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Background: Newborn screening for congenital adrenal hyperplasia (CAH) based on 17-hydroxyprogesterone (17-OHP) concentration in dried blood spots has been taking place in North-Eastern Italy since 2001. Since 2017, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been introduced, for the first time in Italy, as a second-tier test. Aims: Our study aims to evaluate, on the one hand, the effectiveness of the newborn screening for CAH after 20 years of testing and, on the other, the impact that the introduction of the second-tier test had on the diagnostic accuracy of the screening program. Methods: Since 2001 dried blood spots taken from newborns have been screened with a time-resolved fluoroimmunoassay for 17-OHP determination. Over the years, the cut-off levels of 17-OHP were adjusted according to gestational age. Since 2017, a second-tier test in LC-MS/MS was introduced for samples displaying fluoroimmunoassay 17-OHP exceeding the cut-off. Results: In total, 862,521 newborns have been screened over a period of 20 years. The total incidence of 21-hydroxylase deficiency (21-OHD) was 1:25,368, moreover, a case of 11-β-hydroxylase deficiency was identified. All these diagnoses were genetically confirmed. The sensitivity and specificity of the screening program were 97% and 99.4%, respectively. The use of LC-MS/MS as a second-tier test significantly reduced the recall rate and increased the positive predictive value. Conclusions: Screening for CAH is useful in the neonatal diagnosis of a classic form of 21-OHD, allowing a precocious treatment of affected children. The introduction of an LC-MS/MS second-tier reduced the recall rate, avoiding unnecessary blood withdrawal and medical evaluations and preventing stress to families. Furthermore, it helped identify rarer forms of CAH.

Introduction Infants of mothers with autoimmune hypothyroidism (AH) are at risk of developing late-onset hypothyroidism, often escaping at newborn screening. This condition might be caused both by the action of maternal antibodies and/or by maternal treatment. Objectives The aim of this study is to evaluate the prevalence of AH in the mothers of children born in Veneto region, Italy, and to define what is the most appropriate management for these newborns. Methods Newborns of six different hospitals with a mother suffering from AH and with negative neonatal screening for congenital hypothyroidism (CH) were included in the study. Between 15 and 20 days of life, we collected a serum sample for the evaluation of thyroid function (thyroid-stimulating hormone (TSH), free thyroxine (FT4), free triiodothyronine (FT3)) and anti-thyroid antibodies. On the same occasion, a capillary blood sampling was performed for a second screening test. Results Maternal AH has a prevalence of 3.5%. A total of 291 newborns were enrolled from November 2019 to May 2021. Whereas the 11.4% of infants had a slight elevated serum TSH (>6 mU/L) and required a follow-up, only 2 children presented an elevated TSH level at the second screening test. One of these, with the gland in situ, showed persistently elevated serum TSH levels and required treatment with levothyroxine. Conclusions Maternal AH rarely caused neonatal thyroid dysfunction. We suggest to reassess newborns from mothers with AH 15 days after birth by means of a second neonatal screening test. This procedure avoids false negatives due to maternal thyroid status, is less invasive and cheaper than the serum TSH evaluation, and prevents a long follow-up.

BCKDK is an important key regulator of branched-chain ketoacid dehydrogenase complex activity by phosphorylating and so inactivating branched-chain ketoacid dehydrogenases, the rate-limiting enzyme of the branched-chain amino acid metabolism. We identified, by whole exome-sequencing analysis, the p.His162Gln variant of the

