Exome Sequencing has a high diagnostic rate in sporadic congenital hypopituitarism and reveals novel candidate genes.

CONTEXT: The pituitary gland is key for childhood growth, puberty, and metabolism. Pituitary dysfunction is associated with a spectrum of phenotypes, from mild to severe. Congenital Hypopituitarism (CH) is the most commonly reported pediatric endocrine dysfunction with an incidence of 1:4000, yet low rates of genetic diagnosis have been reported. OBJECTIVE: We aimed to unveil the genetic etiology of CH in a large cohort of patients from Argentina. METHODS: We performed whole exome sequencing of 137 unrelated cases of CH, the largest cohort examined with this method to date. RESULTS: Of the 137 cases, 19.1% and 16% carried pathogenic or likely pathogenic variants in known and new genes, respectively, while 28.2% carried variants of uncertain significance. This high yield was achieved through the integration of broad gene panels (genes described in animal models and/or other disorders), an unbiased candidate gene screen with a new bioinformatics pipeline (including genes high loss of function intolerance), and analysis of copy number variants. Three novel findings emerged. First, the most prevalent affected gene encodes the cell adhesion factor ROBO1. Affected children had a spectrum of phenotypes, consistent with a role beyond pituitary stalk interruption syndrome. Second, we found that CHD7 mutations also produce a phenotypic spectrum, not always associated with full CHARGE syndrome. Third, we add new evidence of pathogenicity in the genes PIBF1 and TBC1D32, and report 13 novel candidate genes associated with CH (e.g. PTPN6, ARID5B). CONCLUSION: Overall, these results provide an unprecedented insight into the diverse genetic etiology of hypopituitarism.

Case report: Novel SIN3A loss-of-function variant as causative for hypogonadotropic hypogonadism in Witteveen-Kolk syndrome.

Pubertal delay can be due to hypogonadotropic hypogonadism (HH), which may occur in association with anosmia or hyposmia and is known as Kallmann syndrome (OMIM #308700). Recently, hypogonadotropic hypogonadism has been suggested to overlap with Witteveen-Kolk syndrome (WITKOS, OMIM #613406) associated with 15q24 microdeletions encompassing SIN3A. Whether hypogonadotropic hypogonadism is due to haploinsufficiency of SIN3A or any of the other eight genes present in 15q24 is not known. We report the case of a female patient with delayed puberty associated with intellectual disability, behavior problems, dysmorphic facial features, and short stature, at the age of 14 years. Clinical, laboratory, and imaging assessments confirmed the diagnosis of Kallmann syndrome. Whole-exome sequencing identified a novel heterozygous frameshift variant, NM_001145358.2:c.3045_3046dup, NP_001138830.1:p.(Ile1016Argfs*6) in SIN3A, classified as pathogenic according to the American College of Medical Genetics and Genomics (ACMG/AMP) criteria. Reverse phenotyping led to the clinical diagnosis of WITKOS. No other variant was found in the 96 genes potentially related to hypogonadotropic hypogonadism. The analysis of the other contiguous seven genes to SIN3A in 15q24 did not reveal any clinically relevant variant. In conclusion, these findings point to SIN3A as the gene in 15q24 related to the reproductive phenotype in patients with overlapping WITKOS and Kallmann syndrome.

Diagnosing and treating anterior pituitary hormone deficiency in pediatric patients.

Hypopituitarism, or the failure to secrete hormones produced by the anterior pituitary (adenohypophysis) and/or to release hormones from the posterior pituitary (neurohypophysis), can be congenital or acquired. When more than one pituitary hormone axis is impaired, the condition is known as combined pituitary hormone deficiency (CPHD). The deficiency may be primarily due to a hypothalamic or to a pituitary disorder, or concomitantly both, and has a negative impact on target organ function. This review focuses on the pathophysiology, diagnosis and management of anterior pituitary hormone deficiency in the pediatric age. Congenital hypopituitarism is generally due to genetic disorders and requires early medical attention. Exposure to toxicants or intrauterine infections should also be considered as potential etiologies. The molecular mechanisms underlying the fetal development of the hypothalamus and the pituitary are well characterized, and variants in the genes involved therein may explain the pathophysiology of congenital hypopituitarism: mutations in the genes expressed in the earliest stages are usually associated with syndromic forms whereas variants in genes involved in later stages of pituitary development result in non-syndromic forms with more specific hormone deficiencies. Tumors or lesions of the (peri)sellar region, cranial radiation therapy, traumatic brain injury and, more rarely, other inflammatory or infectious lesions represent the etiologies of acquired hypopituitarism. Hormone replacement is the general strategy, with critical periods of postnatal life requiring specific attention.

