Pressure ulcers after prone positioning in patients undergoing extracorporeal membrane oxygenation: A cross-sectional study.

BACKGROUND: The combination of prone positioning and extracorporeal membrane oxygenation (ECMO) in patients with acute respiratory distress syndrome (ARDS) is recognized as safe but its use has been limited due to potential complications. AIM: To report the prevalence of pressure ulcers and other complications due to prone positioning in adult patients receiving veno-venous ECMO. STUDY DESIGN: This cross-sectional study was conducted in a tertiary level intensive care unit (ICU) in Milan (Italy), between January 2015 and December 2019. The study population was critically ill adult patients undergoing veno-venous ECMO. Statistical association between pressure ulcers and the type of body positioning (prone versus supine) was explored fitting a logistic model. RESULTS: In the study period, 114 patients were treated with veno-venous ECMO and 62 (54.4%) patients were placed prone for a total of 130 prone position cycles. ECMO cannulation was performed via femoro-femoral configuration in the majority of patients (82.4%, 94/114). Pressure ulcers developed in 57.0% of patients (95%CI: 44.0%-72.6%), most often arising on the face and the chin (37.1%, 23/62), particularly in those placed prone. The main reason of prone positioning interruption was the decrease of ECMO blood flow (8.1%, 5/62). The fitted model showed no association between body position during ECMO and occurrence of pressure ulcers (OR 1.3, 95%CI: 0.5-3.6, p = .532). CONCLUSIONS: Facial pressure ulcers were the most frequent complications of prone positioning. Nurses should plan and implement evidence-based care to prevent such pressure injuries in patients undergoing ECMO. RELEVANCE TO CLINICAL PRACTICE: The combination of prone positioning and ECMO shows few life-threating complications. This manoeuvre during ECMO is feasible and safe when performed by experienced ICU staff.

Selenoprotein deficiency disorder predisposes to aortic aneurysm formation.

Aortic aneurysms, which may dissect or rupture acutely and be lethal, can be a part of multisystem disorders that have a heritable basis. We report four patients with deficiency of selenocysteine-containing proteins due to selenocysteine Insertion Sequence Binding Protein 2 (SECISBP2) mutations who show early-onset, progressive, aneurysmal dilatation of the ascending aorta due to cystic medial necrosis. Zebrafish and male mice with global or vascular smooth muscle cell (VSMC)-targeted disruption of Secisbp2 respectively show similar aortopathy. Aortas from patients and animal models exhibit raised cellular reactive oxygen species, oxidative DNA damage and VSMC apoptosis. Antioxidant exposure or chelation of iron prevents oxidative damage in patient's cells and aortopathy in the zebrafish model. Our observations suggest a key role for oxidative stress and cell death, including via ferroptosis, in mediating aortic degeneration.

Short-Term Exposure to Benzo(a)Pyrene Causes Disruption of GnRH Network in Zebrafish Embryos.

Benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon, is considered a common endocrine disrupting chemical (EDC) with mutagenic and carcinogenic effects. In this work, we evaluated the effects of BaP on the hypothalamo-pituitary-gonadal axis (HPG) of zebrafish embryos. The embryos were treated with 5 and 50 nM BaP from 2.5 to 72 hours post-fertilization (hpf) and obtained data were compared with those from controls. We followed the entire development of gonadotropin releasing hormone (GnRH3) neurons that start to proliferate from the olfactory region at 36 hpf, migrate at 48 hpf and then reach the pre-optic area and the hypothalamus at 72 hpf. Interestingly, we observed a compromised neuronal architecture of the GnRH3 network after the administration of 5 and 50 nM BaP. Given the toxicity of this compound, we evaluated the expression of genes involved in antioxidant activity, oxidative DNA damage and apoptosis and we found an upregulation of these pathways. Consequently, we performed a TUNEL assay and we confirmed an increment of cell death in brain of embryos treated with BaP. In conclusion our data reveal that short-term exposure of zebrafish embryos to BaP affects GnRH3 development likely through a neurotoxic mechanism.

Defective jagged-1 signaling affects GnRH development and contributes to congenital hypogonadotropic hypogonadism.

