Joachim Misselwitz

Mutations in CYP24A1 and Idiopathic Infantile Hypercalcemia

BACKGROUND Vitamin D supplementation for the prevention of rickets is one of the oldest and most effective prophylactic measures in medicine, having virtually eradicated rickets in North America. Given the potentially toxic effects of vitamin D, the recommendations for the optimal dose are still debated, in part owing to the increased incidence of idiopathic infantile hypercalcemia in Britain in the 1950s during a period of high vitamin D supplementation in fortified milk products. We investigated the molecular basis of idiopathic infantile hypercalcemia, which is characterized by severe hypercalcemia, failure to thrive, vomiting, dehydration, and nephrocalcinosis. METHODS We used a candidate-gene approach in a cohort of familial cases of typical idiopathic infantile hypercalcemia with suspected autosomal recessive inheritance. Identified mutations in the vitamin D-metabolizing enzyme CYP24A1 were evaluated with the use of a mammalian expression system. RESULTS Sequence analysis of CYP24A1, which encodes 25-hydroxyvitamin D 24-hydroxylase, the key enzyme of 1,25-dihydroxyvitamin D(3) degradation, revealed recessive mutations in six affected children. In addition, CYP24A1 mutations were identified in a second cohort of infants in whom severe hypercalcemia had developed after bolus prophylaxis with vitamin D. Functional characterization revealed a complete loss of function in all CYP24A1 mutations. CONCLUSIONS The presence of CYP24A1 mutations explains the increased sensitivity to vitamin D in patients with idiopathic infantile hypercalcemia and is a genetic risk factor for the development of symptomatic hypercalcemia that may be triggered by vitamin D prophylaxis in otherwise apparently healthy infants.

Deletion of Complement Factor H–Related Genes CFHR1 and CFHR3 Is Associated with Atypical Hemolytic Uremic Syndrome

Atypical hemolytic uremic syndrome (aHUS) is associated with defective complement regulation. Disease-associated mutations have been described in the genes encoding the complement regulators complement factor H, membrane cofactor protein, factor B, and factor I. In this study, we show in two independent cohorts of aHUS patients that deletion of two closely related genes, complement factor H–related 1 (CFHR1) and complement factor H–related 3 (CFHR3), increases the risk of aHUS. Amplification analysis and sequencing of genomic DNA of three affected individuals revealed a chromosomal deletion of ∼84 kb in the RCA gene cluster, resulting in loss of the genes coding for CFHR1 and CFHR3, but leaving the genomic structure of factor H intact. The CFHR1 and CFHR3 genes are flanked by long homologous repeats with long interspersed nuclear elements (retrotransposons) and we suggest that nonallelic homologous recombination between these repeats results in the loss of the two genes. Impaired protection of erythrocytes from complement activation is observed in the serum of aHUS patients deficient in CFHR1 and CFHR3, thus suggesting a regulatory role for CFHR1 and CFHR3 in complement activation. The identification of CFHR1/CFHR3 deficiency in aHUS patients may lead to the design of new diagnostic approaches, such as enhanced testing for these genes.

Successful withdrawal of steroids in pediatric renal transplant recipients receiving cyclosporine A and mycophenolate mofetil treatment : results after four years