An in vivo functional assay to characterize human STAT5B genetic variants during zebrafish development.

Growth hormone (GH) binding to GH receptor activates janus kinase 2 (JAK2)-signal transducer and activator of transcription 5b (STAT5b) pathway, which stimulates transcription of insulin-like growth factor-1 (IGF1), insulin-like growth factor binding protein 3 (IGFBP3) and insulin-like growth factor acid-labile subunit (IGFALS). Although STAT5B deficiency was established as an autosomal recessive disorder, heterozygous dominant-negative STAT5B variants have been reported in patients with less severe growth deficit and milder immune dysfunction. We developed an in vivo functional assay in zebrafish to characterize the pathogenicity of three human STAT5B variants (p.Ala630Pro, p.Gln474Arg and p.Lys632Asn). Overexpression of human wild-type (WT) STAT5B mRNA and its variants led to a significant reduction of body length together with developmental malformations in zebrafish embryos. Overexpression of p.Ala630Pro, p.Gln474Arg or p.Lys632Asn led to an increased number of embryos with pericardial edema, cyclopia and bent spine compared with WT STAT5B. Although co-injection of WT and p.Gln474Arg and WT and p.Lys632Asn STAT5B mRNA in zebrafish embryos partially or fully rescues the length and the developmental malformations in zebrafish embryos, co-injection of WT and p.Ala630Pro STAT5B mRNA leads to a greater number of embryos with developmental malformations and a reduction in body length of these embryos. These results suggest that these variants could interfere with endogenous stat5.1 signaling through different mechanisms. In situ hybridization of zebrafish embryos overexpressing p.Gln474Arg and p.Lys632Asn STAT5B mRNA shows a reduction in igf1 expression. In conclusion, our study reveals the pathogenicity of the STAT5B variants studied.

Delayed Puberty Phenotype Observed in Noonan Syndrome Is More Pronounced in Girls than Boys.

INTRODUCTION: Pubertal delay is described as one of the clinical features in Noonan syndrome (NS) and it may be one of the factors causing short adult height in those patients. The present study aimed at characterizing pubertal development in NS and identifying pubertal delay predictors. METHODS: We analyzed 133 individuals with a molecular diagnosis of NS and clinical puberty evaluation. We characterized delayed puberty as pubertal onset after 12 years in girls and 13.5 years in boys, according to parameters of the Brazilian population. To investigate its predictors, we correlated the age at onset of puberty with several characteristics and genotype in a multilevel regression model. For comprehending pubertal development in NS, we assessed age and anthropometric measures at each Tanner stage and adult age. RESULTS: The mean age at puberty onset for girls was 11.9 ± 1.9 years and for boys, 12.5 ± 1.7 years, significantly later than the Brazilian population (p = 0.025; p < 0.001). Girls (49.1%) presented delayed puberty more frequently than boys (27.9%, p = 0.031). Body mass index standard deviation scores (SDS) and insulin growth factor 1 SDS at puberty onset significantly predicted later puberty entry. Height gain from the onset of puberty to adult height was lower in children with pubertal delay. CONCLUSION: Pubertal delay is characteristically found in children with NS, more frequently in females. The low weight of patients with NS could modulate the age of puberty, just as the increase in overweight/obesity in the general population has shown an effect on reducing the age of onset of puberty.

High-throughput splicing assays identify missense and silent splice-disruptive POU1F1 variants underlying pituitary hormone deficiency.

Pituitary hormone deficiency occurs in ∼1:4,000 live births. Approximately 3% of the cases are due to mutations in the alpha isoform of POU1F1, a pituitary-specific transcriptional activator. We found four separate heterozygous missense variants in unrelated individuals with hypopituitarism that were predicted to affect a minor isoform, POU1F1 beta, which can act as a transcriptional repressor. These variants retain repressor activity, but they shift splicing to favor the expression of the beta isoform, resulting in dominant-negative loss of function. Using a high-throughput splicing reporter assay, we tested 1,070 single-nucleotide variants in POU1F1. We identified 96 splice-disruptive variants, including 14 synonymous variants. In separate cohorts, we found two additional synonymous variants nominated by this screen that co-segregate with hypopituitarism. This study underlines the importance of evaluating the impact of variants on splicing and provides a catalog for interpretation of variants of unknown significance in POU1F1.