In vertebrate species, fertility is controlled by gonadotropin-releasing hormone (GnRH) neurons. GnRH cells arise outside the central nervous system, in the developing olfactory pit, and migrate along olfactory/vomeronasal/terminal nerve axons into the forebrain during embryonic development. Congenital hypogonadotropic hypogonadism (CHH) and Kallmann syndrome are rare genetic disorders characterized by infertility, and they are associated with defects in GnRH neuron migration and/or altered GnRH secretion and signaling. Here, we documented the expression of the jagged-1/Notch signaling pathway in GnRH neurons and along the GnRH neuron migratory route both in zebrafish embryos and in human fetuses. Genetic knockdown of the zebrafish ortholog of JAG1 (jag1b) resulted in altered GnRH migration and olfactory axonal projections to the olfactory bulbs. Next-generation sequencing was performed in 467 CHH unrelated probands, leading to the identification of heterozygous rare variants in JAG1. Functional in vitro validation of JAG1 mutants revealed that 7 out of the 9 studied variants exhibited reduced protein levels and altered subcellular localization. Together our data provide compelling evidence that Jag1/Notch signaling plays a prominent role in the development of GnRH neurons, and we propose that JAG1 insufficiency may contribute to the pathogenesis of CHH in humans.

Short-Term Exposure Effects of the Environmental Endocrine Disruptor Benzo(a)Pyrene on Thyroid Axis Function in Zebrafish.

Benzo(a)Pyrene (BaP) is one of the most widespread polycyclic aromatic hydrocarbons (PAHs) with endocrine disrupting properties and carcinogenic effects. In the present study, we tested the effect of BaP on thyroid development and function, using zebrafish as a model system. Zebrafish embryos were treated with 50 nM BaP from 2.5 to 72 h post fertilization (hpf) and compared to 1.2% DMSO controls. The expression profiles of markers of thyroid primordium specification, thyroid hormone (TH) synthesis, hypothalamus-pituitary-thyroid (HPT) axis, TH transport and metabolism, and TH action were analyzed in pools of treated and control embryos at different developmental stages. BaP treatment did not affect early markers of thyroid differentiation but resulted in a significant decrease of markers of TH synthesis (tg and nis) likely secondary to defective expression of the central stimulatory hormones of thyroid axis (trh, tshba) and of TH metabolism (dio2). Consequently, immunofluorescence of BaP treated larvae showed a low number of follicles immunoreactive to T4. In conclusion, our results revealed that the short-term exposure to BaP significantly affects thyroid function in zebrafish, but the primary toxic effects would be exerted at the hypothalamic-pituitary level thus creating a model of central hypothyroidism.

Muscle strength and functional outcome after prone positioning in COVID-19 ICU survivors.

OBJECTIVE: To evaluate the muscle strength and functional level of patients discharged from intensive care unit (ICU) in relation to the swimmer position as a nurse intervention during pronation. METHODS: Prospective study conducted in the hub COVID-19 center in Milan (Italy), between March and June 2020. All patients with COVID-19 discharged alive from ICU who received invasive mechanical ventilation were included. Forward continuation ratio model was fitted to explore the statistical association between muscle strength grades and body positioning during ICU stay. RESULTS: Over the 128 patients admitted to ICU, 87 patients were discharged alive from ICU, with available follow-up measures at hospital discharge. Thirty-four patients (39.1%) were treated with prone positioning as rescue therapy, for a total of 106 pronation cycles with a median duration of 72 (IQR 60-83) hours. Prone positioning did not influence the odds of showing particular level of muscle strength, in any of the evaluated districts, namely shoulder (OR 1.34, 95%CI:0.61-2.97), elbow (OR 1.10, 95%CI:0.45-2.68) and wrist (OR 0.97, 95%CI:0.58-1.63). Only in the shoulder district, age showed evidence of association with strength (OR 1.06, 95%CI:1.02-1.10), affecting people as they get older. No significant sequalae related to swimmer position were reported by physiotherapists or nurses. CONCLUSION: Swimmer position adopted during prone ventilation is not associated with worse upper limb strength or poor mobility level in COVID-19 survivors after hospital discharge.

Structure-Guided Approach to Relieving Transcriptional Repression in Resistance to Thyroid Hormone α.