Background. Despite their numerous systemic side effects, glucocorticoids (steroids) still form a cornerstone in immunosuppressive regimens in pediatric renal transplant recipients. The addition of mycophenolate mofetil (MMF) to a cyclosporine A (CsA)-based immunosuppressive regimen after renal transplantation may allow steroid withdrawal and amelioration or avoidance of steroid-specific side effects. Methods. In a retrospective case-control study, covering a mean follow-up period of 46±2.3 months and 40 patients aged 11.4±4.9 years, we analyzed the safety and efficacy of steroid withdrawal in pediatric renal transplant recipients receiving CsA micoroemulsion, MMF, and low-dose prednisone treatment. Results. Steroid withdrawal in all 20 pediatric renal transplant recipients receiving CsA and MMF was successful and not associated with an acute rejection episode; graft function remained stable. At baseline, the degree of growth retardation was comparable between the groups (mean height standard deviation scores [SDSs] −1.60±0.30 [withdrawal group] and −1.32±0.39 [case-control group]). After steroid withdrawal, prepubertal patients exhibited a significant catch-up growth with a mean height gain of 1.47±0.32 SDS, whereas height SDS did not improve in patients receiving steroids. Growth was also improved in pubertal patients who stopped taking steroids. Standardized body mass index in patients who stopped taking steroids decreased significantly by 49% from 0.87±0.31 SDS to 0.45±0.30 SDS. After steroid withdrawal, mean arterial blood pressure SDS decreased significantly by 45%. Moreover, the need for antihypertensive medication declined significantly in patients who stopped taking steroids. The white blood cell counts and hemoglobin levels were comparable between the groups. Conclusions. This study suggests that steroids can be safely and successfully withdrawn in selected pediatric renal transplant recipients receiving immunosuppressive maintenance therapy consisting of CsA and MMF.

Cluster of hemolytic-uremic syndrome caused by Shiga toxin-producing Escherichia coli O26:H11.

Background. The epidemiology and clinical characteristics of the hemolytic-uremic syndrome (HUS) caused by Escherichia coli O157:H7 are well-known, but HUS attributable to non-O157:H7 Shiga toxin (Stx)-producing E. coli (STEC) are less thoroughly described. Here we report a cluster of HUS cases caused by STEC O26:H11 the most common non-O157:H7 STEC isolated from sporadic cases of HUS in Europe. Methods. Three children between 13 and 17 months of age, living in the same small town, developed HUS within an interval of 5 days. We present clinical and microbiologic data on the patients and their infecting isolates. Results. The clinical course ranged from mild uncomplicated HUS to severe HUS complicated by multiorgan involvement. Microbiologic investigation demonstrated STEC of serotype O26:H11 in stools of all the patients. The phenotypic and molecular characterization of the STEC O26:H11 isolates demonstrated that these strains were identical and, unusual for STEC O26, they harbored the stx2 but not the stx1 gene. None of the patients had evidence of STEC O157:H7 infection either by culture or by E. coli O157 serology. The source of the STEC O26:H11 infection was undetermined. Conclusions. Our results demonstrate that diagnostic procedures based on the detection of stx genes and/or Stx production and subsequent subtyping of the isolates using molecular methods are necessary to identify such outbreaks caused by non-O157:H7 STEC.

Pediatric renal transplantation with mycophenolate mofetil-based immunosuppression without induction: results after three years1,2

Background. Mycophenolate mofetil (MMF)-based immunosuppression has reduced the acute rejection rate in adults and in children in the early posttransplantation period. Three-year posttransplantation results have been reported for adults but not for children thus far. In the present open-labeled study, patients 18 years old and younger were evaluated prospectively for up to 3 years after renal transplantation (RTX). Methods. Eighty-six patients receiving MMF in combination with cyclosporine and prednisone without induction were evaluated for patient survival, transplant survival, renal function, arterial blood pressure, adverse events, and opportunistic infections. These patients were compared with a historic control group (n=54) receiving azathioprine (AZA) instead of MMF. Results. Patient survival after 3 years was 98.8% in the MMF group and 94.4% in the AZA group (NS). Intent-to-treat analysis of graft survival demonstrated superiority for MMF (98% vs. 80%; P<0.001). Cumulative acute rejection episodes occurred in 47% of patients in the MMF group versus 61% in the AZA group (P<0.05). Renal function was not significantly different, neither after 3 years nor in the long-term calculation. Antihypertensive medication was administered to 73% to 84% of patients, similar in both groups. Opportunistic infections were recorded only for MMF. Infection rates were comparable to those reported in adults. Conclusions. These results suggest that MMF is safe and beneficial as a longer term maintenance immunosuppressive drug in children and adolescents.

