Outcome of tonsillotomy in children with obstructive sleep-disordered breathing.

OBJECTIVE/BACKGROUND: Most children treated for obstructive sleep disordered breathing (oSDB) are not systematically assessed by polysomnography (PSG) nor by structuredsymptom quantification before surgical treatment. The main objective of this study wasto investigate the effect of adeno-tonsillotomy (ATO) on symptom burden and PSGparameters. METHODS: Children aged 2-10 years with oSDB were selected for ATO based uponclinical findings according to current standards of care in Denmark. Each childunderwent standardized assessment before and 3 months after surgery, including aPSG, tonsil size assessment, and the Pediatric Sleep Questionnaire -Sleep RelatedBreathing Disorder (PSQ) to quantify symptom burden. Obstructive sleep apnea (OSA)was defined as an obstructive apnea-hypopnea index (oAHI) ≥2/h. Successfultreatment was defined as post-surgery oAHI ≤5/h, and complete cure as oAHI ≤2/h. RESULTS: Fifty-two children were included. Mean age was 5.0 years (SD ± 1.76). Thirteen children were identified with baseline oAHI <2/h. Significant improvement inOSA severity was observed in children with moderate-to-severe OSA, in whom oAHI decreased from 15.7/h to 2.6/h (p < 0.001). Treatment success was obtained in 85% and cure was obtained in 42% of children. PSQ-score significantly improved from 0.52 (CI 0.47-0.56) to 0.26 (CI 0.21-0.32) (p < 0.001). Baseline OSA severity was notcorrelated to baseline symptom burden nor to symptom relief after ATO. There were noserious adverse events. CONCLUSIONS: Adeno-tonsillotomy significantly reduced symptom burden in otherwise healthy children with oSDB symptoms. Significant improvement in oAHI was observedonly in children with moderate-to-severe OSA. We recommend combining clinicalevaluation with PSQ and oAHI.

GFR measurements and ultrasound findings in 154 children with a congenital solitary functioning kidney.

BACKGROUND: Multicystic dysplastic kidney (MCDK) and unilateral renal agenesis (URA) are the most common reasons for a congenital solitary functioning kidney (SFK). We aimed to assess the presence of abnormalities in the congenital SFK and evaluate kidney function using chrome EDTA (CrEDTA) measurements. METHODS: We retrospectively reviewed the medical records of 154 children with MCDK and URA in the period from 2005 to 2022 to analyze results from ultrasound scans and CrEDTA glomerular filtration rate (GFR) examinations. RESULTS: Of 154 children with a solitary kidney due to MCDK (62%) or URA (38%), abnormalities on the congenital SFK were found in 13 children (8%). The abnormalities spontaneously resolved in 6 children (46%). The most common abnormality was hydronephrosis. Compensatory hypertrophy was found in 17% of the children within the first 6 months of life. 116 children (90%) had a standard GFR (sdGFR) above 75% of expected for the age. Out of those with a sdGFR below 75% of expected, 3 (23%) had abnormalities in the congenital SFK. There was no difference in sdGFR between children with MCDK and URA. CONCLUSIONS: Our study is the first using CrEDTA for GFR measurements and suggests that most children with a congenital SFK due to MCDK or URA have a kidney function within expected for the age. Compensatory hypertrophy of the SFK is found in a minority of children within the first six months of life, suggesting that this process is developing over time. The prevalence of abnormalities in the SFK seems low, however those with abnormalities (e.g. hydronephrosis) are at higher risk of reduced sdGFR.

Successful low-dose immunotherapy after kidney transplantation in a 10-year-old girl with Schimke immuno-osseous dysplasia.

