoatrophy at the age of 3. The PPARG (p.Tyr173Cys) variant was present in the heterozygous state in her parents.

DISCUSSION

Our project aimed to characterize the multi-tissular phenotype associated with PPARG-related lipodystrophy in the largest series reported to date, involving 26 patients originating from 15 unrelated families. Given the importance of PPAR not only in the regulation of adipogenesis and metabolism [START_REF] Tontonoz | Fat and beyond: the diverse biology of PPARgamma[END_REF][START_REF] Auwerx | PPARgamma, the ultimate thrifty gene[END_REF], but also in placental formation and functions [START_REF] Fournier | PPARs and the placenta[END_REF][START_REF] Nadra | PPARγ in Placental Angiogenesis[END_REF], we studied metabolic features, reproductive functions in women, and perinatal characteristics of the patients. We enlarge the knowledge of the FPLD3 phenotype by showing that the disease is associated with severe and early metabolic complications, including PCOS in women, and could involve growth retardation and increased risk of prematurity during the prenatal life.

Based on our results, we recommend that the multidisciplinal care of patients with FPLD3 should also include prenatal monitoring of offspring in affected families, in either case of maternal or paternal potential transmission of the disease.

Our data suggest that early diabetes is a major characteristic of FPLD3, and is frequently reported before the diagnosis of clinical lipodystrophy. In addition, and in contrast to what is observed in most forms of lipodystrophic syndromes [START_REF] Brown | The Diagnosis and Management of Lipodystrophy Syndromes: A Multi-Society Practice Guideline[END_REF], the majority of the patients with PPARG-related lipodystrophy presented with a BMI greater than 25 kg/m 2 . This could result from increased lean rather than fat mass, since muscular hypertrophy is a prominent feature of the disease, and serum leptin values are lower than expected with regards to BMI. However, a recent study reported that patients with PPARG-related lipodystrophy have a greater amount of adipose tissue than patients with LMNA-related lipodystrophy [START_REF] Vasandani | Phenotypic Differences Among Familial Partial Lipodystrophy Due to LMNA or PPARG Variants[END_REF]. The metabolic phenotype of FPLD3 could be related not only to the consequences of lipodystrophy and adipocyte dysfunction, but also, at least in part, to deleterious roles of PPARG pathogenic variants on other insulin-sensitizing pathways. In addition to its major role on adipogenesis, PPAR is involved in the regulation of multiple pathways modulating energy homeostasis and inflammation (25). High blood pressure, which can also develop early in patients with FPLD3, could be related to the dysregulation of PPAR-mediated vascular functions, including angiogenesis, vascular tone and cellular renin-angiotensin system [START_REF] Martine | Peroxisome Proliferator-Activated Receptor-γ Mutations Responsible for Lipodystrophy With Severe Hypertension Activate the Cellular Renin-Angiotensin System[END_REF][START_REF] Halabi | Interference with PPAR gamma function in smooth muscle causes vascular dysfunction and hypertension[END_REF][START_REF] Montaigne | PPAR control of metabolism and cardiovascular functions[END_REF].

Patients with FPLD3 are also frequently affected with hepatic steatosis, which is a typical feature of lipodystrophic syndromes, and with hypertriglyceridemia, which can be severe, leading to the risk of acute pancreatitis, especially in women.

Adipose tissue homeostasis and insulin response are important determinant of reproductive fonction in women. However, a few studies have described the reproductive phenotype in women with lipodystrophy syndromes [START_REF] Auwerx | PPARgamma, the ultimate thrifty gene[END_REF][START_REF] Huang-Doran | Ovarian Hyperandrogenism and Response to Gonadotropin-releasing Hormone Analogues in Primary Severe Insulin Resistance[END_REF][START_REF] Vantyghem | Fertility and Obstetrical Complications in Women with LMNA-Related Familial Partial Lipodystrophy[END_REF][START_REF] Vatier | Role of insulin resistance on fertilityfocus on polycystic ovary syndrome[END_REF][START_REF] Joy | Prevalence of reproductive abnormalities among women with familial partial lipodystrophy[END_REF][START_REF] Keller | Lipodystrophy: An Unusual Diagnosis in a Case of Oligomenorrhea and Hirsutism[END_REF]. In our cohort, 75% of women with PPARG heterozygous variants present with PCOS and 81.2% with hirsutism. Nevertheless, our data suggest that fertility is preserved in most cases, the requirement to medically-assisted procreation being comparable to the general population in women with FPLD3 from our study. When pregnant, all women with FPLD3 present with diabetes, hypertension, and/or hypertriglyceridemia. Almost 50% of women with FPLD3 had diabetes before pregnancy and more than 25% developped a gestationnel diabetes. This is in accordance with previous results based on a model of pregnancy in PPAR2 knockout mice, showing that PPARG-dependent healthy adipose tissue expansion and function are mandatory for physiological metabolic adaptations during pregnancy [START_REF] Vivas | Peroxisome proliferator activated receptor gamma 2 modulates late pregnancy homeostatic metabolic adaptations[END_REF].

