Hypercalcemia due to CYP24A1 mutations: a systematic descriptive review.

BACKGROUND AND OBJECTIVES: CYP24A1 encodes a 24-hydroxylase involved in vitamin D catabolism, whose loss-of-function results in vitamin D-dependent hypercalcemia. Since the identification of CYP24A1 variants as a cause of idiopathic infantile hypercalcemia, a large body of literature has emerged indicating heterogeneity in penetrance, symptoms, biochemistry, and treatments. The objectives of the present research work were to investigate the clinical heterogeneity of the disease, the possibility of a relevant phenotype for monoallelic carriers, and to compare the hypocalcemic effect of the available therapies. METHODS: Two reviewers searched different databases for studies published between the identification of CYP24A1 variants and December 31, 2020. Eligible studies included clinical trials and reports describing carriers of CYP24A1 variants. RESULTS: Fifty eligible studies were identified, accounting for 221 patients. Genetic data were retrieved and allele frequencies were calculated. Acute hypercalcemia was the typical presentation during the first year of life (76%, P = 0.0005), and nephrocalcinosis was more frequent in infancy (P < 0.0001). Pregnancy was associated with symptomatic hypercalcemia in 81.8% and high rates of obstetric complications. Monoallelic carriers displayed significant rates of nephrolithiasis (19.4%), nephrocalcinosis (4.9%), and symptomatic hypercalcemia (5.6%). CONCLUSIONS: CYP24A1 loss-of-function results in an age-dependent phenotype, which can be exacerbated by triggering factors, such as pregnancy. Although biallelic carriers present more significant clinical and biochemical features, monoallelic carriers have an increased risk of calcium-related conditions. The highly variable tested therapeutic approaches did not allow to draw conclusions on preferable therapeutic regime.

Hypercalcemia due to methadone-induced adrenal insufficiency in a case of oral cancer.

A 31-year-old patient of post-surgical recurrent buccal carcinoma (post-chemo and radiotherapy) on multimodal analgesia with methadone, paracetamol and gabapentin presented to pain clinic with occasional bleeding from tumor area and incidental hypercalcemia. The hypercalcemia was attributed to adrenal insufficiency due to methadone, with no other obvious reasons identified for hypercalcemia or adrenal insufficiency. The patient was managed with the change of opioid, regular aseptic wound dressings and management of hypercalcemia with hydration, calcitonin and steroid therapy. Hypercalcemia in a cancer patient can have multiple other causes like hypercalcemia of malignancy and primary or secondary parathyroid carcinoma. A strong clinical suspicion and appropriate battery of tests may be required to arrive at the diagnosis. Prompt management, including identification and management of the primary pathology along with aggressive hydration with hormonal therapy, may prove to be life-saving.

Metastatic Calcinosis of Gastric Mucosa.

Calcinosis cutis refers to the deposition of calcium salts in the cutaneous and subcutaneous tissue and is frequently associated with inflammation. Gastric calcinosis can be classified into metastatic, dystrophic, and idiopathic; metastatic calcinosis is the most common type. In metastatic calcification, calcium salts are deposited in normal soft tissues in the setting of altered metabolism of serum calcium and phosphorus and is a rare and serious complication of chronic renal failure. The important factors contributing to the development of metastatic calcinosis are hypercalcemia, hyperphosphatemia, and an elevated calcium-phosphate product. The most striking feature of this diagnosis is the calcification around the large joints. While it mostly involves dermis of small and medium-sized vessels, it can rarely affect the mucosal layers of the gastrointestinal (GI) tract. Calcinosis presents as a marker for the presence of calcifications in other organs, such as heart or lung, which can be life-threatening. Patients rarely present with clinical symptoms of GI upset, dyspepsia, or epigastric pain that are attributed to calcinosis. If patients present with GI symptoms, infectious causes remain to be higher on the differential. We present a case of incidental finding of gastric mucosal calcinosis during the workup and treatment of dysphagia.

Elevated levels of circulating fibroblast growth factor 23 with hypercalcemia following discontinuation of denosumab.