BACKGROUND: This case report highlights a successful steroid-free, low-dose immunosuppressive protocol for renal transplantation in a pediatric patient with Schimke immuno-osseous dysplasia with excellent 7-year patient and graft survival. Schimke immuno-osseous dysplasia is a rare multisystem disorder involving the kidney. Renal transplantation is a therapeutic option, but posttransplant mortality is high due to severe infections and posttransplant lymphoproliferative disease. METHODS: A 10-year-old girl diagnosed with Schimke immuno-osseous dysplasia and end-stage renal disease underwent an AB0-compatible living-related kidney transplantation, with no donor-specific antibodies. Our standard immunosuppression protocol was modified due to the risk of infection. Basiliximab was used as induction therapy, and a reduced dose of mycophenolate mofetil and tacrolimus was initiated following transplantation, maintaining the patient on a low tacrolimus target (3-5 µg/L). Mycophenolate mofetil was discontinued after 8 weeks due to neutropenia and the patient was kept on tacrolimus as monotherapy. Five years posttransplant the patient developed acute onset of neurological symptoms, consisting of ataxia, lack of voluntary coordination, balance, aphasia and dysphagia, and diplopia. She recovered without neurological deficits within 6 weeks. Extensive evaluation revealed no pathology. To avoid a possible a component of tacrolimus-induced cerebral vasoconstriction, the immunosuppressive therapy was changed to everolimus. RESULTS: Seven years posttransplant, the patient has experienced no serious infections, no rejections, and had excellent graft function, and no de novo donor-specific antibodies. CONCLUSIONS: The present results indicate that low-dose immunosuppressive therapy after renal transplantation with low immunological risk should be considered for patients with Schimke immuno-osseous dysplasia.

Risk factors for kidney scarring and vesicoureteral reflux in 421 children after their first acute pyelonephritis, and appraisal of international guidelines.

BACKGROUND: Acute pyelonephritis (AP) is a common bacterial infection in childhood. Follow-up guidelines on these children are controversial. This study aimed to identify risk factors for kidney scarring and vesicoureteral reflux (VUR). Furthermore, international follow-up guidelines were used for simulation to evaluate sensitivity and specificity. METHODS: Urinary culture-confirmed first-time AP patients (aged 0-14 years) were enrolled (n = 421) from review of patient charts. All underwent kidney ultrasound (US) and a technetium-99m-dimercaptosuccinic acid (DMSA) scan or technetium-99m-mercaptoacetyltriglycine scinti-renography (MAG3) at 4-6 months of follow-up. The international guidelines used for simulation were from the National Institute of Health UK (NICE), the American Association of Paediatrics (AAP) and the Swedish Paediatric Society (SPS). RESULTS: 17.8% presented with an abnormal DMSA/MAG3 at follow-up, 7.1% were diagnosed with VUR grades III-V and 4.7% were admitted for surgery. Non-Escherichia coli infections, abnormal kidney US, elevated creatinine and delayed response to treatment (>48 h) were risk factors for abnormal DMSA findings and VUR grades III-V. NICE and SPS guidelines showed best sensitivity in diagnosing VUR grades III-V (75%) compared with AAP (56%). CONCLUSIONS: Risk factors are helpful in identifying the children in need of further investigations and minimizing invasive work-up for the rest. International guidelines on follow-up detect a varying number of children with kidney damage and/or significant VUR. Future work must focus on identifying more specific risk factors, better imaging, or specific biomarkers, to enhance sensitivity and specificity in detecting the children at high risk for developing recurrent infections and/or nephropathy.

Acute intravenous acyl ghrelin infusion induces thirst but does not affect sodium excretion: two randomized, double-blind, placebo-controlled crossover studies in hypopituitary patients.

OBJECTIVE: Acyl ghrelin, which is the endogenous ligand for the growth hormone secretagogue receptor, potently stimulates pituitary growth hormone release, and to some degree adrenocorticotropic hormone and prolactin. Ghrelin is also orexigenic and has recently been shown to stimulate renal sodium absorption in rodent models. Increased thirst sensation has been observed as a side effect of acyl ghrelin administration in some human studies. The objective of this clinical trial was to investigate the direct effects of acyl ghrelin on thirst sensation and sodium excretion in hypopituitary patients. DESIGN: Hypopituitary patients on stable replacement with hydrocortisone and growth hormone were investigated in two double-blind and placebo-controlled crossover studies. The patients received a 5-h intravenous infusion of acyl ghrelin (5 pmol/kg/min in the first study and 1 pmol/kg/min in the second study). Thirst sensation was measured on a Visual Analog Scale (VAS). In the second study plasma osmolality, vasopressin, copeptin, water intake, diuresis and urinary excretion of sodium and creatinine were measured. RESULTS: In the initial study, acyl ghrelin (5 pmol/kg/min) increased thirst sensation (time × treatment analysis of variance for the effect of acyl ghrelin infusion P = 0.003). In the second study acyl ghrelin (1 pmol/kg/min) also increased thirst (P = 0.04) but did not affect urinary excretion of either sodium or water. CONCLUSIONS: We demonstrate that acyl ghrelin infusion increases thirst sensation, without affecting sodium excretion or diuresis in human subjects.

