A multi-method approach to the molecular diagnosis of overt and borderline 11p15.5 defects underlying Silver-Russell and Beckwith-Wiedemann syndromes.

BACKGROUND: Multiple (epi)genetic defects affecting the expression of the imprinted genes within the 11p15.5 chromosomal region underlie Silver-Russell (SRS) and Beckwith-Wiedemann (BWS) syndromes. The molecular diagnosis of these opposite growth disorders requires a multi-approach flowchart to disclose known primary and secondary (epi)genetic alterations; however, up to 20 and 30 % of clinically diagnosed BWS and SRS cases remain without molecular diagnosis. The complex structure of the 11p15 region with variable CpG methylation and low-rate mosaicism may account for missed diagnoses. Here, we demonstrate the relevance of complementary techniques for the assessment of different CpGs and the importance of testing multiple tissues to increase the SRS and BWS detection rate. RESULTS: Molecular testing of 147 and 450 clinically diagnosed SRS and BWS cases provided diagnosis in 34 SRS and 185 BWS patients, with 9 SRS and 21 BWS cases remaining undiagnosed and herein referred to as "borderline." A flowchart including complementary techniques and, when applicable, the analysis of buccal swabs, allowed confirmation of the molecular diagnosis in all borderline cases. Comparison of methylation levels by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) in borderline and control cases defined an interval of H19/IGF2:IG-DMR loss of methylation that was distinct between "easy to diagnose" and "borderline" cases, which were characterized by values ≤mean -3 standard deviations (SDs) compared to controls. Values ≥mean +1 SD at H19/IGF2: IG-DMR were assigned to borderline hypermethylated BWS cases and those ≤mean -2 SD at KCNQ1OT1: TSS-DMR to hypomethylated BWS cases; these were supported by quantitative pyrosequencing or Southern blot analysis. Six BWS cases suspected to carry mosaic paternal uniparental disomy of chromosome 11 were confirmed by SNP array, which detected mosaicism till 10 %. Regarding the clinical presentation, borderline SRS were representative of the syndromic phenotype, with exception of one patient, whereas BWS cases showed low frequency of the most common features except hemihyperplasia. CONCLUSIONS: A conclusive molecular diagnosis was reached in borderline methylation cases, increasing the detection rate by 6 % for SRS and 5 % for BWS cases. The introduction of complementary techniques and additional tissue analyses into routine diagnostic work-up should facilitate the identification of cases undiagnosed because of mosaicism, a distinctive feature of epigenetic disorders.

Recommendations of the Scientific Committee of the Italian Beckwith-Wiedemann Syndrome Association on the diagnosis, management and follow-up of the syndrome.

UNLABELLED: Beckwith-Wiedemann syndrome (BWS) is the most common (epi)genetic overgrowth-cancer predisposition disorder. Given the absence of consensual recommendations or international guidelines, the Scientific Committee of the Italian BWS Association (www.aibws.org) proposed these recommendations for the diagnosis, molecular testing, clinical management, follow-up and tumor surveillance of patients with BWS. The recommendations are intended to allow a timely and appropriate diagnosis of the disorder, to assist patients and their families, to provide clinicians and caregivers optimal strategies for an adequate and satisfactory care, aiming also at standardizing clinical practice as a national uniform approach. They also highlight the direction of future research studies in this setting. With recent advances in understanding the disease (epi)genetic mechanisms and in describing large cohorts of BWS patients, the natural history of the disease will be dissected. In the era of personalized medicine, the emergence of specific (epi)genotype-phenotype correlations in BWS will likely lead to differentiated follow-up approaches for the molecular subgroups, to the development of novel tools to evaluate the likelihood of cancer development and to the refinement and optimization of current tumor screening strategies. CONCLUSIONS: In this article, we provide the first comprehensive recommendations on the complex management of patients with Beckwith-Wiedemann syndrome.

Triglycerides-to-HDL cholesterol ratio as screening tool for impaired glucose tolerance in obese children and adolescents.