We report a case of a 47-year-old woman with hypercalcemia 6 months after discontinuation of denosumab. She underwent right mastectomy for breast cancer and had received aromatase inhibitor and denosumab therapy for 5 years. Thirst, appetite loss, and bilateral ankle pain began few months after cessation of denosumab. She was admitted to the hospital for hypercalcemia and hyperthyroidism 6 months after the last dose of denosumab. Laboratory investigations revealed hypercalcemia, normophosphatemia, normal renal function, and elevated levels of fibroblast growth factor 23 (FGF-23). Serum tartrate-resistant acid phosphatase 5b and urine N-terminal cross-linked telopeptide of type I collagen were both elevated, and bone scintigraphy revealed increase of whole bone uptake. Radiological examinations showed no recurrence of breast cancer or tumors that secrete intact PTH or FGF-23. Hypercalcemia, which lasted for 1 month, was refractory to discontinuation of the aromatase inhibitor, normalization of thyroid hormone levels, saline hydration, and calcitonin administration, but was effectively treated with zoledronic acid. Abnormal uptake on bone scintigraphy and ankle pain both resolved a few months after treatment, and hypercalcemia has not recurred in the ensuing 2 years. In conclusion, we found elevated levels of circulating FGF-23 with hypercalcemia following the discontinuation of denosumab. FGF-23 might be a surrogate marker for massive bone resorption triggered by discontinuation of long-term denosumab treatment.

Renal involvement in adrenal insufficiency (Addison disease): can we always recognize it?

Addison disease is due to the destruction or dysfunction of the entire adrenal cortex. Nowadays, the causes of adrenal insufficiency are autoimmune disease for 70-90% and tuberculosis for 7-20%. Many typical signs and symptoms, such as hyponatremia, hyperkalaemia, or renal insufficiency can represent the reasons for a nephrology consultation, especially in conditions of urgency, and they can easily be confused with other causes. Moreover, the fact that in a short time range we have diagnosed the three cases described as a guide in this review, has aroused our attention as nephrologists on a disease in which we have probably already encountered but without recognizing it. The blood tests showed in all three patients severe electrolyte disorders and acute renal failure which will be discussed in their physiopathogenetic mechanisms. In a peculiar way, these alterations were not controlled with repolarizing solutions, fluid replacement and increased volemia, but only after steroid administration. In conclusion, in this review all the known pathogenic mechanisms causing disorders of nephrological interest in adrenal insufficiency are discussed.

A clinical perspective of parathyroid hormone related hypercalcaemia.

There are many causes of hypercalcaemia including hyperparathyroidism, drugs, granulomatous disorders and malignancy. Parathyroid hormone (PTH) related hypercalcaemia is most commonly caused by primary hyperparathyroidism (PHPT) and more rarely by familial hypocalciuric hypercalcaemia (FHH). Algorithms for diagnosis of PTH related hypercalcaemia require assessment of a 24-h urinary calcium and creatinine excretion to calculate calcium/creatinine clearance ratio and radiological investigations including ultrasound scan and 99mTc-sestamibi-SPECT/CT. To illustrate investigations and management of parathyroid-related hypercalcaemia, we present a selection of distinct cases of PHPT due to eutopic and ectopic parathyroid adenomas, as well as a case with a syndromic form of PHPT (multiple endocrine neoplasia type 1), and a case with FHH type 1 due to a CASR inactivating mutation. Additional cases with normocalcaemic hyperparathyroidism and secondary hyperparathyroidism are included for completeness of differential diagnosis. The common eutopic parathyroid adenomas are easily treated with parathyroidectomy while the less common ectopic parathyroid adenomas require more complex investigations and operative procedures such as video-assisted thoracoscopic surgery. On the other hand, the much less common FHH does not require treatment. Assessment of kin with FHH is important to identify members with this inherited condition in order to prevent unnecessary interventions.

Hypercalcemia Heralding Pneumocystis jirovecii Pneumonia in an HIV-Seronegative Patient with Diffuse Cutaneous Systemic Sclerosis.

Pneumocystis pneumonia (PCP) is a life-threatening fungal infection occurring in immunocompromised patients such as HIV-positive patients with low CD4 cell count or patients under heavy immunosuppressive therapy. We report the case of a 59-year-old male with severe diffuse cutaneous systemic sclerosis presenting with asthenia, dry cough and worsening shortness of breath for the last 15 days. Biological studies were remarkable for PTH-independent severe hypercalcemia with low 25-hydroxyvitamin D and a paradoxically elevated 1,25-dihydroxyvitamin D. Early bronchoalveolar lavage allowed for PCP diagnosis and targeted treatment. We discuss the underlying physiopathology and difficulties regarding prophylaxis and treatment.