Macrophage Markers Are Poorly Associated With Liver Histology in Children With Nonalcoholic Fatty Liver Disease.

OBJECTIVES: We have previously demonstrated associations between the macrophage activation marker soluble (s)CD163 and histology of nonalcoholic fatty liver disease (NAFLD) in adults, and elevated sCD163 levels in children with obesity with NAFLD. Macrophage activation has, however, not been investigated in children with biopsy-proven NAFLD, which was the objective of the present study. METHODS: We used in-house enzyme-linked immunosorbent assays to measure sCD163 and the novel macrophage marker soluble mannose receptor (sMR) in a cross-sectional (n = 155) pediatric NAFLD cohort, and a cohort of NAFLD children (n = 36) undergoing a randomized trial by the probiotic VSL#3. We included 56 healthy nonobese children for comparison. RESULTS: Levels of sCD163 and sMR were higher in both of the NAFLD cohorts compared with controls (P < 0.001). In the cross-sectional cohort, sCD163 only showed trends toward association with ballooning (rho = 0.14, P = 0.08) and portal inflammation (rho = 0.17, P = 0.08). sMR showed similar associations with liver histology. In the VSL#3 cohort, sCD163 correlated inversely with steatosis (rho = -0.35, P = 0.04), and lobular (rho = -0.57, P < 0.001) and portal inflammation (rho = -0.38, P = 0.02); sMR was not associated with any histological scores. Neither sCD163 nor sMR changed significantly during intervention, and without association with NAFLD resolution. CONCLUSIONS: The macrophage activation markers sCD163 and sMR showed poor associations with liver histology in 2 different cohorts of children with biopsy-proven NAFLD, and none of the markers decreased during successful intervention. These results are in contrast with studies of adult NAFLD and may suggest a possibility of different roles for macrophages in the pathogenesis of adult and pediatric NAFLD.

The Novel Ser18del AVP Variant Causes Inherited Neurohypophyseal Diabetes Insipidus by Mechanisms Shared with Other Signal Peptide Variants.

BACKGROUND/AIM: Variability in the severity and age at onset of autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) may be associated with certain types of variants in the arginine vasopressin (AVP) gene. In this study, we aimed to describe a large family with an apparent predominant female occurrence of polyuria and polydipsia and to determine the underlying cause. METHODS: The family members reported their family demography and symptoms. Two subjects were diagnosed by fluid deprivation and dDAVP challenge tests. Eight subjects were tested genetically. The identified variant along with 3 previously identified variants in the AVP gene were investigated by heterologous expression in a human neuronal cell line (SH-SY5Y). RESULTS: Both subjects investigated clinically had a partial neurohypophyseal diabetes insipidus phenotype. A g.276_278delTCC variant in the AVP gene causing a Ser18del deletion in the signal peptide (SP) of the AVP preprohormone was perfectly co-segregating with the disease. When expressed in SH-SY5Y cells, the Ser18del variant along with 3 other SP variants (g.227G>A, Ser17Phe, and Ala19Thr) resulted in reduced AVP mRNA, impaired AVP secretion, and partial AVP prohormone degradation and retention in the endoplasmic reticulum. Impaired SP cleavage was demonstrated directly in cells expressing the Ser18del, g.227G>A, and Ala19Thr variants, using state-of-the-art mass spectrometry. CONCLUSION: Variants affecting the SP of the AVP preprohormone cause adFNDI with variable phenotypes by a mechanism that may involve impaired SP cleavage combined with effects at the mRNA, protein, and cellular level.