Introduction Mitochondrial fatty acid oxidation disorders (FAODs) are a heterogeneous group of hereditary autosomal recessive diseases included in newborn screening (NBS) program in Italy. The aim of this study was to analyse FAODs cases, identified either clinically or by NBS,for clinical and genetic characterization and to evaluate a five years' experience of NBS, in the attempt to figure out the complexity of genotype-phenotype correlation and to confirm the clinical impact of NBS in our centre experience. Materials and methods We analysed FAODs patients diagnosed either by NBS or clinically, followed since February 2014 to April 2019 at the Regional Screening Centre and Inherited Metabolic Diseases Unit of Verona. Diagnosis was confirmed by plasma acylcarnitines, urinary organic acids, enzymatic and genetic testing. For not clear genotypes due to the presence of variants of uncertain significance, in silico predictive tools have been used as well as enzymatic activity assays. Patients underwent clinical, nutritional and biochemical follow up. Results We diagnosed 30 patients with FAODs. 20 by NBS: 3 CUD, 6 SCADD, 5 MCADD, 4 VLCADD, 2 MADD. Overall incidence of FAODs diagnosed by NBS was 1:4316 newborns. No one reported complications during the follow up period. 10 patients were diagnosed clinically: 2 CUD, 2 CPT2D, 1 VLCADD, 5 MADD. Mean age at diagnosis was 29.3 years. Within this group, complications or symptoms were reported at diagnosis, but not during follow-up. 12 mutations not previously reported in literature were found, all predicted as pathogenic or likely pathogenic. Discussion and conclusions Our study highlighted the great phenotypic variability and molecular heterogeneity of FAODs and confirmed the importance of a tailored follow up and treatment. Despite the short duration of follow up, early identification by NBS prevented diseases related complications and resulted in normal growth and psycho-motor development as well., Highlights • Early identification by newborn screening prevents disease related complications. • Newborn screening is changing prevalence clinical and molecular heterogeneity of FAODs. • Genotype-phenotype correlation helps to achieve personalized follow-up and treatment. • Enzymatic assay may be pivotal in predicting phenotype and symptoms severity. • Diagnosis on clinical grounds is anyway important to change disease course.

Multiple acyl-CoA dehydrogenase deficiency, or glutaric aciduria type II, is an autosomal recessive disorder of fatty acid oxidation due to defects in electron transfer flavoprotein (ETF) encoded by ETFA and ETFB, or in electron transfer flavoprotein dehydrogenase (ETFDH) encoded by the ETFDH gene. The disease may present as a severe neonatal onset form and a mild late-onset form which is heterogeneous for the age at onset and clinical presentation. We describe two patients in their seventies, referred for a nonspecific myopathy, which resulted to manifest carriers of ETFDH gene mutation. Treatment with riboflavin and L-carnitine improved the clinical picture and the biochemical profile. This condition should be included in the differential diagnosis of myopathies even at an old age.

BCKDK is an important key regulator of branched-chain ketoacid dehydrogenase complex activity by phosphorylating and so inactivating branched-chain ketoacid dehydrogenases, the rate-limiting enzyme of the branched-chain amino acid metabolism. We identified, by whole exome-sequencing analysis, the p.His162Gln variant of the BCKDK gene in a neonate, picked up by newborn screening, with a biochemical phenotype of a mild form of maple syrup urine disease (MSUD). The same biochemical and genetic picture was present in the father. Computational analysis of the mutation was performed to better understand its role. Extensive atomistic molecular dynamics simulations showed that the described mutation leads to a conformational change of the BCKDK protein, which reduces the effect of inhibitory binding bound to the protein itself, resulting in its increased activity with subsequent inactivation of BCKDC and increased plasmatic branched-chain amino acid levels. Our study describes the first evidence of the involvement of the BCKDK gene in a mild form of MSUD. Although further data are needed to elucidate the clinical relevance of the phenotype caused by this variant, awareness of this regulatory activation of BCKDK is very important, especially in newborn screening data interpretation.

ObjectiveBreast-feeding is an unequalled way of providing optimal food for infants’ healthy growth and development and the WHO recommends that infants should be exclusively breast-fed for the first 6 months of life. For mothers who are unable to breast-feed or who decide not to, infant formulas are the safest alternative. Despite recommendations, it is possible that parents make potentially harmful nutritional choices for their children because of cultural beliefs or misinformation on infant nutrition. We describe a possible health risk of not breast-feeding, highlighting a potentially dangerous dietetic practice.Design/Setting/SubjectsWe report the case of a newborn who was fed with undiluted goat’s milk because her mother could not breast-feed and was not aware of infant formulas.ResultsThe dietary mistake was detected because of a positive expanded newborn screening result, characterized by severe hypertyrosinaemia with high methionine and phenylalanine levels, a pattern suggestive of severe liver impairment. The pattern of plasma amino acids was related to a goat’s milk diet, because of its very different composition compared with human milk and infant formula.ConclusionsOur experience demonstrates that, when breast-feeding is not possible or is not exclusive, infants may be at risk of dangerous nutritional practices, including diets with very high protein content, such as a goat’s milk diet. Families of not breast-fed infants may need appropriate advice on safe alternatives for infant nutrition to avoid the risks of inappropriate diets.