Eficacia y seguridad de cinacalcet en el tratamiento de pacientes adultos con hiperparatiroidismo primario con hipercalcemia persistente pese a terapia quirúrgica / Efficacy and safety of cinacalcet in the treatment of adult patients with primary hyperparathyroidism with persistent hypercalcaemia despite surgical therapy

INTRODUCCIÓN: El presente dictamen expone la evaluación de la eficacia y seguridad de cinacalcet 30mg administrada VO, para el tratamiento de pacientes adultos con diagnóstico de Hiperparatiroidismo Primario (HPTP) con hipercalcemia persistente pese a terapia quirúrgica (Paratiroidectomía). El hiperparatiroidismo primario (HPTP) es una enfermedad que involucra a una o más glándulas paratiroides, responsables de la producción de la hormona paratiroidea (PTH) y que afecta directamente el control normal de los niveles de calcio en sangre. Un paciente con HPTP produce altos niveles de PTH y calcio en sangre (hipercalcemia) así como altos niveles de calcio en orina. Durante la hipercalcemia, el calcio liberado de los huesos con el tiempo puede desencadenar en osteoporosis, cálculos renales y reducción en la función renal. Este trastorno se da en aproximadamente 1 % de la población adulta, pero afecta a más del 2 % de la misma después de los 55 años y especialmente a mujeres. La paratiroidectomía (PTx) es la terapia de elección para el HPTP con una tasa de curación cercana al 95 %. Esta consiste en una intervención quirúrgica para extraer una o más glándulas paratiroides, realizando previamente un diagnóstico y evaluación por imágenes. No obstante, existe una proporción de pacientes que no responden a la terapia quirúrgica, persistiendo con la sintomatología propia de la enfermedad y la hipercalcemia, los cuales son diagnosticados como pacientes con terapia quirúrgica fallida (PTx fallida) o fracaso quirúrgico. Actualmente, en EsSalud no se cuenta con un tratamiento farmacológico alternativo para dichos pacientes, en quienes la terapia convencional y de mayor beneficio (i.e., PTx) no funciona. Es por ello que existe la necesidad de evaluar otras alternativas farmacológicas que podrían ser de beneficio para éstos pacientes. METODOLOGÍA: Se llevó a cabo una búsqueda sistemática de la literatura con respecto a la eficacia y seguridad de Cinacalcet para el tratamiento de pacientes adultos con hiperparatiroidismo primario con hipercalcemia persistente pese a terapia quirúrgica. Se empleó además un motor de búsqueda para las bases de datos de PubMed-Medline, the Cochrane Library, LILACS y SciELO. La búsqueda se enfocó en guías de práctica clínica (GPC), evaluaciones de tecnologías sanitarias (ETS) y revisiones sistemáticas (RS) con o sin meta-análisis (MA), identificándose términos en lenguaje simple, así como términos MeSH2 relacionados a la población de interés, la intervención según la pregunta PICO especificada". RESULTADOS: Los resultados se han obtenido a partir de los desenlaces que se encuentran en la pregunta PICO. No se incluyeron, por ningún motivo, resultados que no están enmarcados en la pregunta PICO. De acuerdo con la pregunta PICO, se llevó a cabo una búsqueda de evidencia científica relacionada al uso de Cinacalcet como tratamiento de pacientes con HPTP con Hipercalcemia persistente pese a terapia quirúrgica. En la presente sinopsis se describe la evidencia disponible según el tipo de publicación, siguiendo lo indicado en los criterios de elegibilidad (GPC, ETS, RS, MA y ECA fase III). CONCLUSIONES: El presente dictamen preliminar tuvo como objetivo evaluar la evidencia disponible en relación a la eficacia y seguridad de cinacalcet 30 mg VO para el tratamiento de pacientes con HPTP con hipercalcemia persistente pese a terapia quirúrgica.  Luego de realizar una búsqueda sistemática de la literatura, se identificaron y consideraron para el presente dictamen 4 GPC, una ETS y un solo ECA que evaluaron la eficacia de cinacalcet en pacientes con HPTP y con hipercalcemia no quirúrgicos. Tres de estas GPC coinciden en que cinacalcet es una opción terapéutica para el tratamiento de pacientes con HPTP con hipercalcemia que no se someten a terapia quirúrgica por algunas de las razones antes mencionada; y una GPC no reportó ninguna recomendación sobre el uso de cinacalcet u otro tratamiento más allá que realizar procedimientos más invasivos a fin evaluar mejor al paciente y buscar otras causas de hiperparatiroidismo secundario. Así mismo, la ETS elaborada por el NHS del Reino Unido, aprueba la disponibilidad del uso de cinacalcet para pacientes con HPTP con PTx fallida como opción terapéutica frente a la falla de la terapia convencional. El ECA desarrollado por Peacock, muestra la eficacia de cinacalcet comparada con placebo para la disminución de los niveles de calcio sérico llevándolo a niveles normales (8.5 ­ 10.5 mg/dl) y una reducción de 0.5 mg/dl (0.12 mmol/litro) del valor basal, estadísticamente significativos, mostrando que en el grupo que recibió cinacalcet los niveles de calcio bajaron hasta estar dentro de ,os valores normales dentro de las primeras 2 semanas y manteniéndose constante a lo largo de todo el periodo de seguimiento a diferencia del grupo placebo que mantuvo la hipercalcemia a lo largo del seguimiento (10.9 mng/dl). Se reportaron efectos adversos leves como náuseas y vómitos, los cuales fueron de igual proporción entre el grupo de intervención y el grupo control. Y que a pesar de las limitaciones existentes cinacalcet ofrece una alternativa de tratamiento para estos pacientes. Existe una proporción de pacientes que, a pesar de someterse a PTx, la terapia de elección para HPTP con una tasa de curación cercana al 95%, persiste con hipercalcemia. Frente a ello, actualmente EsSalud no cuenta con alguna alternativa de tratamiento farmacológico para controlar los niveles de calcio de manera gradual y segura, cuando la PTx es fallida, más allá de un seguimiento y evaluación constante del paciente por lo que surge la necesidad de evaluar otras alternativas de tratamientos. Consideramos en ese sentido, que a pesar de las limitaciones que pueda presentar la evidencia encontrada y teniendo el antecedente de aprobación de uso de cinacalcet para el tratamiento de hipercalcemia en pacientes con HPTS en la institución, se tiene experiencia de uso de este fármaco. Así podemos concluir que, cinacalcet supondría una alternativa de tratamiento para el control de los pacientes con HPTP que no responden a terapia quirúrgica. Por lo expuesto, el Instituto de Evaluaciones de Tecnologías en Salud e Investigación ­ IETSI aprueba el uso de cinacalcet para el manejo de los pacientes con diagnóstico de HPTP con hipercalcemia persistente pese a terapia quirúrgica. La vigencia del presente dictamen preliminar es de un año a partir de la publicación. Así, la continuación de dicha aprobación estará sujeta a los resultados obtenidos de los pacientes que reciban este tratamiento, a los reportes de seguridad que puedan surgir durante farmacovigilancia activa y nueva evidencia que pueda surgir en el tiempo.