The follicle-stimulating hormone (FSH) and luteinizing hormone (LH) response to a gonadotropin-releasing hormone analogue test in healthy prepubertal girls aged 10 months to 6 years.

OBJECTIVE: Premature thelarche and precocious puberty are frequently diagnosed in girls even below 6 years of age and may be difficult to differentiate in the early stages. A GnRH test is often included in the diagnostic work-up, although interpretation of the GnRH test in girls below 6 years of age is challenging, as no reference interval exists for this age group. The objective is to determine the normal FSH and LH response to a GnRH test in healthy prepubertal girls below 6 years of age. DESIGN AND METHODS: A standardized GnRH test, baseline reproductive hormones, clinical evaluation and bone age were determined in all participants. Forty-eight healthy normal-weight girls aged 3.5 ± 0.2 years (range: 0.8-5.9 years) were included. Serum concentrations of LH and FSH were measured before and 30 min after the gonadorelin injection. RESULTS: The 30-min LH responses (mean ± 2 s.d.) were 5.2 ± 4.0 and 2.9 ± 2.5 IU/L and the FSH responses were 23.3 ± 16.2 and 14.5 ± 10.3 IU/L in girls aged 0.8-3.0 years and 3.0-5.9 years respectively. This corresponds to upper cut-off limits for LH of 9.2 IU/L (<3 years) and 5.3 IU/L (3-6 years). The stimulated LH/FSH ratio was 0.23 ± 0.19 (range 0.06-0.43) and did not correlate with age. CONCLUSIONS: We found that LH increases up to 9.2 IU/L during GnRH test in healthy normal-weight girls below 3 years of age and that the stimulated LH/FSH ratio did not exceed 0.43. Our findings have important implications for appropriate diagnosis of central precocious puberty in girls below 6 years of age.

Urinary Neutrophil Gelatinase-associated Lipocalin in the evaluation of Patent Ductus Arteriosus and AKI in Very Preterm Neonates: a cohort study.

BACKGROUND: A patent ductus arteriosus (PDA) is frequently found in very preterm neonates and is associated with increased risk of morbidity and mortality. A shunt across a PDA can result in an unfavorable distribution of the cardiac output and may in turn result in poor renal perfusion. Urinary Neutrophil Gelatinase-associated Lipocalin (U-NGAL) is a marker of renal ischemia and may add to the evaluation of PDA. Our primary aim was to investigate if U-NGAL is associated with PDA in very preterm neonates. Secondary, to investigate whether U-NGAL and PDA are associated with AKI and renal dysfunction evaluated by fractional excretion of sodium (FENa) and urine albumin in a cohort of very preterm neonates. METHODS: A cohort of 146 neonates born at a gestational age less than 32 weeks were consecutively examined with echocardiography for PDA and serum sodium, and urine albumin and sodium were measured on postnatal day 3 and U-NGAL and serum creatinine day 3 and 6. AKI was defined according to modified neonatal Acute Kidney Injury Network (AKIN) criteria. The association between U-NGAL and PDA was investigated. And secondly we investigated if PDA and U-NGAL was associated with AKI and renal dysfunction. RESULTS: U-NGAL was not associated with a PDA day 3 when adjusted for gestational age and gender. A PDA day 3 was not associated with AKI when adjusted for gestational age and gender; however, it was associated with urine albumin. U-NGAL was not associated with AKI, but was found to be associated with urine albumin and FENa. CONCLUSIONS: Based on our study U-NGAL is not considered useful as a diagnostic marker to identify very preterm neonates with a PDA causing hemodynamic changes resulting in early renal morbidity. The interpretation of NGAL in preterm neonates remains to be fully elucidated.

Combination treatment of nocturnal enuresis with desmopressin and indomethacin.

