Berthold P. Hauffa

Recommendations for the diagnosis and management of Prader-Willi syndrome

OBJECTIVE The objective of the study was to provide recommendations for the diagnosis and management of Prader-Willi syndrome throughout the life span to guide clinical practice. PARTICIPANTS An open international multidisciplinary expert meeting was held in October 2006 in Toulouse, France, with 37 invited speakers and session chairs (see Acknowledgments) and 85 additional registered participants. The meeting was supported by an unrestricted educational grant from Pfizer. EVIDENCE Invited participants with particular expertise reviewed the published evidence base for their specialist topic and unpublished data from personal experience, previous national and international PWS conferences, and PWS Association clinical advisory groups. Sessions covered epidemiology, psychiatric, and behavioral disorders; breathing and sleep abnormalities; genetics; endocrinology; and management in infancy, childhood, transition, and adulthood. CONSENSUS PROCESS This included group meetings including open discussion after each session. The guidelines were written by the Scientific Committee (authors), using the conclusions provided by the sessions chairs and summary provided by each speaker, including incorporation of changes suggested after review by selected meeting participants (see Acknowledgments). CONCLUSIONS The diagnosis and management of this complex disorder requires a multidisciplinary approach with particular emphasis on the importance of early diagnosis using accredited genetic testing, use and monitoring of GH therapy from early childhood, control of the food environment and regular exercise, appropriate management of transition, consideration of group home placement in adulthood, and distinction of behavioral problems from psychiatric illness.

Bone Mineral Density in Children and Adolescents with Juvenile Diabetes: Selective Measurement of Bone Mineral Density of Trabecular and Cortical Bone Using Peripheral Quantitative Computed Tomography

Bone mineral density (BMD) was studied in 21 children and adolescents with type I diabetes and in age- and sex-matched healthy controls. BMD was selectively measured in trabecular and total bone using peripheral quantitative computed tomography (pQCT). Cortical bone density was calculated. There was a decrease of trabecular bone density (-18.9%, p < 0.01), total bone density (-9.0%, NS) and cortical bone density (-5.1%, NS) in diabetes. Trabecular bone density was inversely correlated with the duration of diabetes and the concentration of glycosylated hemoglobin (HbA1) (r = -0.48, p = 0.027 and r = -0.63, p = 0.002, respectively). Total BMD correlated inversely with HbA1 (r = -0.52, p = 0.017). pQCT allows the selective measurement of metabolically active trabecular bone where changes of mineralization first occur. We conclude that pQCT is a useful method for investigating BMD in diabetes.

Prenatal diagnosis of P450 oxidoreductase deficiency (ORD): A disorder causing low pregnancy estriol, maternal and fetal virilization, and the Antley–Bixler syndrome phenotype

We report studies on the second pregnancy of a woman who had previously given birth to a virilized female infant. The cause of the virilization had not been established, but common forms of congenital adrenal hyperplasia (CAH) were excluded. Longitudinal monitoring of the second pregnancy revealed that estriol excretion failed to increase normally, reaching a maximum 0.7 mg/24 hr at the end of pregnancy (normal mean 30 mg/24 hr). The mother showed signs of virilization by the 23rd week of gestation and aromatase deficiency was suspected. However, predicted urinary metabolites for diagnosis of aromatase deficiency (for example, 16α‐hydroxyandrosterone) were not increased significantly during the pregnancy. Interestingly, excretion of the androgen metabolite androsterone increased rapidly at the beginning of pregnancy and peaked around the 20th week, suggesting increased production of testosterone and 5αDHT, probably the cause of maternal virilization. Urine steroid analysis by GC/MS showed gradually increasing excretion (9 mg/24 hr) of the normally minor metabolite 5α‐pregnane‐3β,20α‐diol (epiallopregnanediol), an epimer of the dominant progesterone metabolite pregnanediol (5β‐pregnane‐3α,20α‐diol). We believe epiallopregnanediol is largely the maternal urinary excretion product of fetal 5‐pregnene‐3β,20α‐diol, the principal metabolite of pregnenolone, implying a build‐up of the latter steroid in the fetal adrenal. These findings suggested that the ‘block’ in the estriol biosynthetic pathway occurs at an early stage with 17‐hydroxylation of pregnenolone being affected. The male baby born of this pregnancy had normal genitalia but showed a urinary steroid profile indicating partial deficiencies of P450c17 and P450c21. However, no mutations in the corresponding CYP17 and CYP21 genes were identified. Urinary steroid analysis carried out on his virilized older sibling showed the same pattern of metabolites. Recently, we determined that this disorder is caused by mutations in P450 oxidoreductase (OR), the essential redox partner for CYP17 and CYP21 hydroxylases. The novel metabolic profile has now been seen in many patients, most diagnosed with the skeletal dysplasia Antley–Bixler syndrome. We propose that excessive excretion of epiallopregnanediol together with low estriol may be prenatally diagnostic for OR deficiency (ORD).