AIMS: To identify metabolic phenotypes at increased risk of impaired glucose tolerance (IGT) in Italian overweight/obese children (n = 148, age 5-10 years) and adolescents (n = 531, age 10-17.9 year). METHODS: Phenotypes were defined as follows: obesity by the 95th cut-points of the Center for Disease Control body mass index reference standards, impaired fasting glucose (fasting plasma glucose ≥100 mg/dl), high circulating triglycerides (TG), TG/HDL cholesterol ≥2.2, waist-to-height ratio (WTHR) >0.6, and combination of the latter with high TG or TG/HDL cholesterol ≥2.2. RESULTS: In the 148 obese children, TG/HDL-C ≥ 2.2 (OR 20.19; 95 % CI 2.50-163.28, p = 0.005) and the combination of TG/HDL-C ≥ 2.2 and WTHR > 0.60 (OR 14.97; 95 % CI 2.18-102.76, p = 0.006) were significantly associated with IGT. In the 531 adolescents, TG/HDL-C ≥ 2.2 (OR 1.991; 95 % CI 1.243-3.191, p = 0.004) and the combination with WTHR > 0.60 (OR 2.24; 95 % CI 1.29-3.87, p = 0.004) were associated with significantly increased risk of IGT. In the whole sample, having high TG levels according to the NIH National Heart, Lung and Blood Institute Expert Panel was not associated with an increased risk of presenting IGT. CONCLUSIONS: TG/HDL-C ratio can be useful, particularly in children, to identify obese young patients at risk of IGT. Its accuracy as screening tool in a general population needs to be verified. The combination of TG/HDL-C ratio and WTHR > 0.6 did not improve prediction. Having high TG according to the NIH definition was not associated with increased risk of developing IGT.

Obstructive sleep apnea in Prader-Willi syndrome: risks and advantages of adenotonsillectomy.

Obstructive sleep apnea is a well-known clinical manifestation of Prader-Willi syndrome. The aim of our study is to evaluate the efficacy of adenotonsillectomy for the treatment of the disorder as well as the improvement of their post-operative quality of life. Five patients with moderate to severe obstructive sleep apneas and adenotonsillar hypertrophy of grade III-IV underwent adenotonsillectomy. Pre- and postoperative apneas and Quality of Life were assessed respectively with a polysomnography with multi-sleep latency test and with the pediatric Quality of Life questionnaire, performed before and 6 months after surgery. A decrease of apnea/hypopnea index values has been detected between pre- and post-surgery (t=2.64, P=0.005), as well as oxygen desaturation index values (t=5.51, P=0.005), multi-sleep latency test (t=4.54, P=0.01), and of the values of pediatric Quality of Life questionnaire. No correlation has been detected between body mass index and apnea/hypopnea index, oxygen desaturation index and multi-sleep latency test values pre- and post-adenotonsillectomy. A correlation has been found between multi-sleep latency test and oxygen desaturation index values post-surgery (P=0.04). No post-operative complications were observed. Our data underline the efficacy of surgery in Prader-Willi patients with adenotonsillar hypertrophy in order to improve their quality of life.

Growth hormone therapy and respiratory disorders: long-term follow-up in PWS children.

CONTEXT: Adenotonsillar tissue hypertrophy and obstructive sleep apnea have been reported during short-term GH treatment in children with Prader-Willi syndrome (PWS). OBJECTIVE: We conducted an observational study to evaluate the effects of long-term GH therapy on sleep-disordered breathing and adenotonsillar hypertrophy in children with PWS. DESIGN: This was a longitudinal observational study. PATIENTS AND METHODS: We evaluated 75 children with genetically confirmed PWS, of whom 50 fulfilled the criteria and were admitted to our study. The patients were evaluated before treatment (t0), after 6 weeks (t1), after 6 months (t2), after 12 months (t3), and yearly (t4-t6) thereafter, for up to 4 years of GH therapy. The central apnea index, obstructive apnea hypopnea index (OAHI), respiratory disturbance index, and minimal blood oxygen saturation were evaluated overnight using polysomnography. We evaluated the adenotonsillar size using a flexible fiberoptic endoscope. RESULTS: The percentage of patients with an OAHI of >1 increased from 3 to 22, 36, and 38 at t1, t4, and t6, respectively (χ(2) = 12.2; P < .05). We observed a decrease in the respiratory disturbance index from 1.4 (t0) to 0.8 (t3) (P < .05) and the central apnea index from 1.2 (t0) to 0.1 (t4) (P < .0001). We had to temporarily suspend treatment for 3 patients at t1, t4, and t5 because of severe obstructive sleep apnea. The percentage of patients with severe adenotonsillar hypertrophy was significantly higher at t4 and t5 than at t0. The OAHI directly correlated with the adenoid size (adjusted for age) (P < .01) but not with the tonsil size and IGF-1 levels. CONCLUSION: Long-term GH treatment in patients with PWS is safe; however, we recommend annual polysomnography and adenotonsillar evaluation.

Whole genome SNP genotyping and exome sequencing reveal novel genetic variants and putative causative genes in congenital hyperinsulinism.