Comprehensive Identification of Pathogenic Gene Variants in Patients With Neuroendocrine Disorders.

PURPOSE: Congenital hypopituitarism (CH) can present in isolation or with other birth defects. Mutations in multiple genes can cause CH, and the use of a genetic screening panel could establish the prevalence of mutations in known and candidate genes for this disorder. It could also increase the proportion of patients that receive a genetic diagnosis. METHODS: We conducted target panel genetic screening using single-molecule molecular inversion probes sequencing to assess the frequency of mutations in known hypopituitarism genes and new candidates in Argentina. We captured genomic deoxyribonucleic acid from 170 pediatric patients with CH, either alone or with other abnormalities. We performed promoter activation assays to test the functional effects of patient variants in LHX3 and LHX4. RESULTS: We found variants classified as pathogenic, likely pathogenic, or with uncertain significance in 15.3% of cases. These variants were identified in known CH causative genes (LHX3, LHX4, GLI2, OTX2, HESX1), in less frequently reported genes (FOXA2, BMP4, FGFR1, PROKR2, PNPLA6) and in new candidate genes (BMP2, HMGA2, HNF1A, NKX2-1). CONCLUSION: In this work, we report the prevalence of mutations in known CH genes in Argentina and provide evidence for new candidate genes. We show that CH is a genetically heterogeneous disease with high phenotypic variation and incomplete penetrance, and our results support the need for further gene discovery for CH. Identifying population-specific pathogenic variants will improve the capacity of genetic data to predict eventual clinical outcomes.

A novel heterozygous STAT5B variant in a patient with short stature and partial growth hormone insensitivity (GHI).

BACKGROUND: The most frequent monogenic causes of growth hormone insensitivity (GHI) include defects in genes encoding the GH receptor itself (GHR), the signal transducer and activator of transcription (STAT5B), the insulin like-growth factor type I (IGF1) and the acid-labile subunit (IGFALS). GHI is characterized by a continuum of mild to severe post-natal growth failure. OBJECTIVE: To characterize the molecular defect in a patient with short stature and partial GHI. PATIENT AND METHODS: The boy was born at term adequate for gestational age from non-consanguineous normal-stature parents. At 2.2 years, he presented proportionate short stature (height -2.77 SDS), wide forehead and normal mental development. Whole-exome analysis and functional characterization (site-directed mutagenesis, dual luciferase reporter assay, immunofluorescence and western immunoblot) were performed. RESULTS: Biochemical and endocrinological evaluation revealed partial GH insensitivity with normal stimulated GH peak (7.8 ng/mL), undetectable IGF1 and low IGFBP3 levels. Two heterozygous variants in the GH-signaling pathway were found: a novel heterozygous STAT5B variant (c.1896G>T, p.K632N) and a hypomorphic IGFALS variant (c.1642C>T, p.R548W). Functional in vitro characterization demonstrated that p.K632N-STAT5b is an inactivating variant that impairs STAT5b activity through abolished phosphorylation. Remarkably, the patient's immunological evaluation displayed only a mild hypogammaglobulinemia, while a major characteristic of STAT5b deficient patients is severe immunodeficiency. CONCLUSIONS: We reported a novel pathogenic inactivating STAT5b variant, which may be associated with partial GH insensitivity and can present without severe immunological complications in heterozygous state. Our results contribute to expand the spectrum of phenotypes associated to GHI.

Development and Validation of a Prediction Rule for Growth Hormone Deficiency Without Need for Pharmacological Stimulation Tests in Children With Risk Factors.