Microarray analysis reveals differential gene expression patterns and regulation of single target genes contributing to the opposing phenotype of TrkA- and TrkB-expressing neuroblastomas

Expression of neurotrophin receptors of the tyrosine kinase receptor (Trk) family is an important prognostic factor in solid tumors including neuroblastoma. High expression of TrkA (NTRK1) is associated with a favorable biology and outcome of neuroblastoma, whereas TrkB (NTRK2) is expressed on aggressive neuroblastomas with unfavorable outcome. To gain new insights into the global gene expression program resulting in these divergent biological phenotypes, we stably expressed either TrkA or TrkB in the human SH-SY5Y neuroblastoma cell line. Gene expression profiles were obtained from parental cells and transfectants activated by their ligands in a time course over 24 h using oligonucleotide microarrays. Basal activation of Trk receptors in the absence of exogenous ligand was sufficient to induce broad and divergent genetic changes. Global gene regulation following external ligand stimulation was surprisingly similar in SY5Y-TrkA and SY5Y-TrkB cells except for the differential expression of distinct novel target genes. Consistent with their divergent biological phenotype, SY5Y-TrkA cells were characterized by upregulation of proapoptotic genes and angiogenesis inhibitors, whereas SY5Y-TrkB cells demonstrated upregulation of genes involved in invasion or therapy resistance. We suggest that the transcriptional program of neuroblastoma cells is modulated by Trk-receptor expression and basal activation rather than by ligand-induced activation. Fine-tuning of the malignant phenotype may be achieved by additional ligand stimulation with subsequent activation of a few specific genes.

Biochemical diagnosis of Antley-Bixler syndrome by steroid analysis.

Antley–Bixler syndrome (ABS, MIM 207410) is a skeletal abnormality syndrome primarily affecting head and limbs. Little is known of the origin of the condition but inactivating mutations in the fibroblast growth factor receptor (FGFR2) has been found in some patients. Genital ambiguity is seen occasionally in this condition, suggesting possible disordered steroidogenesis in early pregnancy. We report the steroid excretion of eight patients diagnosed with the syndrome and one with a related condition, a mild phenotype of the disorder since skeletal and genital abnormalities were not evident. The steroid excretion pattern was consistent and very distinctive in all nine patients. Metabolites of the two primary precursors of steroid hormones, pregnenolone and progesterone, were elevated as were the classical diagnostic metabolites for 17‐ and 21‐hydroxylase deficiencies. Cortisol production was typically within the normal range but generally had blunted response to ACTH. Androgen metabolite excretion tends to be low in patients over 2 months of age, but may be elevated in the newborn period. The metabolome suggested attenuated steroid hydroxylation (including 17,20‐lyase activity) although underlying cause is yet to be established. Mutations in CYP17 and CYP21 have not been found and currently the prime suspect is an abnormality in an essential redox partner (P450 oxidoreductase). This paper proposes use of the distinctive steroid metabolome as the primary biochemical parameter for diagnosis of ABS, at least the form not associated with FGFR2 mutations.