Congenital hyperinsulinism of infancy (CHI) is a rare disorder characterized by severe hypoglycemia due to inappropriate insulin secretion. The genetic causes of CHI have been found in genes regulating insulin secretion from pancreatic ß-cells; recessive inactivating mutations in the ABCC8 and KCNJ11 genes represent the most common events. Despite the advances in understanding the molecular pathogenesis of CHI, specific genetic determinants in about 50 % of the CHI patients remain unknown, suggesting additional locus heterogeneity. In order to search for novel loci contributing to the pathogenesis of CHI, we combined a family-based association study, using the transmission disequilibrium test on 17 CHI patients lacking mutations in ABCC8/KCNJ11, with a whole-exome sequencing analysis performed on 10 probands. This strategy allowed the identification of the potential causative mutations in genes implicated in the regulation of insulin secretion such as transmembrane proteins (CACNA1A, KCNH6, KCNJ10, NOTCH2, RYR3, SCN8A, TRPV3, TRPC5), cytosolic (ACACB, CAMK2D, CDKAL1, GNAS, NOS2, PDE4C, PIK3R3) and mitochondrial enzymes (PC, SLC24A6), and in four genes (CSMD1, SLC37A3, SULF1, TLL1) suggested by TDT family-based association study. Moreover, the exome-sequencing approach resulted to be an efficient diagnostic tool for CHI, allowing the identification of mutations in three causative CHI genes (ABCC8, GLUD1, and HNF1A) in four out of 10 patients. Overall, the present study should be considered as a starting point to design further investigations: our results might indeed contribute to meta-analysis studies, aimed at the identification/confirmation of novel causative or modifier genes.

Genetic analysis of Italian patients with congenital hyperinsulinism of infancy.

BACKGROUND/AIMS: Congenital hyperinsulinism of infancy is a rare disease that needs prompt treatment to avoid brain damage. There are currently no data regarding the clinical and molecular features of Italian patients. METHODS: Thirty-three patients with HI and their parents were included. Consanguinity was reported in six patients. Half of patients were macrosomic at birth. None had raised 3-hydroxybutyrylcarnitine or hyperammonemia. Molecular analysis of ABCC8 and KCNJ11 genes was performed in all patients, and subjects with no mutation underwent analysis of HNF4A and GCK. GLUD1 and HADH genes were analyzed in a patient with leucine sensitivity. RESULTS: Mutations in the ABCC8 and KCNJ11 genes were found in 45% of the patients (6 novel). No mutations in HNF4A, GLUD1 and GCK genes were found. Recessive mode of inheritance was found in 21% of patients. A single heterozygous mutation was identified in 24% of probands. 72% of the patients were responsive to medical treatment, and 44% of the 17 patients with no identified mutation achieved spontaneous remission. Nine children, unresponsive to medical therapy, underwent pancreatectomy. CONCLUSION: This is the first report on hyperinsulinism of infancy in Italy, confirming the complexity of the clinical forms and the heterogeneity of the genetic causes of the disease.

Central adrenal insufficiency in young adults with Prader-Willi syndrome.

OBJECTIVE: A high prevalence (60%) of central adrenal insufficiency (CAI) has been reported in Prader-Willi syndrome (PWS) using the metyrapone test. We have assessed CAI in adults with PWS using the low-dose short synacthen test (LDSST). DESIGN: Basal cortisol and ACTH, and 30-min cortisol after the administration of 1 µg synacthen, were determined in 53 PWS adults (33 females). A peak cortisol value of ≥500 nmol/l was taken as normal. Hormonal profiles were analysed in relation to gender, genotype and phenotype. Deficient patients were retested by high-dose short synachten test (HDSST) or a repeat LDSST. RESULTS: Mean ± SD basal cortisol and ACTH were 336·6 ± 140·7 nmol/l and 4·4 ± 3·7 pmol/l respectively. Cortisol rose to 615·4 ± 135·0 nmol/l after LDSST. Eight (15·1%) patients had a peak cortisol response <500 nmol/l, with a lower mean ± SD (range) basal cortisol of 184·9 ± 32·0 (138·0-231·7) compared with 364·1 ± 136·6 (149·0-744·5) in normal responders (P < 0·001). Seven of the eight patients underwent retesting, with 4 (7·5%) showing persistent suboptimal responses. Basal and peak cortisol correlated in females (r = 0·781, P < 0·001). Logistic regression revealed that only female gender and baseline cortisol were predictors of cortisol peaks (adjusted R square 0·505). CONCLUSIONS: Although CAI can be part of the adult PWS phenotype, it has a lower prevalence (7·5%) than previously reported. Clinicians are advised to test PWS patient for CAI. Our study also shows that basal cortisol is closely correlated with adrenal response to stimulation, indicating that its measurement may be helpful in selecting patients for LDSST.