Introduction: Practice guidelines cannot recommend establishing a diagnosis of growth hormone deficiency (GHD) without performing growth hormone stimulation tests (GHST) in children with risk factors, due to the lack of sufficient evidence. Objective: Our goal was to generate an evidence-based prediction rule to diagnose GHD in children with growth failure and clinically identifiable risk factors. Methods: We studied a cohort of children with growth failure to build the prediction model, and a second, independent cohort to validate the prediction rule. To this end, we assessed the existence of: pituitary dysgenesis, midline abnormalities, (supra)sellar tumor/surgery, CNS infection, traumatic brain injury, cranial radiotherapy, chemotherapy, genetic GHD, pituitary hormone deficiencies, and neonatal hypoglycemia, cholestasis, or hypogenitalism. Selection of variables for model building was performed using artificial intelligence protocols. Specificity of the prediction rule was the main outcome measure in the validation set. Results: In the first cohort (n=770), the resulting prediction rule stated that a patient would have GHD if (s)he had: pituitary dysgenesis, or two or more anterior pituitary deficiencies, or one anterior pituitary deficiency plus: neonatal hypoglycemia or hypogenitalism, or diabetes insipidus, or midline abnormalities, or (supra)sellar tumor/surgery, or cranial radiotherapy ≥18 Gy. In the validation cohort (n=161), the specificity of the prediction rule was 99.2% (95% CI: 95.6-100%). Conclusions: This clinical rule predicts the existence of GHD with high specificity in children with growth disorders and clinically identifiable risk factors, thus providing compelling evidence to recommend that GHD can be safely diagnosed without recurring to GHST in neonates and children with growth failure and specific comorbidities.

p.R209H GH1 variant challenges short stature assessment.

OBJECTIVE: to describe the marked variability in clinical and biochemical patterns that are associated with a p.R209H GH1 missense variant in a large Argentinean pedigree, which makes the diagnosis of GHD elusive. DESIGN: We describe a non-consanguineous pedigree composed by several individuals with short stature, including 2 pediatric patients with typical diagnosis of isolated growth hormone deficiency (IGHD) and 4 other siblings with severe short stature, low serum IGF-1 and IGFBP-3, but normal stimulated GH levels, suggesting growth hormone insensitivity (GHI) in the latter group. RESULTS: Patients with classical IGHD phenotype carried a heterozygous variant in GH1: c.626G>A (p.R209H). Data from the extended pedigree suggested GH1 as the initial candidate gene, which showed the same pathogenic heterozygous GH1 variant in the four siblings with short stature and a biochemical pattern of GHI. CONCLUSIONS: We suggest considering GH1 sequencing in children with short stature associated to low IGF-1 and IGFBP-3 serum levels, even in the context of normal response to growth hormone provocative testing (GHPT).

A homozygous mutation in the highly conserved Tyr60 of the mature IGF1 peptide broadens the spectrum of IGF1 deficiency.

BACKGROUND: IGF1 is a key factor in fetal and postnatal growth. To date, only three homozygous IGF1 gene defects leading to complete or partial loss of IGF1 activity have been reported in three short patients born small for gestational age. We describe the fourth patient with severe short stature presenting a novel homozygous IGF1 gene mutation. RESULTS: We report a boy born from consanguineous parents at 40 weeks of gestational age with intrauterine growth restriction and severe postnatal growth failure. Physical examination revealed proportionate short stature, microcephaly, facial dysmorphism, bilateral sensorineural deafness and mild global developmental delay. Basal growth hormone (GH) fluctuated from 0.2 to 29 ng/mL, while IGF1 levels ranged from -1.15 to 2.95 SDS. IGFBP3 was normal-high. SNP array delimited chromosomal regions of homozygosity, including 12q23.2 where IGF1 is located. IGF1 screening by HRM revealed a homozygous missense variant NM_000618.4(IGF1):c.322T>C, p.(Tyr108His). The change of the highly conserved Tyr60 in the mature IGF1 peptide was consistently predicted as pathogenic by multiple bioinformatic tools. Tyr60 has been described to be critical for IGF1 interaction with type 1 IGF receptor (IGF1R). In vitro, HEK293T cells showed a marked reduction of IGF1R phosphorylation after stimulation with serum from the patient as compared to sera from age-matched controls. Mutant IGF1 was also less efficient in inducing cell growth. CONCLUSION: The present report broadens the spectrum of clinical and biochemical presentation of homozygous IGF1 defects and underscores the variability these patients may present depending on the IGF/IGF1R pathway activity.