A practical self-made device in parathyroid autotransplantation for patients with secondary hyperparathyroidism.

OBJECTIVE: Secondary hyperparathyroidism (sHPT) is one of the most serious complications in patients on long-term hemodialysis. These patients may suffer from metabolic bone diseases, severe atherosclerosis, and undesirable cardiovascular events. Endoscopic parathyroidectomy with autotransplantation is a treatment option for those who do not respond to clinical management. This study aimed to investigate practical use of a self-made device in parathyroid autotransplantation for patients with sHPT, and to compare this device with ordinary surgical scissors. METHODS: A total of 15 patients with sHPT were treated with endoscopic parathyroidectomy and autotransplantation. Pieces of parathyroid tissue were squeezed in our self-made device and injected into the brachioradialis muscle. Sixteen patients with sHPT who were treated with traditional parathyroid transplantation served as controls. Serum levels of parathyroid hormone, alkaline phosphatase, calcium, phosphorus and intact parathyroid hormone were measured before and after surgery. RESULTS: Preoperative symptoms were alleviated, and serum parathyroid hormone and alkaline phosphatase levels, hyperphosphatemia, and hypercalcemia were improved or normalized in all of the patients in both groups. Pathological examinations showed that parathyroid cells remained active. CONCLUSION: Application of our squeezing device is an economic, effective, and safe method in endoscopic parathyroidectomy and autotransplantation for patients with sHPT.