Functional characterization of novel loss-of-function mutations in the vasopressin type 2 receptor gene causing nephrogenic diabetes insipidus.

BACKGROUND: X-linked nephrogenic diabetes insipidus (NDI) is a rare polyuric disorder caused by inactivating mutations in the arginine vasopressin receptor Type 2 (AVPR2) gene. METHODS: NDI patients from six unrelated families were subjected to mutational analysis of the AVPR2 gene. In-depth in vitro characterization of novel AVPR2 mutants by a combination of functional and immunological techniques provided further insight into molecular mechanisms causing receptor dysfunction. RESULTS: Mutational analysis revealed four novel (A89P, G107R, Q174R, W208X) and three recurrent (V277A, R337X, ΔR247-G250) mutations within the AVPR2 gene. One family carried the missense mutation R337X and a 12-bp deletion (ΔR247-G250), corresponding to a fragment in the third intracellular loop (ICL3), which was not genetically linked to R337X. The functionally tested missense mutations A89P, G107R and Q174R led to reduced receptor cell surface expression in transfected COS-7 cells, most probably due to misfolding and intracellular retention, and consequently to reduction or loss of agonist-mediated cyclic adenosine monophosphate formation. Deletion of R247-G250 had no effect on receptor function in vitro. Comparison with other mammalian AVPR2 orthologs showed that this part of the ICL3 is structurally not conserved and, therefore, less relevant for receptor function. In contrast, all missense mutations (A89P, G107R, Q174R, V277A) affect receptor positions that were fully preserved during mammalian evolution. CONCLUSION: Our results provide valuable information about residues critical for AVPR2 folding, trafficking and function and proof that these mutations are responsible for causing NDI in the affected subjects.

Assessment of central adrenal insufficiency in children and adolescents with Prader-Willi syndrome.

OBJECTIVE: A recent study evidenced by metyrapone test a central adrenal insufficiency (CAI) in 60% of Prader-Willi syndrome (PWS) children. These results were not confirmed in investigations with low [Low-Dose Tetracosactrin Stimulation Test (LDTST), 1 µg] or standard-dose tetracosactrin stimulation tests. We extended the research by LDTST in paediatric patients with PWS. DESIGN: Cross-sectional evaluation of adrenal stress response to LDTST in a PWS cohort of a tertiary care referral centre. PATIENTS: Eighty-four children with PWS. MEASUREMENTS: Assessment of adrenal response by morning cortisol and ACTH dosage, and 1-µg tetracosactrin test. Response was considered appropriate when cortisol reached 500 nm; below this threshold, patients were submitted to a second test. Responses were correlated with the patients' clinical and molecular characteristics to assess genotype-phenotype correlation. RESULTS: Pathological cortisol peak responses to the LDTST were registered in 12 patients (14.3%) who had reduced basal (169.4 ± 83.3 nm) and stimulated (428.1 ± 69.6 nm) cortisol levels compared to patients with normal responses (367.1 ± 170.6 and 775.9 ± 191.3 nm, P < 0.001). Body mass index standard deviation score was negatively correlated with basal and peak cortisol levels (both P < 0.001), and the patients' ages (P < 0.001). In patients with deletion on chromosome 15, the cortisol peak was significantly lower than that in uniparental disomy (UPD) cases (P = 0.030). At multiple regression analysis, the predictors of peak response were basal cortisol, age, and UPD subclass (r(2) = 0.353, P < 0.001). Standard-dose (250 µg) tetracosactrin test confirmed CAI in 4/12 patients (4.8% of the cohort). CONCLUSIONS: Our results support the hypothesis that, albeit rare, CAI may be part of the PWS in childhood.

Fasting plasma glucose (FPG) and the risk of impaired glucose tolerance in obese children and adolescents.