Genotype-phenotype analysis in congenital adrenal hyperplasia due to P450 oxidoreductase deficiency

Context: P450 oxidoreductase deficiency (PORD) is a unique congenital adrenal hyperplasia variant that manifests with glucocorticoid deficiency, disordered sex development (DSD), and skeletal malformations. No comprehensive data on genotype-phenotype correlations in Caucasian patients are available. Objective: The objective of the study was to establish genotype-phenotype correlations in a large PORD cohort. Design: The design of the study was the clinical, biochemical, and genetic assessment including multiplex ligation-dependent probe amplification (MLPA) in 30 PORD patients from 11 countries. Results: We identified 23 P450 oxidoreductase (POR) mutations (14 novel) including an exonic deletion and a partial duplication detected by MLPA. Only 22% of unrelated patients carried homozygous POR mutations. p.A287P was the most common mutation (43% of unrelated alleles); no other hot spot was identified. Urinary steroid profiling showed characteristic PORD metabolomes with variable impairment of 17α-hydroxylase and 21-hydroxylase. Short cosyntropin testing revealed adrenal insufficiency in 89%. DSD was present in 15 of 18 46,XX and seven of 12 46,XY individuals. Homozygosity for p.A287P was invariably associated with 46,XX DSD but normal genitalia in 46,XY individuals. The majority of patients with mild to moderate skeletal malformations, assessed by a novel scoring system, were compound heterozygous for missense mutations, whereas nearly all patients with severe malformations carried a major loss-of-function defect on one of the affected alleles. Conclusions: We report clinical, biochemical, and genetic findings in a large PORD cohort and show that MLPA is a useful addition to POR mutation analysis. Homozygosity for the most frequent mutation in Caucasians, p.A287P, allows for prediction of genital phenotype and moderate malformations. Adrenal insufficiency is frequent, easily overlooked, but readily detected by cosyntropin testing.

Removal of metabolites, cytokines and hepatic growth factors by extracorporeal liver support in children.

Background: Molecular Adsorbents Recirculating System (MARS)-mini has recently been approved and applied in children with hepatic failure. However, its indication, efficacy and capability to induce liver regeneration remain unclear. The aim of our pilot study in children was to analyse the impact of MARS on markers of detoxification and regeneration. Methods: In children with fulminant Wilsons disease and bridged with MARSmini for liver transplantation, we analyzed toxic metabolites (bile acids, bilirubin, lactate, ammonia, tryptophan and copper), regulators of the inflammatory cascade [nitrate, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), methionine, cystine and hyaluronic acid] and hepatic growth factors [hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor-β1 (TGF-β1), cortisol, corticosteroid-binding globulin (CBG), insulin-like growth factor-1 (IGF-1), angiogenin, vascular endothelial growth factor (VEGF), IL-6 and TNF-α] from blood, albumin circuit and haemodialysate from four applications. Results: In all four applications, transfer of toxic metabolites (6/6) and inflammatory mediators (6/6), but also of hepatic growth factors (9/10), into the albumin circuit of MARS was consistently detected. Corresponding blood levels were decreased for 3/6 metabolites, 3/6 inflammatory mediators and 1/10 growth factors and increased for 1/10 growth factors. Bridging for liver transplantation was successful with MARS. Conclusions: In our prospective study, substantial extraction of albumin-bound and water-soluble candidate substances was detected with variable effect on respective blood levels. Notably, essential factors inducing liver regeneration were simultaneously removed. These data provide a basis for evaluation of liver restoration and efficacy of liver support in children with liver failure to devise a collaborative, multicentre trial.