Insuficiencia Hipofisaria Congénita / Congenital Pituitary Hormone Deficiency

La insuficiencia hipofisaria congénita es un trastorno originado en la alteración de la ontogenia de la glándula hipofisaria que determina la disminución o falta de trofinas hipofisarias: adrenocorticotropina, tirotropina, hormona de crecimiento, prolactina, gonadotrofinas y/u hormona antidiurética. Es una patología compleja e infrecuente que, debido a su signo sintomatología inespecífica, suele ser difícil de reconocer a edades tempranas, derivando en aumento de la morbilidad y eventualmente de la mortalidad. Durante el periodo neonatal, es característica la ictericia colestática asociada a hipoglucemias recurrentes. Puede formar parte de un cuadro sindrómico, siendo el más frecuente la displasia septoóptica, que asocia defectos de línea media y alteraciones oculares. La mayoría presenta anomalías anatómicas de la región selar y supraselar evidenciables en la Resonancia Magnética. El diagnóstico bioquímico tiene especificaciones particulares para la evaluación de cada trofina hipofisaria y de acuerdo a la edad del paciente. El tratamiento consiste en la terapia de reemplazo hormonal observándose buena respuesta en la mayoría de los pacientes. La detección precoz de los niños con insuficiencia hipofisaria permite la activación rápida y efectiva de una estrategia diagnóstica con la toma de muestras bioquímicas apropiadas, la consulta temprana al endocrinólogo infantil y la instauración del tratamiento específico

Congenital pituitary hormone deficiency is a disorder originated in pituitary gland ontogeny generating decrease or lack of pituitary hormones: adrenocorticotropin, thyrotropin, growth hormone, prolactin, gonadotropins and/or antidiuretic hormone. It is a complex and infrequent disease usually difficult to recognize at an early age due to its non-specific symptomatology, resulting in increased morbidity and eventual mortality. During the neonatal period, cholestatic jaundice associated with recurrent hypoglycaemia is frequent. Pituitary hormone deficiency can be part of a syndrome; the most frequent is septo-optic dysplasia, associating midline defects and ocular disorders. Most have anatomical anomalies of the sellar and suprasellar region seen in magnetic resonance imaging. Biochemical diagnosis has particular specifications for the evaluation of each pituitary hormone and varies according to patient´s age. The treatment consists in hormone replacement therapy and generally with good results. The early detection of children with pituitary hormone deficiency allows the rapid and effective activation of a diagnostic strategy, facilitates the appropriate biochemical samples, the early contact with the pediatric endocrinologist and the establishment of specific treatment

Síndrome de Turner: variaciones en la constitución cromosómica y sus efectos en la expresión fenotípica / Turner Syndrome: variations in the chromosomal constitution and its effects on the phenotypic expression

El Síndrome de Turner es un desorden cromosómico causado por haploinsuficiencia completa o parcial de uno de los cromosomas sexuales. Incidencia 1: 2500 recién nacidas vivas. Clínicamente las pacientes presentan talla baja, un espectro amplio de anomalías somáticas y disgenesia gonadal. Desde el año 1968 hasta el presente se estudiaron clínica y citogenéticamente 630 niñas con fenotipo de Turner, sin ambigüedad genital y con cariotipos anormales, quienes consultaron en la División de Endocrinología del Hospital de Niños "Ricardo Gutiérrez". Se realizó cariotipo en sangre, al inicio con metodología estándar, luego con diferentes bandeos convencionales y de alta resolución. En casos especiales se aplicó la técnica FISH y el análisis molecular de los cromosomas X e Y. El número de metafases analizadas también varió con el tiempo, permitiendo evidenciar más de una línea celular. En casos de alta sospecha clínica, la lectura de 100 metafases permitió poner en evidencia mosaicismos bajos conteniendo la línea 45,X. En nuestra serie la monosomía de cromosoma sexual 45, X fue la más frecuente siguiendo los mosaicos numéricos y estructurales de uno de los cromosomas sexuales. Los diferentes hallazgos cromosómicos nos han permitido establecer una correlación fenotipo-cariotipo en regiones específicas de los cromosomas sexuales