Blood Pressure, Renal Function, and Proteinuria in Children with Unilateral Renal Agenesis

Background/Aim: Unilateral renal agenesis (URA) is a model for a reduced nephron number that is believed to be a risk factor for blood pressure (BP) elevation and reduced renal function. The aim of the study was to investigate BP and renal function in children with URA. Methods: Data on children with URA from two pediatric nephrology centers were firstly retrospectively reviewed (renal ultrasound and scintigraphy, clinical BP, creatinine clearance, urinalysis). Children with normal renal ultrasound and scintigraphy were thereafter investigated using ambulatory BP monitoring. Results: Twenty-nine children with URA were investigated – 14 children with an abnormal kidney (mostly scarring) and 15 children with healthy kidneys. Hypertension was diagnosed on the basis of clinical BP in 57% of the children with abnormal kidneys and on the basis of ambulatory BP monitoring in 1 child (7%) with healthy kidneys. The mean ambulatory BP in children with normal kidneys was not significantly different from that in controls. Forty-three percent of the children with abnormal kidneys had a reduced renal function, but none of children with normal kidneys. Conclusions: Children with abnormalities of a solitary kidney have often hypertension, proteinuria, or a reduced renal function. In contrast, children with healthy solitary kidneys have BP and renal function similar to those of healthy children.

Clinical features of unilateral multicystic renal dysplasia in children

Abstract A clinical study of 204 patients with unilateral multicystic renal dysplasia referred to 30 nephrology departments was undertaken to assess the frequency of complications in children who underwent nephrectomy (n=40) versus those who were treated conservatively (n=164). Six patients required antihypertensive treatment, 30 (13% of girls, 17% of boys) had at least one episode of urinary tract infection. The number of clinical complications did not differ in patients who underwent nephrectomy in comparison to those who did not. The dysplastic kidney decreased in size in 65% of kidneys with repeated ultrasound values; no change occurred in 16%, while an increase in maximal diameter was observed in 19%. Contralateral kidney length of more than 2 standard deviation scores (SDS) was seen in 30% of patients. There is evidence for a slight impairment of renal function in the whole study group given by a median of serum creatinine level of 0.63 SDS in all patients available for analysis. Among those 35 patients with contralateral anomalies (mainly obstructive changes and vesico-ureteral reflux), all 3 patients with contralateral changes suggestive of hypoplasia and 22% with obstruction, but only 1/7 (14%) with reflux showed elevated serum creatinine level >2 SDS. Conclusion Renal function seems to be slightly impaired in patients with unilateral multicystic renal dysplasia. The apparent tendency to regression of the dysplastic kidney and no difference in the number of complications justify a conservative management rather than operative intervention.

Efficacy and safety of basiliximab in pediatric renal transplant patients receiving cyclosporine, mycophenolate mofetil, and steroids

Background. Basiliximab, a monoclonal CD25 antibody has proofed effective in reducing acute rejection episodes in adults in various immunosuppressive regimens. The effect of basiliximab in the pediatric population is controversial. Methods. In a 12-month, double-blind, placebo-controlled trial, renal transplant patients aged 1 to 18 years were randomized to basiliximab or placebo with cyclosporine microemulsion, mycophenolate mofetil, and corticosteroids. The intent-to-treat population comprised 192 patients (100 basiliximab and 92 placebo). Results. The primary efficacy endpoint, time to first biopsy-proven acute rejection episode, or treatment failure by month 6, occurred in 16.7% of basiliximab-treated patients and 21.7% of placebo-treated patients (Kaplan-Meier estimates; hazard ratio 0.72, two-sided 90% confidence interval 0.416–1.26, n.s.). The rate and severity of subclinical rejections in protocol biopsies performed at 6 months posttransplant was higher in the basiliximab group (25.0%) than in the placebo group (11.7%). Patient and death-censored graft survival at 12 months was 97% and 99%, respectively, in the basiliximab cohort, and 100% and 99% among placebo-treated patients (n.s.). Renal function was similar in both treatment groups, and there were no significant between-treatment differences in the incidence of adverse events or infections. Conclusions. Addition of basiliximab induction to a regimen of cyclosporine microemulsion, mycophenolate mofetil, and steroids resulted in a numerically lower but not significant incidence of biopsy-proven acute rejection versus placebo and excellent graft and patient survival at 1 year in pediatric renal transplant recipients. Whether this numerical difference is a true therapeutic benefit in view of the higher rate and severity of subclinical rejections in the basiliximab group in the protocol biopsy will be investigated in a long-term follow-up study.