Mutations in thyroid hormone receptor α (TRα), a ligand-inducible transcription factor, cause resistance to thyroid hormone α (RTHα). This disorder is characterized by tissue-specific hormone refractoriness and hypothyroidism due to the inhibition of target gene expression by mutant TRα-corepressor complexes. Using biophysical approaches, we show that RTHα-associated TRα mutants devoid of ligand-dependent transcription activation function unexpectedly retain the ability to bind thyroid hormone. Visualization of the ligand T3 within the crystal structure of a prototypic TRα mutant validates this notion. This finding prompted the synthesis of different thyroid hormone analogues, identifying a lead compound, ES08, which dissociates corepressor from mutant human TRα more efficaciously than T3. ES08 rescues developmental anomalies in a zebrafish model of RTHα and induces target gene expression in TRα mutation-containing cells from an RTHα patient more effectively than T3. Our observations provide proof of principle for developing synthetic ligands that can relieve transcriptional repression by the mutant TRα-corepressor complex for treatment of RTHα.

Clinical Consequences of Variable Results in the Measurement of Free Thyroid Hormones: Unusual Presentation of a Family with a Novel Variant in the THRB Gene Causing Resistance to Thyroid Hormone Syndrome.

INTRODUCTION: Resistance to thyroid hormone ß (RTHß) is an inherited syndrome caused by dominant negative variants in the THRB gene (NM_000461.5). The clinical picture of RTHß is variable, and patients harboring the same variant may display different degrees of disease severity. CASE PRESENTATION: A 30-year-old man presented with thyrotoxicosis and central hyperthyroidism and was found to have a novel variant in the exon 10 of THRB gene (c.C1282G, p.L428V), located within the third hot spot region of the C-terminal of the receptor. Surprisingly, the same variant was found in two other relatives with an apparent normal thyroid function at initial screening. After exclusion of a TSH-secreting adenoma and serum interference in the proband, and the finding that exogenous levothyroxine failed to suppress the TSH in the brother affected by nodular goiter, relatives' thyroid function tests (TFTs) were reassessed with additional analytical method revealing biochemical features consistent with RTHß in all carriers of the p.L428V variant. Functional studies showed a slightly impaired in vitro transcriptional activity of p.L428V. Interestingly' the expression of the human p.L428V thyroid hormone receptor beta in the zebrafish embryo background generated a phenotype consistent with RTHß. CONCLUSION: Variable results of TFTs on some immunoassays can be a cause of RTHß diagnostic delay, but the genotype-phenotype correlation in this family and functional studies support p.L428V as a novel THRB variant expanding the spectrum of gene variants causing RTHß. In vivo, rather than in vitro, functional assays may be required to demonstrate the dominant negative action of THRB variants.

Complications of prone positioning in patients with COVID-19: A cross-sectional study.

OBJECTIVE: To determine the prevalence of complications in patients with COVID-19 undergone prone positioning, focusing on the development of prone-related pressure ulcers. METHODS: Cross-sectional study conducted in the hub COVID-19 centre in Milan (Italy), between March and June 2020. All patients with COVID-19 admitted to intensive care unit on invasive mechanical ventilation and treated with prone positioning were included. Association between prone-related pressure ulcers and selected variables was explored by the means of logistic regression. RESULTS: A total of 219 proning cycles were performed on 63 patients, aged 57.6 (10.8) and predominantly obese males (66.7%). The main complications recorded were: prone-related pressure ulcers (30.2%), bleeding (25.4%) and medical device displacement (12.7%), even if no unplanned extubation was recorded. The majority of patients (17.5%) experienced bleeding of upper airways. Only 15 prone positioning cycles (6.8%) were interrupted, requiring staff to roll the patient back in the supine position. The likelihood of pressure ulcers development was independently associated with the duration of prone positioning, once adjusting for age, hypoxemic level, and nutritional status (OR 1.9, 95%CI 1.04-3.6). CONCLUSION: The use of prone positioning in patients with COVID-19 was a safe and feasible treatment, also in obese patients, who might deserve more surveillance and active prevention by intensive care unit staff.

From Endoderm to Progenitors: An Update on the Early Steps of Thyroid Morphogenesis in the Zebrafish.