In vitro and in vivo studies showed an essential role of PPARG in placental development in mice [START_REF] Nadra | PPARγ in Placental Angiogenesis[END_REF][START_REF] Fournier | PPARγ and human trophoblast differentiation[END_REF][START_REF] Fournier | PPARgamma and early human placental development[END_REF]. Strikingly, PPARG inactivation in mice results in defects in trophoblast differentiation and uterine vascular remodeling leading to embryonic lethality [START_REF] Barak | PPAR gamma is required for placental, cardiac, and adipose tissue development[END_REF][START_REF] Kubota | PPARγ Mediates High-Fat Diet-Induced Adipocyte Hypertrophy and Insulin Resistance[END_REF], which is prevented by trophoblastic re-expression of PPARG [START_REF] Nadra | PPARγ in Placental Angiogenesis[END_REF]. PPARG was shown to control major processes allowing the vascular exchange interface between maternal and fetal blood in humans [START_REF] Lane | Pharmacological activation of peroxisome proliferatoractivated receptor γ (PPAR-γ) protects against hypoxia-associated fetal growth restriction[END_REF][START_REF] Fournier | PPARγ and human trophoblast differentiation[END_REF][START_REF] Fournier | PPARgamma and early human placental development[END_REF]. Some PPARG variants have been associated with an increased genetic susceptibility of abruptio placentae [START_REF] Workalemahu | Abruptio placentae risk and genetic variations in mitochondrial biogenesis and oxidative phosphorylation: replication of a candidate gene association study[END_REF] and preterm birth [START_REF] Capece | Pathway Analysis of Genetic Factors Associated with Spontaneous Preterm Birth and Pre-Labor Preterm Rupture of Membranes[END_REF] in the general population. In addition, gene expression of PPARG in placentas from women who gave birth to babies born small for gestational age was reported to be two-fold lower as compared to controls, and to be associated to fetal and placental weights [START_REF] Díaz | Placental Expression of Peroxisome Proliferator-Activated Receptor γ (PPARγ): Relation to Placental and Fetal Growth[END_REF]. Nevertheless, the pathophysiological role of PPARG during human pregnancy is largely unknown. As the placenta is a post-zygotic organ, it shares with the fetus the same genetic heritage. In an attempt to study the placental consequences of the lipodystrophy-related PPARG variants, we collected the perinatal data of 18 patients recruited in our national reference center for lipodystrophic diseases. Our results took into account the prenatal metabolic environment, which depends on the maternal PPARG genetic status, and impacts the perinatal outcomes. All patients known to carry a maternallyinherited variant, but none of the patients carrying a paternally-inherited variant, were exposed to an intrauterine dysmetabolic environment. However, the prevalence of prematurity was high in patients carrying a heterozygous PPARG variant (52.9%), and did not differ according to the prenatal metabolic environment. In addition, while maternal metabolic alterations were, as expected, major risk factors for increased birth weight with regards to gestational age, 85.7% of patients without dysmetabolic prenatal exposure were small for gestationnel age. Taken together, our results suggest that growth restriction and prematurity could be linked to defective placental development and functions induced by lipodystrophyrelated PPARG variants. They are in line with our previous in vitro study, which showed that the lipodystrophy-associated PPARG (p.Glu352Gln) variant, expressed in human trophoblast cells, inhibits the differentiation of villous cytotrophoblasts into syncytiotrophoblasts [START_REF] Shoaito | Peroxisome proliferator-activated receptor gamma-ligand-binding domain mutations associated with familial partial lipodystrophy type 3 disrupt human trophoblast fusion and fibroblast migration[END_REF]. In addition, PPARγ was shown to promote uteroplacental perfusion by increasing nitric oxide and decreasing downregulating endothelin-1 production [START_REF] Lane | Pharmacological activation of peroxisome proliferatoractivated receptor γ (PPAR-γ) protects against hypoxia-associated fetal growth restriction[END_REF]. Defects in foetal vascularization impairing availibility of nutrients could thus play a role in FPLD3-associated growth restriction and prematurity. Whatever the mechanism involved, our results underline the importance of a close metabolic and obstetrical follow-up of pregnant women either diagnosed with FPLD3, or susceptible to bear a child with a paternally-transmitted PPARG variant.