Turner Syndrome is a common chromosomal disorder caused by total or partial haploinsufficiency of one of the sex chromosomes. Incidence: 1: 2500. Clinically is characterized by short stature, several typical somatic features, and gonadal dysgenesis. This is a retrospective study involving 630 girls with Turner phenotype and abnormal karyotype, evaluated at the Endocrinology Division of Children´s Hospital "Ricardo Gutiérrez" between 1968 and 2018. The karyotype was done in leucocytes from peripheral blood and the metaphases were analyzed at the beginning with standard methodology and then with different banding techniques, standard and high resolution. In special cases, the FISH technique and the molecular analysis of the X and Y chromosomes were applied. The number of metaphases analyzed also changed with time, allowing the finding of more than one cellular line. In cases where the clinical suspicion was strong, the analysis of 100 metaphases allowed us to put in evidence low mosaicisms containing the 45,X line. In our study the monosomy of sexual chromosome 45, X was the most frequent following the numerical and structural mosaics of one of the sex chromosomes. The different chromosomal constitutions have contributed to establish in our patients phenotype-karyotype correlation with specific regions of the sex chromosomes

Next generation sequencing panel based on single molecule molecular inversion probes for detecting genetic variants in children with hypopituitarism.

BACKGROUND: Congenital Hypopituitarism is caused by genetic and environmental factors. Over 30 genes have been implicated in isolated and/or combined pituitary hormone deficiency. The etiology remains unknown for up to 80% of the patients, but most cases have been analyzed by limited candidate gene screening. Mutations in the PROP1 gene are the most common known cause, and the frequency of mutations in this gene varies greatly by ethnicity. We designed a custom array to assess the frequency of mutations in known hypopituitarism genes and new candidates, using single molecule molecular inversion probes sequencing (smMIPS). METHODS: We used this panel for the first systematic screening for causes of hypopituitarism in children. Molecular inversion probes were designed to capture 693 coding exons of 30 known genes and 37 candidate genes. We captured genomic DNA from 51 pediatric patients with CPHD (n = 43) or isolated GH deficiency (IGHD) (n = 8) and their parents and conducted next generation sequencing. RESULTS: We obtained deep coverage over targeted regions and demonstrated accurate variant detection by comparison to whole-genome sequencing in a control individual. We found a dominant mutation GH1, p.R209H, in a three-generation pedigree with IGHD. CONCLUSIONS: smMIPS is an efficient and inexpensive method to detect mutations in patients with hypopituitarism, drastically limiting the need for screening individual genes by Sanger sequencing.

Estilos parentales y estrategias de afrontamiento en pacientes con hipotiroidismo congénito detectado y tratado en forma temprana / Parenting styles and coping strategies among patients with early detected and treated congenital hypothyroidism

El hipotiroidismo congénito (HC) como enfermedad crónica impacta en la vinculación padres-hijo y en recursos del niño para afrontar situaciones conflictivas. Objetivos. Describir estilos parentales desde la percepción del hijo con HC y sus estrategias de afrontamiento. Población y métodos. Niños de entre 9 y 10 años con HC detectado por pesquisa neonatal y adecuadamente tratado y un grupo sin HC (grupo control). Se utilizó el cuestionario argentino de afrontamiento y la escala argentina de percepción de la relación con los padres y el subtest comprensión de la Wechsler Intelligence Scale for Children III (WISC III). Se compararon los resultados con el análisis multivariante de la varianza (multivariate analysis of variance; MANOVA, por sus siglas en inglés). Resultados. Se incluyeron 60 niños con HC; percibían a su madre con una modalidad de control estricto y a su padre con más aceptación. Buscaban mayor apoyo y se paralizaban más ante situaciones conflictivas que los 60 niños sin patología. Conclusión. Estos hallazgos podrían asociarse a mayor dependencia. Deben considerarse en la atención del HC.

Congenital hypothyroidism (CH), as any chronic disease, has an impact on the parent-child relationship and on the child's resources to cope with conflicting situations. Objectives. To describe parenting styles according to the perception of children with CH and their coping strategies. Population and methods. Children aged 9-10 years who had CH detected by newborn screening and had received adequate treatment and a group without CH (control group). The Argentine Coping Questionnaire, the Argentine Scale for the Perception of Parent Relations, and the comprehension subtest of the Wechsler Intelligence Scale for Children III (WISC III) were used. Results were compared using a multivariate analysis of variance (MANOVA). Results. Sixty children with CH were included; they perceived that their mothers exercised a strict control and that their fathers showed more acceptance. They sought more support and became paralyzed more often in conflicting situations than the 60 children without CH. Conclusion. These findings may be associated with a higher level of dependence. They should be taken into consideration in CH care.