The mechanisms underlying thyroid gland development have a central interest in biology and this review is aimed to provide an update on the recent advancements on the early steps of thyroid differentiation that were obtained in the zebrafish, because this teleost fish revealed to be a suitable organism to study the early developmental stages. Physiologically, the thyroid precursors fate is delineated by the appearance among the endoderm cells of the foregut of a restricted cell population expressing specific transcription factors, including pax2a, nkx2.4b, and hhex. The committed thyroid primordium first appears as a thickening of the pharyngeal floor of the anterior endoderm, that subsequently detaches from the floor and migrates to its final location where it gives rise to the thyroid hormone-producing follicles. At variance with mammalian models, thyroid precursor differentiation in zebrafish occurs early during the developmental process before the dislocation to the eutopic positioning of thyroid follicles. Several pathways have been implicated in these early events and nowadays there is evidence of a complex crosstalk between intrinsic (coming from the endoderm and thyroid precursors) and extrinsic factors (coming from surrounding tissues, as the cardiac mesoderm) whose organization in time and space is probably required for the proper thyroid development. In particular, Notch, Shh, Fgf, Bmp, and Wnt signaling seems to be required for the commitment of endodermal cells to a thyroid fate at specific developmental windows of zebrafish embryo. Here, we summarize the recent findings produced in the various zebrafish experimental models with the aim to define a comprehensive picture of such complicated puzzle.

Nursing Management of Prone Positioning in Patients With COVID-19.

BACKGROUND: At the height of the coronavirus disease 2019 (COVID-19) pandemic, Italy had the highest number of deaths in Europe; most occurred in the Lombardy region. Up to 4% of patients with COVID-19 required admission to an intensive care unit because they developed a critical illness (eg, acute respiratory distress syndrome). Numerous patients with acute respiratory distress syndrome who had been admitted to the intensive care unit required rescue therapy like prone positioning. OBJECTIVE: To describe the respiratory management of and the extensive use of prone positioning in patients with COVID-19 at the intensive care unit hub in Lombardy, Italy. METHODS: A total of 89 patients (67% male; median age, 59 years [range, 23-80 years]) with confirmed COVID-19 who were admitted between February 23 and March 31, 2020, were enrolled in this quality improvement project. RESULTS: Endotracheal intubation was required in 86 patients (97%). Prone positioning was used as rescue therapy in 43 (48%) patients. Significantly more younger patients (age ≤ 59 years) were discharged alive (43 of 48 [90%]) than were older patients (age ≥ 60 years; 26 of 41 [63%]; P < .005). Among the 43 patients treated with prone ventilation, 15 (35% [95% CI, 21%-51%]) died in the intensive care unit, of which 10 (67%; P < .001) were older patients. CONCLUSIONS: Prone positioning is one strategy available for treating acute respiratory distress syndrome in patients with COVID-19. During this pandemic, prone positioning can be used extensively as rescue therapy, per a specific protocol, in intensive care units.

LGR4 deficiency results in delayed puberty through impaired Wnt/ß-catenin signaling.

The initiation of puberty is driven by an upsurge in hypothalamic gonadotropin-releasing hormone (GnRH) secretion. In turn, GnRH secretion upsurge depends on the development of a complex GnRH neuroendocrine network during embryonic life. Although delayed puberty (DP) affects up to 2% of the population, is highly heritable, and is associated with adverse health outcomes, the genes underlying DP remain largely unknown. We aimed to discover regulators by whole-exome sequencing of 160 individuals of 67 multigenerational families in our large, accurately phenotyped DP cohort. LGR4 was the only gene remaining after analysis that was significantly enriched for potentially pathogenic, rare variants in 6 probands. Expression analysis identified specific Lgr4 expression at the site of GnRH neuron development. LGR4 mutant proteins showed impaired Wnt/ß-catenin signaling, owing to defective protein expression, trafficking, and degradation. Mice deficient in Lgr4 had significantly delayed onset of puberty and fewer GnRH neurons compared with WT, whereas lgr4 knockdown in zebrafish embryos prevented formation and migration of GnRH neurons. Further, genetic lineage tracing showed strong Lgr4-mediated Wnt/ß-catenin signaling pathway activation during GnRH neuron development. In conclusion, our results show that LGR4 deficiency impairs Wnt/ß-catenin signaling with observed defects in GnRH neuron development, resulting in a DP phenotype.

Publisher Correction: Prokineticin receptor 2 affects GnRH3 neuron ontogeny but not fertility in zebrafish.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Knocking-down of the Prokineticin receptor 2 affects reveals its complex role in the regulation of the hypothalamus-pituitary-gonadal axis in the zebrafish model.