The calcium-sensing receptor in physiology and in calcitropic and noncalcitropic diseases.

The Ca2+-sensing receptor (CaSR) is a dimeric family C G protein-coupled receptor that is expressed in calcitropic tissues such as the parathyroid glands and the kidneys and signals via G proteins and ß-arrestin. The CaSR has a pivotal role in bone and mineral metabolism, as it regulates parathyroid hormone secretion, urinary Ca2+ excretion, skeletal development and lactation. The importance of the CaSR for these calcitropic processes is highlighted by loss-of-function and gain-of-function CaSR mutations that cause familial hypocalciuric hypercalcaemia and autosomal dominant hypocalcaemia, respectively, and also by the fact that alterations in parathyroid CaSR expression contribute to the pathogenesis of primary and secondary hyperparathyroidism. Moreover, the CaSR is an established therapeutic target for hyperparathyroid disorders. The CaSR is also expressed in organs not involved in Ca2+ homeostasis: it has noncalcitropic roles in lung and neuronal development, vascular tone, gastrointestinal nutrient sensing, wound healing and secretion of insulin and enteroendocrine hormones. Furthermore, the abnormal expression or function of the CaSR is implicated in cardiovascular and neurological diseases, as well as in asthma, and the CaSR is reported to protect against colorectal cancer and neuroblastoma but increase the malignant potential of prostate and breast cancers.

Normocalcemic Primary Hyperparathyroidism: A Comparison with the Hypercalcemic Form in a Tertiary Referral Population.

Normocalcemic primary hyperparathyroidism (NPHPT) is a formally recognized variant of primary hyperparathyroidism (PHPT), characterized by normal total and ionized serum calcium concentrations and elevated parathyroid hormone (PTH) levels, in the absence of secondary causes for hyperparathyroidism. NPHPT has been studied previously, but data are limited and confounded. We aimed to compare the clinical and biochemical data of normocalcemic and hypercalcemic subjects in a hospital-based population.We retrospectively analysed the medical records of 131 subjects diagnosed with PHPT at the university hospital Brussels (UZ Brussel) between January 1st 2007 and December 31st 2016, including 25 normocalcemic and 106 hypercalcemic subjects.The mean values of age, BMI, sex, serum 25-OH vitamin D levels and urinary phosphate excretion were comparable between both groups. Subjects diagnosed with NPHPT had significantly lower plasma PTH levels, lower urinary calcium excretion and lower serum creatinine levels compared to the hypercalcemic subjects with PHPT. Corresponding eGFR values were higher in the normocalcemic group. Normocalcemic subjects (NPHPT) presented with a high prevalence of nephrolithiasis (36%), fragility fractures (12%) and osteoporosis (25%). Clinical manifestations and BMD measurements revealed no statistically significant differences between both groups.Our data show a relative prevalence of 19% NPHPT in PHPT. NPHPT may present the earliest form of PHPT with an extension in time, but is not an indolent disease state. Normocalcemic subjects should be managed as hypercalcemic subjects with PHPT. Further research regarding the pathophysiology and natural course of NPHPT is required.

Potassium conservation is impaired in mice with reduced renal expression of Kir4.1.

To better understand the role of the inward-rectifying K channel Kir4.1 (KCNJ10) in the distal nephron, we initially studied a global Kir4.1 knockout mouse (gKO), which demonstrated the hypokalemia and hypomagnesemia seen in SeSAME/EAST syndrome and was associated with reduced Na/Cl cotransporter (NCC) expression. Lethality by ~3 wk, however, limits the usefulness of this model, so we developed a kidney-specific Kir4.1 "knockdown" mouse (ksKD) using a cadherin 16 promoter and Cre-loxP methodology. These mice appeared normal and survived to adulthood. Kir4.1 protein expression was decreased ~50% vs. wild-type (WT) mice by immunoblotting, and immunofluorescence showed moderately reduced Kir4.1 staining in distal convoluted tubule that was minimal or absent in connecting tubule and cortical collecting duct. Under control conditions, the ksKD mice showed metabolic alkalosis and relative hypercalcemia but were normokalemic and mildly hypermagnesemic despite decreased NCC expression. In addition, the mice had a severe urinary concentrating defect associated with hypernatremia, enlarged kidneys with tubulocystic dilations, and reduced aquaporin-3 expression. On a K/Mg-free diet for 1 wk, however, ksKD mice showed marked hypokalemia (serum K: 1.5 ± 0.1 vs. 3.0 ± 0.1 mEq/l for WT), which was associated with renal K wasting (transtubular K gradient: 11.4 ± 0.8 vs. 1.6 ± 0.4 in WT). Phosphorylated-NCC expression increased in WT but not ksKD mice on the K/Mg-free diet, suggesting that loss of NCC adaptation underlies the hypokalemia. In conclusion, even modest reduction in Kir4.1 expression results in impaired K conservation, which appears to be mediated by reduced expression of activated NCC.