Our cohort includes the first reported patient carrying an homozygous lipodystrophyassociated PPARG variant. She presented with congenital generalized lipodystrophy, as reported in the only case previously described with biallelic PPARG pathogenic variants [START_REF] Dyment | Biallelic mutations at PPARG cause a congenital, generalized lipodystrophy similar to the Berardinelli-Seip syndrome[END_REF].

The PPARG variant identified in the patient from our study affects the DNA binding domain of the protein (p.Tyr173Cys), which was also the case for the two variants previously reported in the compound heterozygous state [PPARG (p.Glu138Valfs*168) and (p.Arg164Trp)].

Metabolic complications appeared early in life with hypertriglyceridemia and hepatosplenomegaly at birth, and diabetes diagnosed at the age of 6 years. Biallelic lipodystrophy-associated PPARG variants probably induce important prenatal adverse effects, since the patient described here was born preterm and small for gestational age (610g, 28 weeks of amenorrhea), and the previous case also suffered from intrauterine growth restriction (birthweight of 940g at 32 weeks) [START_REF] Dyment | Biallelic mutations at PPARG cause a congenital, generalized lipodystrophy similar to the Berardinelli-Seip syndrome[END_REF].

We present here the largest cohort of patient carrying a PPARG variant with study of lipodystrophy, metabolic, reproductive and perinatal aspects. Our results hightlight specific features of the disease, with early onset of diabetes, before the clinical recognition of lipodystrophy, which could contribute to disease underdiagnosis, especially since patients are frequently overweighted. Precocious high blood pressure, muscular hypertrophy, hypertriglyceridemia and familial history are important signs to help diagnosis. For the first time, we show that lipodystrophy-related PPARG variants are likely to impact fetal development and parturition, with risk of impaired fetal growth and prematurity. This justifies a close metabolic and obstetrical follow-up of women with FPLD3, and the monitoring of fetal growth of the descendants of all subjects, male or female, bearing PPARG pathogenic variants. This study also justifies further investigations regarding the pathophysiological role and potential therapeutic modulation of PPAR in human pregnancy.

Declaration of interest:

All authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding sources: This work was supported by the French Ministry of Solidarity and Health,

  lipodystrophy. All other patients with a heterozygous PPARG variant present with a clinical lipodystrophy, associating in most of them limb lipoatrophy, muscular hypertrophy, android fat distribution and acanthosis nigricans. Most patients (73.9%) have an increased BMI (median value: 25.8 kg/m 2 , Q25: 22.25) : eleven patients are overweight (BMI 25 to 30), and three are obese (BMI 30). Serum leptin levels are low-to-normal with regards to BMI (median 8.4 ng/ml). Diabetes is reported in all patients except a 16-year-old man (Patient 3) and a 55-year-old woman (Patient 12), both diagnosed with hypertriglyceridemia, hepatic steatosis and insulin resistance. Most patients with diabetes are treated with insulin (56.5%), with non-optimal glucose control (median HbA1c 8.1%). Diabetes developed early in life (median age at diagnosis 24 years), and lipodystrophy later on (median age at diagnosis 37 years old), without any statistical difference between females and males regarding prevalence and age at diagnosis of diabetes and lipodystrophy. Hypertension is frequent (58.3% of the patients), diagnosed early in the course of the disease (at a median age of 28 years old). Most patients (78.2%) present with hypertriglyceridemia (median triglycerides 5.6 mmol/L). Six of them, all women (25% of the whole cohort and 37.3% of the female patients) have experienced at least one episode of acute pancreatitis. Hepatic cytolysis is observed in 70.8% of the patients, all of them being diagnosed with hepatic steatosis by echography.