Prokineticin receptors (PROKR1 and PROKR2) are G protein-coupled receptors which control human central and peripheral reproductive processes. Importantly, allelic variants of PROKR2 in humans are associated with altered migration of GnRH neurons, resulting in congenital hypogonadotropic hypogonadism (CHH), a heterogeneous disease characterized by delayed/absent puberty and/or infertility. Although this association is established in humans, murine models failed to fully recapitulate the reproductive and olfactory phenotypes observed in patients harboring PROKR2 mutations. Here, taking advantage of zebrafish model we investigated the role of prokr1b (ortholog of human PROKR2) during early stages of GnRH neuronal migration. Real-Time PCR and whole mount in situ hybridization assays indicate that prokr1b spatial-temporal expression is consistent with gnrh3. Moreover, knockdown and knockout of prokr1b altered the correct development of GnRH3 fibers, a phenotype that is rescued by injection of prokr1b mRNA. These results suggest that prokr1b regulates the development of the GnRH3 system in zebrafish. Analysis of gonads development and mating experiments indicate that prokr1b is not required for fertility in zebrafish, although its loss determine changes also at the testis level. Altogether, our results support the thesis of a divergent evolution in the control of vertebrate reproduction and provide a useful in vivo model for deciphering the mechanisms underlying the effect of PROKR2 allelic variants on CHH.

Glis3 as a Critical Regulator of Thyroid Primordium Specification.

Background: GLIS3 (GLI-Similar protein 3) is a transcription factor involved in several cellular processes. Homozygous mutations in the GLIS3 gene have been typically associated with neonatal diabetes and congenital hypothyroidism (CH) in a syndrome called NDH. NDH patients present developmental abnormalities including endocrine pancreas defects and a spectrum of thyroid abnormalities, mainly including thyroid dysgenesis (TD). The mouse models revealed a key role of Glis3 in pancreatic islets but not in early thyroid development, as Glis3 was described to retain a role in regulating thyroid hormone synthesis downstream the thyrotropin (TSH)/TSHR signaling pathway and in postnatal follicle proliferation. Hence, in this study, we have been taking advantage of the zebrafish model to gain insights on the Glis3 activity during thyroid organogenesis. Methods: Transient glis3-knockdown zebrafish embryos (called glis3 morphants) were generated by the microinjection of specific glis3 morpholinos at one- to two-cell stage to analyze the thyroid phenotype in vivo. Several additional analyses (in situ hybridization, immunohistochemistry, and pharmacological treatments) were performed for further molecular characterization. Results: The analysis of thyroid embryonic development revealed that Glis3 is involved in early steps of thyroid specification. glis3 morphants exhibited a reduced expression of the early transcription factors nkx2.4 and pax2a at the thyroid primordium level, which is not caused by changes in proliferation or apoptosis of the pharyngeal endoderm. As a result, the differentiated thyroid tissue in morphants appeared reduced in size with decreased expression of tg and slc5a5, a low number of thyroxine (T4)-producing follicles, associated with an elevation of tshba (homologous of the human TSHß), thus resembling the clinical and biochemical manifestations of patients with TD. Interestingly, glis3 morphants have pancreatic ß-cell defects, but not liver defects. In vitro and in vivo data also demonstrated that Glis3 is an effector of the Sonic Hedgehog (SHH) pathway. Molecular and pharmacological inhibition of SHH reproduced the thyroid defects observed in glis3 morphant. Conclusions: Our results demonstrate that glis3, within the SHH pathway, appears to determine the number of endodermal cells committed to a thyroid fate. This is the first evidence of the involvement of Glis3 in TD, thereby expanding the understanding of the genetic basis of thyroid development and CH.

Thyroid Hormone Hyposensitivity: From Genotype to Phenotype and Back.

Thyroid hormone action defects (THADs) have been classically considered conditions of impaired sensitivity to thyroid hormone (TH). They were originally referring to alterations in TH receptor genes (THRA and THRB), but the discovery of genetic mutations and polymorphisms causing alterations in cell membrane transport (e.g., MCT8) and metabolism (e.g., SECISBP2, DIO2) led recently to a new and broader definition of TH hyposensitivity (THH), including not only THADs but all defects that could interfere with the activity of TH. Due to the different functions and tissue-specific expression of these genes, affected patients exhibit highly variable phenotypes. Some of them are characterized by a tissue hypothyroidism or well-recognizable alterations in the thyroid function tests (TFTs), whereas others display a combination of hypo- and hyperthyroid manifestations with normal or only subtle biochemical defects. The huge effort of basic research has greatly aided the comprehension of the molecular mechanisms underlying THADs, dissecting the morphological and functional alterations on target tissues, and defining the related-changes in the biochemical profile. In this review, we describe different pictures in which a specific alteration in the TFTs (TSH, T4, and T3 levels) is caused by defects in a specific gene. Altogether these findings can help clinicians to early recognize and diagnose THH and to perform a more precise genetic screening and therapeutic intervention. On the other hand, the identification of new genetic variants will allow the generation of cell-based and animal models to give novel insight into thyroid physiology and establish new therapeutic interventions.