Hypercalcemic crisis as a prodromal feature of Pneumcystis jirovecii pneumonia.

We describe a patient who had developed hypercalcemic crisis, with altered mental status and renal failure, one year following aggressive corticosteroid-therapy for lupus nephritis. Her disease relapsed after successful live-related kidney transplantation 11 years ago. She had normal parathyroid hormone and 25-hydroxyvitamin D yet high 1,25 dihydroxyvitamin D. Four weeks later, she developed severe dyspnea and hypoxia with a reticulonodular pattern on chest computed tomography. Bacteriological and serological tests were negative for pathogens. However, bronchoalveolar lavage established the diagnosis of Pneumocystis jiroviceii pneumonia (PJP). Her pneumonia and hypercalcemia improved with Co-trimoxazole. The case indicates that severe hypercalcemia can herald PJP.

Primary Hyperparathyroidism.

Background: Primary hyperparathyroidism (PHPT), the most common cause of hypercalcemia, is most often identified in postmenopausal women. The clinical presentation of PHPT has evolved over the past 40 years to include three distinct clinical phenotypes, each of which has been studied in detail and has led to evolving concepts about target organ involvement, natural history, and management. Methods: In the present review, I provide an evidence-based summary of this disorder as it has been studied worldwide, citing key concepts and data that have helped to shape our concepts about this disease. Results: PHPT is now recognized to include three clinical phenotypes: overt target organ involvement, mild asymptomatic hypercalcemia, and high PTH levels with persistently normal albumin-corrected and ionized serum calcium values. The factors that determine which of these clinical presentations is more likely to predominate in a given country include the extent to which biochemical screening is used, vitamin D deficiency is present, and whether parathyroid hormone levels are routinely measured in the evaluation of low bone density or frank osteoporosis. Guidelines for parathyroidectomy apply to all three clinical forms of the disease. If surgical guidelines are not met, parathyroidectomy can also be an appropriate option if no medical contraindications are present. If either the serum calcium or bone mineral density is of concern and surgery is not an option, pharmacological approaches are available and effective. Conclusions: Advances in our knowledge of PHPT have guided new concepts in diagnosis and management.

Effect of Continuous Intravenous Calcium Loading on Fibroblast Growth Factor 23 in Normal and Uremic Rats.

Fibroblast growth factor 23 (FGF23) is associated with mortality in patients with CKD. However, the mechanisms underlying stimulation of FGF23 remain to be investigated. We examined whether hypercalcemia induced by continuous intravenous (CIV) calcium (Ca) infusion regulates FGF23 levels in normal rats (Normal) and 5/6 nephrectomized uremic rats (Nx). Microinfusion pumps were implanted in the Normal and Nx rats for CIV Ca infusion, and blood, urine, kidney, and tibia were collected. The results showed an increase in serum Ca-stimulated FGF23 independently of serum phosphate (P) and creatinine levels in Normal and Nx rats. FGF23 mRNA from the tibia was also increased by the Ca infusion. Despite high FGF23 levels after Ca infusion, urinary P excretion was decreased. Renal α-Klotho expression was significantly reduced by Ca infusion. These results suggest that intravenous Ca loading might stimulate FGF23 production from bone in normal and uremic rats. Reduction of renal P excretion suggests that the bioactivity of FGF23 is inhibited, and the decrease in renal α-Klotho expression might have a role in this pathological process. In conclusion, CIV Ca loading increased FGF23 in normal and uremic rats, and renal α-Klotho is necessary to maintain the bioactivity of FGF23 as a phosphaturic factor.