  35.7%) were large for gestational age. Two newborns experienced neonatal hypoglycemia: Patient 2 was born from a mother with diabetes during pregnancy, and Patient 4 was born premature and small for gestational age without dysmetabolic maternal context. Amniotic fluid disorders were reported in three cases. Oligohydramnios was diagnosed during the fetal life of Patient 3, Patient 6 and Patient 15 (in the context of maternal diabetes for Patient 6 and 15).

  women and 8 men) from 15 families. Their mean age was 40.1 years old (SD 19.1, range 0-83) at investigation and 33.3 years old (SD 19.2, range 0-83) at molecular diagnosis (Table1).All patients carry a heterozygous PPARG variant except Patient 17, issued from an endogamy marriage, who is homozygous for the PPARG (p.Tyr173Cys) variant. Patients from two unrelated families were diagnosed with the same PPARG (p.Arg425Cys) variant, whereas other families all harbored different PPARG variants. Three variants are located in exons encoding the DNA binding domain and 11 variants in exons encoding the ligand binding domain of PPARγ. The PPARG variants p.Arg165Thr, p.Gly211Glu, p.Arg240Trp, p.Arg262Gly, p.Val318Met, p.Leu339*, p.Glu352Gln, p.Arg425Cys, p.Gln443*, and p.Pro495Leu were considered as pathogenic based on ACMG criteria, ClinVar database, or cosegregation and/or functional studies[START_REF] Barroso | Dominant negative mutations in human PPARγ associated with severe insulin resistance, diabetes mellitus and hypertension[END_REF][START_REF] Richards | Standards and Guidelines for the Interpretation of Sequence Variants: A Joint Consensus Recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology[END_REF][START_REF] Majithia | Prospective functional classification of all possible missense variants in PPARG[END_REF][START_REF] Agarwal | A novel heterozygous mutation in peroxisome proliferatoractivated receptor-gamma gene in a patient with familial partial lipodystrophy[END_REF][START_REF] Jeninga | Impaired Peroxisome Proliferator-Activated Receptor γ Function through Mutation of a Conserved Salt Bridge (R425C) in Familial Partial Lipodystrophy[END_REF][START_REF] Martine | Peroxisome Proliferator-Activated Receptor-γ Mutations Responsible for Lipodystrophy With Severe Hypertension Activate the Cellular Renin-Angiotensin System[END_REF][START_REF] Shoaito | Peroxisome proliferator-activated receptor gamma-ligand-binding domain mutations associated with familial partial lipodystrophy type 3 disrupt human trophoblast fusion and fibroblast migration[END_REF]. The PPARG variants p.Tyr173Cys, p.Cys180Tyr, p.Pro234Thr and p.Ile434Thr are considered as deleterious, probably or possibly damaging by the SIFT and/or Polyphen-2 prediction softwares. Patients were not found to carry any additional pathogenic or likely pathogenic variant in the complete coding sequence and intronic junctions of PPARG, nor in 22 other genes known to be involved in lipodystrophic and/or insulin resistance syndromes (i.e. AGPAT2, AKT2, BSCL2,

	CAV1, CIDEC, DYRK1B, INSR, LIPE, LMF1, LMNA, LMNB2, NSMCE2, PCYT1A, PIK3R1,
	PLD3, PLIN1, POC1A, POLD1, PSMB8, PTRF, TBC1D4, ZMPSTE4).

Assistance-Publique Hôpitaux de Paris, Sorbonne University, the Institut National de la Santé et de la Recherche Médicale (Inserm), the Institut Hospitalo-Universitaire de Cardiométabolisme et Nutrition (ICAN) and the Fondation pour la Recherche Médicale, France.

Acknowledgements

We thank the patients who participated in these studies, and Prof. Jean-Philippe Bastard and Dr Soraya Fellahi (AP-HP, Department of Biochemistry, Henri Mondor Hospital, Paris, France) for leptin measurements. Bruno Fève, Camille Vatier and Corinne Vigouroux are members of the European Reference Network on Rare Endocrine Conditions -Project ID No 739527. The authors do not declare any conflict of interest.

Data sharing/ availibility statement

The datasets presented in this article are not readily available because of the sensitive nature of the data and possible high risks associated with patient confidentiality. Requests to access the datasets should be directed to camille.vatier@aphp.fr.

Author Contribution

CG, CVi and CVa designed and conducted the research, analysed the data and wrote the manuscript. All authors made substantial contributions to conception, design, acquisition of data, data analyses and data interpretation. OL, IL and CVi generated and/or analyzed the genetic data. IM, FA, XD, CD,BF, SG, BG, SH, YH, EN, CPi, CPo, JPT referred some patients and contribute to the phenotypic description. All authors revised the manuscript critically for important intellectual content and gave their approval of the final version to be published.. CVa and CVi are the guarantor of this work and takes responsibility for the integrity of the data and the accuracy of data analysis.