GLIS3 and Thyroid: A Pleiotropic Candidate Gene for Congenital Hypothyroidism.

Variations in the transcription factor Gli-similar 3 (GLIS3) gene have been associated to variable congenital endocrine defects, including both morphogenetic and functional thyroid alterations. Evidence from Glis3 knockout mice indicates a relevant role for GLIS3 in thyroid hormone biosynthesis and postnatal thyroid gland growth, with a mechanism of action downstream of the TSH/TSHR interaction. However, the pathophysiological role of this transcription factor during the embryonic thyroid development remains unexplored. In this manuscript, we will provide an overview of the current knowledge on GLIS3 function during development. As a perspective, we will present preliminary evidence in the zebrafish model in support of a potential role for this pleiotropic transcription factor in the early stages of thyroid gland development.

Role of TRs in Zebrafish Development.

In recent years, the zebrafish has become a powerful model not only for the developmental biology studies, but also for genetic analyses and drug screenings, mostly thanks to the ease with which its embryos can be manipulated and to its translucent body, which allows in vivo imaging. In this chapter, we will provide an overview of the current knowledge about the role of thyroid hormone receptors during zebrafish embryonic development. Moreover, we will explore the methodologies applied to zebrafish biology to knock down a gene of interest and to analyze in vivo the molecular mechanisms of the mutated receptors.

How zebrafish research has helped in understanding thyroid diseases.

Next-generation sequencing technologies have revolutionized the identification of disease-causing genes, accelerating the discovery of new mutations and new candidate genes for thyroid diseases. To face this flow of novel genetic information, it is important to have suitable animal models to study the mechanisms regulating thyroid development and thyroid hormone availability and activity. Zebrafish ( Danio rerio), with its rapid external embryonic development, has been extensively used in developmental biology. To date, almost all of the components of the zebrafish thyroid axis have been characterized and are structurally and functionally comparable with those of higher vertebrates. The availability of transgenic fluorescent zebrafish lines allows the real-time analysis of thyroid organogenesis and its alterations. Transient morpholino-knockdown is a solution to silence the expression of a gene of interest and promptly obtain insights on its contribution during the development of the zebrafish thyroid axis. The recently available tools for targeted stable gene knockout have further increased the value of zebrafish to the study of thyroid disease. All of the reported zebrafish models can also be used to screen small compounds and to test new drugs and may allow the establishment of experimental proof of concept to plan subsequent clinical trials.

A frequent oligogenic involvement in congenital hypothyroidism.

Congenital hypothyroidism (CH), the most frequent form of preventable mental retardation, is predicted to have a relevant genetic origin. However, CH is frequently reported to be sporadic and candidate gene variations were found in <10% of the investigated patients. Here, we characterize the involvement of 11 candidate genes through a systematic Next Generation Sequencing (NGS) analysis. The NGS was performed in 177 unrelated CH patients (94 gland-in-situ; 83 dysgenesis) and in 3,538 control subjects. Non-synonymous or splicing rare variants (MAF < 0.01) were accepted, and their functional impact was predicted by a comprehensive bioinformatic approach and co-segregation studies. The frequency of variations in cases and controls was extended to 18 CH-unrelated genes. At least one rare variant was accepted in 103/177 patients. Monogenic recessive forms of the disease were found in five cases, but oligogenic involvement was detected in 39 patients. The 167 variations were found to affect all genes independently of the CH phenotype. These findings were replicated in an independent cohort of additional 145 CH cases. When compared to 3,538 controls, the CH population was significantly enriched with disrupting variants in the candidate genes (P = 5.5 × 10-7), but not with rare variations in CH-unrelated genes. Co-segregation studies of the hypothyroid phenotype with multiple gene variants in several pedigrees confirmed the potential oligogenic origin of CH. The systematic NGS approach reveals the frequent combination of rare variations in morphogenetic or functional candidate genes in CH patients independently of phenotype. The oligogenic origin represents a suitable explanation for the frequent sporadic CH occurrence.