BACKGROUND: We investigated the effect of combining indomethacin and desmopressin in treating children with monosymptomatic nocturnal enuresis (MNE) and desmopressin-resistant nocturnal polyuria. METHODS: Twenty-three children with MNE, nocturnal polyuria, and partial or no response to desmopressin were recruited from incontinence clinics of our tertiary referral center. We used a randomized single-arm crossover placebo-controlled study design consisting of two 3-week treatment periods with a combination of desmopressin (0.4 mg) and indomethacin (50 mg) or desmopressin and placebo at bedtime. Home recordings at baseline and for the final 2 weeks of each treatment period were performed and included nocturnal urine output measurements. The number of dry nights achieved and reduction in the nocturnal urine output were the main effect parameters. Student's t test and Pearson's correlation coefficient were used for statistical analysis. RESULTS: The addition of indomethacin to desmopressin significantly reduced nocturnal urine output (from 324 ± 14 ml to 258 ± 13 ml, p < 0.001). This did not lead to more dry nights in all children, and we found no statistically significant reduction in enuresis frequency (from 68 % ± 0.1 to 56 % ± 0.1, p = 0.24). CONCLUSIONS: Addition of indomethacin to desmopressin can further reduce nocturnal urine output in children with MNE and desmopressin-resistant nocturnal polyuria. The combination treatment does not, however, improve outcome in terms of frequency of nights with enuresis. The dissociation of antidiuretic and antienuretic effect may reflect nocturnal bladder reservoir dysfunction in children who present with normal daytime bladder function.

Protracted Clinical Course of Postinfectious Glomerulonephritis in a Previously Healthy Child.

Acute postinfectious glomerulonephritis (PIGN) affects children typically after upper respiratory tract or skin infections with streptococci but can complicate the course of other infections. In children, it is generally a self-limiting disease with excellent prognosis. This paper reports a previously healthy 4-year-old boy who experienced a protracted course of PIGN with persisting episodes of gross haematuria, proteinuria, decreased complement C3c levels but normal P-creatinine levels. Due to the protracted course and the nephrotic-range proteinuria, a renal biopsy was performed 6 months after the initial presentation and the overall pathology was consistent with acute endocapillary glomerulonephritis.

ANKS6 is a central component of a nephronophthisis module linking NEK8 to INVS and NPHP3.

Nephronophthisis is an autosomal recessive cystic kidney disease that leads to renal failure in childhood or adolescence. Most NPHP gene products form molecular networks. Here we identify ANKS6 as a new NPHP family member that connects NEK8 (NPHP9) to INVS (NPHP2) and NPHP3. We show that ANKS6 localizes to the proximal cilium and confirm its role in renal development through knockdown experiments in zebrafish and Xenopus laevis. We also identify six families with ANKS6 mutations affected by nephronophthisis, including severe cardiovascular abnormalities, liver fibrosis and situs inversus. The oxygen sensor HIF1AN hydroxylates ANKS6 and INVS and alters the composition of the ANKS6-INVS-NPHP3 module. Knockdown of Hif1an in Xenopus results in a phenotype that resembles loss of other NPHP proteins. Network analyses uncovered additional putative NPHP proteins and placed ANKS6 at the center of this NPHP module, explaining the overlapping disease manifestation caused by mutation in ANKS6, NEK8, INVS or NPHP3.

Tubulointerstitial nephritis in a patient with probable autoimmune lymphoproliferative syndrome.

Autoimmune lymphoproliferative syndrome (ALPS) is caused by a nonmalignant defective Fas-mediated apoptosis. The main clinical manifestations are chronic lymphadenopathy, splenomegaly, and autoimmune cytopenia. Most patients with ALPS have a FAS germline mutation. ALPS has occasionally been associated with glomerulonephritis and we present the first report of tubulointerstitial nephritis associated with probable ALPS. A 5-year-old girl presented with fever, vomiting, hypertension, and azotemia. No autoantibodies, viral, or streptococcal antibodies were detected. A renal biopsy showed small-vessel vasculitis with normal glomeruli and inflammation in the interstitium. The patient responded to prednisolone treatment and obtained a full renal recovery. Symptoms of connective tissue disorder supervened and after the development of more pronounced splenomegaly, a diagnosis of ALPS was confirmed.

[Family occurrence of steroid-sensitive idiopathic nephrotic syndrome]. / Familiaer forekomst af steroidsensitivt idiopatisk nefrotisk syndrom.