Advanced glycation end products in children with chronic renal failure and type 1 diabetes

Abstract Serum levels of advanced glycation end products (AGEs) are markedly elevated in adults with chronic renal failure (CRF) and diabetes mellitus. Accumulation of AGEs in tissues contributes to the development of long-term complications. Up to now little has been known about the formation of AGEs in childhood. We determined serum levels of the well known AGEs pentosidine and Nɛ-carboxymethyllysine (CML) in children with CRF (n=12), end-stage renal disease (ESRD) (n=9), renal transplantation (n=12), and type 1 diabetes mellitus (n=42) and in healthy children (n=20). Pentosidine was measured by high-performance liquid chromatography (HPLC), CML by a competitive enzyme-linked immunosorbent assay (ELISA) system. Serum levels of pentosidine and CML were significantly higher in the children with CRF and ESRD than in controls (P<0.001), but nearly within the normal range after transplantation. Both AGEs showed a significant negative correlation with creatinine clearance (P<0.001). During a single session of low-flux hemodialysis, total pentosidine and CML levels did not change. Free pentosidine, however, was reduced by 78% (P=0.04). Diabe-tic children showed significantly elevated pentosidine levels (P<0.001) despite normal renal function. We conclude that, similar to adults, increased formation and accumulation of AGEs also exist in children with CRF and type 1 diabetes mellitus. At present the best prevention of AGE-related complications is an early renal transplantation in children with ESRD, as well as a careful metabolic monitoring of diabetics.

Ambulatory blood pressure monitoring in children with unilateral multicystic dysplastic kidney

Abstract Multicystic dysplastic kidney (MCDK) is one of the most common congenital renal anomalies. Arterial hypertension is a potential complication of MCDK. Blood pressure (BP) has so far been measured only casually and the frequency of hypertension has been estimated to be between 0%–8%. Ambulatory blood pressure monitoring (ABPM) provides more precise information on BP than the casual BP measurement. The aim of this study was to investigate the BP profile in children with MCDK using ABPM. A group of 25 children (16 girls), with a mean age of 7.8 years (range 3.8–17.7 years) were investigated. ABPM was performed using the oscillometric SpaceLabs 90207 device. Hypertension was defined as mean systolic and/or diastolic BP during the day and/or in the night exceeding 95th percentile for ABPM. Five (20%) children showed hypertension, two of them had combined daytime and night-time hypertension and three had isolated nocturnal hypertension, although daytime BP was between the 90th–95th percentile in two of them. Children with ultrasonographical and/or laboratory signs of contralateral kidney abnormalities showed a higher incidence of hypertension than those without abnormalities (two of four versus 3 of 21). The mean night-time systolic and diastolic BP of children with MCDK was significantly higher than in healthy children (+0.50 and +0.54 SDS, respectively, P=0.012 and 0.03, respectively). Three of the hypertensive children were already nephrectomised. All five hypertensive children showed ultrasonographical and/or laboratory signs of contralateral kidney abnormalities. Hypertensive children had significantly higher microalbuminuria than normotensive children (6.9 ± 3.2 mg/mmol creatinine versus 1.8 ± 0.7, P=0.03). The nocturnal BP fall (dip) was attenuated in five children, only one of whom was hypertensive. Conclusion Arterial hypertension in children with multicystic dysplastic kidney is seen more often if based on ambulatory blood pressure monitoring than on casual blood pressure recordings. The main risk factor for developing hypertension is contralateral kidney damage. Ambulatory blood pressure monitoring should be performed in children with multicystic dysplastic kidney, especially in those with contralateral kidney abnormalities.