Dehydroepiandrosterone Sulphate and Corticotropin Levels Are High in Young Male Patients with Conduct Disorder: Comparisons for Growth Factors, Thyroid and Gonadal Hormones

Childhood conduct disorder (CD) may originate in a stressful upbringing, and be associated with unusual physical or sexual development and thyroid dysfunction. We therefore explored circulating levels of hormones from adrenal, gonadal and growth hormone axes associated with stress, aggression and development in 28 CD patients and 13 age-matched healthy children (10–18 years old). The CD group had higher levels of dehydroepiandrosterone sulphate (DHEA-S), corticotropin (ACTH) and free tri-iodothyronine (fT3) if under 14 years. There were no differences for gonadal hormones or maturity ratings which were not associated with aggression. Smaller physical measures in CD children correlated with DHEA-S and growth factors (e.g. insulin-like growth factor I) increased ACTH and fT3 correlated with restless-impulsive ratings, and DHEA-S with ‘disruptive behaviour’. Imbalances in the adrenal and growth axes may have neurotropic repercussions in development.

Association of parvovirus B19 infection and Hashimoto's thyroiditis in children.

Hashimotos thyroiditis is a common autoimmune disorder of the thyroid gland. It has been linked to infections with hepatitis C, EBV, HTLV-1, and Yersinia enterocolitica. As parvovirus B19 has been associated with a wide spectrum of autoimmune diseases, we investigated the potential role of B19 infection in inducing Hashimotos thyroiditis. Serum samples derived from 73 children and adolescents with Hashimotos thyroiditis and from 73 age-matched controls were included in the study. The mean age of disease manifestation was 10 y 7 mo. All samples were analyzed for the presence of viral DNA and for antibodies against VP1, VP2, and NS1 proteins. VP1- and VP2-specific antibodies were present in 38 patients (52%) and 43 controls (59%; N.S.). NS1-specific antibodies were detectable in 23 patients (32%) and 19 controls (26%; N.S.). Parvovirus B19 DNA was detectable in 9 patients (12%) and 2 controls (3%; p < 0.03), indicating recent B19-infection. A negative correlation between disease duration and the detection of viral DNA was seen. The mean disease duration in B19-DNA-positive patients was 6 mo, compared to 29 mo in the remainder (p < 0.01). There is strong evidence that acute parvovirus B19 infections are involved in the pathogenesis of some cases of Hashimotos thyroiditis.

Effects of RANK-Ligand Antibody (Denosumab) Treatment on Bone Turnover Markers in a Girl With Juvenile Paget's Disease

CONTEXT Juvenile Pagets disease (JPD) is an extremely rare, yet painful and debilitating bone disease with onset occurring during early childhood. JPD can be caused by loss of function of osteoprotegerin, resulting in subsequent stimulation of osteoclasts via the receptor activator of nuclear factor-κB (RANK) pathway. Increased bone turnover and lack of bone modeling lead to severe deformities, frequent fractures, short stature, and loss of hearing. SETTING The treatment for JPD is challenging and has previously been based on administration of either calcitonin or bisphosphonates. However, with the development of denosumab, a receptor activator of nuclear factor-κB-ligand (RANKL) antibody, a treatment targeting the pathophysiology of JPD may be available. We report the effects of denosumab treatment on an 8-year-old girl with a severe form of JPD. PATIENT Before starting the denosumab treatment regimen, the patient had been treated for 3.5 years with iv pamidronate. INTERVENTION AND OUTCOME The administration of denosumab resulted in improved disease control compared with bisphosphonate, as assessed by monitoring markers of bone turnover. Alkaline phosphatase levels dropped within the normal range and remained at normal levels for 5 months after the final dose of denosumab. Additionally, bone pain was more efficiently controlled with denosumab. However, concomitant with the first injection, severe hypocalcemia developed, for which the patient was hospitalized and iv calcium supplementation was required for 13 days. CONCLUSIONS Denosumab appears to be significantly effective for osteoclast inhibition for the treatment of JPD. However, in our patient, denosumab administration was associated with severe hypocalcemia, indicating that close monitoring of calcium levels is required during treatment.