A timely diagnosis of impaired glucose tolerance (IGT) is desirable in obesity. The oral glucose tolerance test (OGTT), the gold standard to diagnose this condition, may not be realistically performed in all patients due to discomfort, labor, and cost. The aim of this study was to assess whether one or more biochemical indexes measured in fasting conditions could be used to identify obese children at risk of IGT. A cohort of 563 white obese children and adolescents (M/F: 315/248; aged 4-17 years) was recruited and underwent anthropometric evaluation and OGTT. Anthropometric parameters, fasting plasma glucose (FPG), fasting serum insulin (FSI), and homeostasis model assessment of insulin resistance (HOMA(IR)) were tested in pursuit of a possible threshold to be used as a predictor of IGT. Thirty-seven children (6.9%) had IGT and one child (0.1%) had type 2 diabetes (T2D). FPG, FSI, and HOMA(IR) were all significantly higher in children with IGT than in children without IGT. Receiver-operating characteristic (ROC) curve analyses run for gender and puberty-adjusted FPG, FSI, and HOMA(IR) were all significant: area under the curve (95% confidence interval) equaled 0.68 (0.59-0.76), 0.66 (0.56-0.76), and 0.68 (0.59-0.78), respectively. The three parameters did not show significantly different sensitivity/specificity in the pooled population or in the gender/puberty subgroups. Thresholds varied among gender/puberty subgroups for FSI and HOMA(IR), but not for FPG, which showed a fixed threshold of 86 mg/dl. A gender/puberty independent cutoff of FPG may be considered a screening tool to narrow clinical indication to OGTT in obese white children and adolescents.

Identification of a diffuse form of hyperinsulinemic hypoglycemia by 18-fluoro-L-3,4 dihydroxyphenylalanine positron emission tomography/CT in a patient carrying a novel mutation of the HADH gene.

OBJECTIVE: Congenital hyperinsulinism is the most common cause of persistent hypoglycemia in infancy (HI), leading to severe neurologic disabilities if not promptly treated. The recent application of positron emission tomography (PET)/computed tomography (CT) scanning with 18-fluoro-l-3,4 dihydroxyphenylalanine improved the ability to distinguish the two histopathologic forms of HI (focal and diffuse), whose differentiation heavily influences the therapeutic management of the patient. CASE REPORT: We describe the case of a patient presenting with severe hypoglycemia from infancy. High concentration of insulin suggested the diagnosis of congenital hyperinsulinism. No metabolic disorders related to amino acid, organic acids or fatty acid oxidation were detected. Medical treatment was able to obtain a satisfactory metabolic response. RESULTS: The patient underwent PET/CT scanning, revealing a diffuse form of the disease. The absence of mutations in KCNJ11 and ABCC8 genes (responsible for 50% of HI cases), and whole genome single nucleotide polymorphisms analysis by microarray suggested the HADH gene as a likely candidate. Sequence analysis revealed a novel homozygous nonsense mutation (R236X) in HADH gene. CONCLUSIONS: This case indicates that mutations of the HADH gene should be sought in hyperinsulinemic patients in whom diffuse form of HI and autosomal recessive inheritance can be presumed when KCNJ11 and ABCC8 genes mutational screening is negative, even in the absence of altered organic acids and acylcarnitines concentration.

Persistent mild hypothyroidism associated with novel sequence variants of the DUOX2 gene in two siblings.

One of the steps in thyroid hormone biosynthesis is the generation of hydrogen peroxide by dual oxidases (DUOX). Only one study reported mutations in DUOX2 gene in congenital hypothyroidism (CH) associated with total iodide organification defect (TIOD) in homozygosity or with partial iodide organification defect (PIOD) in heterozygous patients. We report genetic and phenotypic characterization of a family affected with isolated CH. The proband was positive at neonatal TSH screening. High serum TSH with low FT4 confirmed the diagnosis. At 4 years, TSH was high after L-T(4) withdrawal and (123)I scintigraphy with perchlorate discharge test revealed a PIOD. His brother was negative at TSH screening, but perinatal iodine overload was documented by urinary test. Serum TSH was elevated at postnatal day 11 and progressively increased together with a decline in urinary iodine. Reevaluation at 4 years confirmed a persistent hyperthyrotropinemia associated with PIOD. Both siblings resulted compound heterozygotes for two novel DUOX2 variants, a nonsense mutation (c.2524C>T, p.Arg842X) and a missense substitution (c.1126C>T, p.Arg376Trp), undetected in 140 control alleles. The parents had normal thyroid function and were heterozygous carriers of mutant alleles. In conclusion, we report two novel sequence variants in DUOX2 gene that are associated with persistent mild hypothyroidism and PIOD in two siblings. Different neonatal iodine supply apparently acted as disease modifier, justifying the discrepant results at TSH screening in the two siblings with same DUOX2 genotype and suggesting that mild dyshormonogenic defects may remain undisclosed in areas characterized by elevated iodine intake.