Nephrotic syndrome (NS) presented within three weeks in siblings aged six and ten years. Both children experienced proteinuria, hypoalbuminaemia and oedema, with the most pronounced symptoms in the older. Standard treatment with prednisolone led to remission of the nephrotic syndrome in the younger, whereas the older required additional therapy with tacrolismus before remission. In view of the low incidence of NS in children, a near simultaneously onset in two siblings must lead to genetic elucidation. Genetic disorders and other causes of childhood NS are discussed.

Diverse vasopressin V2 receptor functionality underlying partial congenital nephrogenic diabetes insipidus.

X-linked congenital nephrogenic diabetes insipidus (CNDI) is characterized by a defective renal response to the antidiuretic hormone (AVP) due to variations in the arginine vasopressin receptor 2 (AVPR2) gene. In a unique group of patients, the renal insensitivity to the effects of AVP is incomplete resulting in a partial phenotype. To investigate the molecular defects, two previously published variations in the AVPR2 gene, known to cause a partial CNDI phenotype, were expressed in transiently transfected human embryonic kidney cells. One variation (p.Arg104Cys) is located in the first extracellular loop and the other variation (p.Ser329Arg) is located in the intracellular COOH terminal of the receptor protein. Western blotting showed almost equal amounts of WT-V2R and Arg104Cys-V2R protein at steady state, whereas the level of Ser329Arg-V2R protein was lower. Confocal microscopy established that WT-V2R and Arg104Cys-V2R are localized on the cellular surface while the Ser329Arg-V2R primarily accumulates within the endoplasmic reticulum resulting in reduced surface expression. Ligand binding analysis demonstrated that the B(max) for cells expressing Arg104Cys-V2R and Ser329Arg-V2R were 14.8- and 2.5-fold lower than B(max) for WT-V2R, respectively. AVP affinity (1/K(d)) for WT-V2R and the Ser329Arg-V2R was similar while 1/K(d) for Arg104Cys-V2R was increased. cAMP assay revealed that cells expressing p.Arg104Cys-V2R or p.Ser329Arg-V2R produced 1.7- and 6.8-fold lower amounts of cAMP compared with WT-V2R, respectively. In conclusion, ligand binding and signal transduction capability are dependent on localization of the amino acid variation. Striking divergences at the level of receptor functionality may thus underlie similar clinical phenotypes in CNDI.

Transcutaneous electrical nerve stimulation for refractory daytime urinary urge incontinence.

PURPOSE: We studied the effect of transcutaneous electrical nerve stimulation in children with overactive bladder and treatment refractory daytime urinary incontinence. MATERIALS AND METHODS: We recruited 27 children 5 to 14 years old with daytime urge incontinence refractory to timer assisted standard urotherapy and anticholinergics who had normal urinalysis, and unremarkable urinary tract ultrasound and physical examination. Study exclusion criteria were bladder underactivity, lower urinary tract obstruction, ongoing defecation disorders, lower urinary tract surgery and previous transcutaneous electrical nerve stimulation. After a 2-week run-in of standard urotherapy the children underwent natural fill ambulatory urodynamics to confirm detrusor overactivity. Subsequently they were randomly allocated to 4 weeks of 2 hours of daily active or placebo S2-S3 transcutaneous electrical nerve stimulation. The severity of incontinence and urgency, and 48-hour bladder diaries were recorded before randomization and during intervention week 4. Children withdrew from anticholinergics throughout the study period. RESULTS: Two children were excluded from randomization due to urodynamic signs of lower urinary tract obstruction. After 4 weeks of intervention 8 children (61%) in the active group showed a significant decrease in incontinence severity but this occurred in only 2 (17%) in the sham treated group (p <0.05). The active group had a significantly greater decrease in daily incontinence episodes compared to the sham treated group (p <0.01). Transcutaneous electrical nerve stimulation did not alter maximal and average voided volumes. CONCLUSIONS: Sacral transcutaneous electrical nerve stimulation seems superior to placebo for refractory daytime incontinence in children with overactive bladder. This effect does not seem to be a consequence of improved bladder reservoir function.

[Prenatal diagnosed hydronephrosis and other urological anomalies]. / Praenatalt diagnosticeret hydronefrose og andre urologiske anomalier.

By renal ultrasound examination, urological anomalies may be demonstrated in 1-2% of fetuses and in about 0.5% of newborns. Boys have about twice the frequency of girls. Surgical treatment is indicated in about one fourth of these urological anomalies. If all pregnant women in Denmark were to have fetal ultrasound examination of the kidneys and the urinary tract, about 70 children would be born each year with a prenatally diagnosed urological anomaly for which surgical procedure is or will be indicated. This paper provides Danish guidelines for prenatal diagnosis, follow-up and intervention in cases of urological anomalies and guidelines for post-natal diagnosis, follow-up and treatment of these anomalies, especially hydronephrosis.

Expression of three different mutations in the arginine vasopressin gene suggests genotype-phenotype correlation in familial neurohypophyseal diabetes insipidus kindreds.

OBJECTIVE AND STUDY DESIGN: The autosomal dominant form of familial neurohypophyseal diabetes insipidus (adFNDI) is a rare disease characterized by a severe and progressive deficiency of AVP secondary to mutations in the gene encoding the AVP precursor. Whereas a number of studies have investigated the pathogenetic mechanisms behind the disease only few studies have included detailed clinical characterization of the affected patients, thereby making genotype-phenotype correlations difficult. The aims of the present study were to investigate the cellular effects of three different adFNDI mutations (A19T, L81P and C110X) by heterologous expression in a neurogenic cell line and to correlate these findings to the corresponding clinical phenotype as determined by extensive clinical tests. RESULTS: The clinical studies showed a later age of onset in the family carrying the A19T mutation (3.4 years, range 2-9 years) compared with families with the L81P and C110X mutations [0.75 year, range 0.5-1 year and 1.0 year (n = 1), respectively]. No other differences could be demonstrated in the clinical phenotype between families. Expression studies showed that each of the three mutant genes caused significant reduction of the amount of immunoreactive AVP in the cell culture medium and severe impairment of the intracellular trafficking and processing of the AVP prohormone, supporting the disease causing nature of all three mutations. However, the A19T mutation was associated with some capacity for processing and trafficking consistent with the clinical observations. Immunoflourescence studies provided evidence of reticular accumulation of protein within the ER in the A19T and C110X mutants but a unique accumulation of much larger aggregates in the L81P, which were localized both within and immediately outside the ER. CONCLUSION: The study suggests a genotype-phenotype correlation with regard to age of onset of diabetes insipidus symptoms and provides support by expression studies.

Impaired trafficking of mutated AVP prohormone in cells expressing rare disease genes causing autosomal dominant familial neurohypophyseal diabetes insipidus.

OBJECTIVE AND STUDY DESIGN: Two different mutations in the arginine vasopressin (AVP) gene associated with autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) predict Y21H (AVP2) and V67A (NP36) amino acid substitutions of the AVP prohormone. They are unique in that they change, respectively, the AVP moiety and a region of the neurophysin II domain not so far affected by any mutations. To test whether they affect the cellular handling of the AVP prohormone in a similar manner to previously investigated mutations, they were examined by heterologous expression in cell lines. RESULTS: Both mutations resulted in significantly reduced amounts of immunoreactive AVP in the cell culture medium as determined by radioimmunoassay analysis. Metabolic labelling combined with immunoprecipitation demonstrated that processing and secretion of the mutant prohormones was reduced but not prevented. Finally, confocal laser scanning microscopy showed that normal AVP prohormone and/or its processed products were localized in the tips of the cellular processes, whereas both mutant prohormones were accumulated in the endoplasmic reticulum (ER) and in the case of the V67A prohormone, also in perinuclear structures outside the ER. CONCLUSION: Both mutations result in reduced AVP prohormone processing and secretion probably due to retention in the ER. This supports, at least partly, the hypothesis that the mutations lead to the production of a mutant hormone precursor that fails to fold and/or dimerize properly and, as a consequence, is retained by the ER protein quality control machinery. Perinuclear accumulation of the V67A prohormone outside the ER indicates that additional mechanisms could be involved.