Background: Iodine supplementation during pregnancy in areas with mild-to-moderate iodine deficiency is still debated. Methods: A single-center, randomized, single-blind and placebo-controlled (3:2) trial was conducted. We enrolled 90 women before 12 weeks of gestation. From enrollment up until 8 weeks after delivery, 52 women were given an iodine supplement (225 ug/day, potassium iodide tablets) and 38 were given placebo. At recruitment (T0), in the second (T1) and third trimesters (T2), and 8 weeks after delivery (T3), we measured participants&rsquo, urinary iodine-to-creatinine ratio (UI/Creat), thyroid function parameters (thyroglobulin (Tg), TSH, FT3, and FT4), and thyroid volume (TV). The newborns&rsquo, urinary iodine concentrations were evaluated in 16 cases. Results: Median UI/Creat at recruitment was 53.3 ug/g. UI/Creat was significantly higher in supplemented women at T1 and T2. Tg levels were lower at T1 and T2 in women with UI/Creat &ge, 150 ug/g, and in the Iodine group at T2 (p = 0.02). There was a negative correlation between Tg and UI/Creat throughout the study (p = 0.03, r = &minus, 0.1268). A lower TSH level was found in the Iodine group at T3 (p = 0.001). TV increased by +&Delta, 7.43% in the Iodine group, and by +&Delta, 11.17% in the Placebo group. No differences were found between the newborns&rsquo, TSH levels on screening the two groups. Conclusion: Tg proved a good parameter for measuring iodine intake in our placebo-controlled series. Iodine supplementation did not prove harmful to pregnancy in areas of mild-to-moderate iodine deficiency, with no appreciable harmful effect on thyroid function.

Background Neonatal screening for 21 hydroxylase deficiency is designed to detect classical form of congenital adrenal hyperplasia (CAH). It is still unclear whether newborns who result false positives at neonatal screening might later develop signs of androgen excess. The aim of this study is to verify whether a slightly elevated 17-OHP at newborn screening is a predictive factor for premature pubarche. Methods We evaluated all infants born between 2001 and 2014 with premature pubarche. In case of increased bone age, they were submitted to functional tests to find out the cause of their symptoms. Their 17-OHP values at newborn screening for CAH were reconsidered. Results We identified 330 patients (269 females, 61 males) with premature pubarche. All these children had a normal 17-OHP at newborn screening with the exception of a child, born preterm and not affected by CAH. Conclusions An elevated 17-OHP at newborn screening is not a predictive factor for premature pubarche. A likely cause of increased 17-OHP level at screening is an immaturity of adrenal gland or a neonatal stress. Therefore a strict follow up of these neonates during childhood is not necessary.

Cobalamin A deficiency (cblA) is an inherited disorder of intracellular cobalamin metabolism, caused by impaired 5'-deoxy-adenosylcobalamin (AdoCbl) synthesis. Hydroxocobalamin (OHCbl) is the cornerstone of cblA treatment because vitamin B12 may completely restore AdoCbl deficiency. Parenteral administration, intravenous, subcutaneous or intramuscular, is generally required to achieve effect. Daily injections represent a problem for the parents and the caregivers, and this may lead to poor compliance and scarce adherence to the long-term treatment.Our report describes the case of a patient with cblA deficiency, diagnosed by newborn screening, positively treated with daily OHCbl administration by a subcutaneous injection port (i-port advanceTM). After the insertion of the device, we checked methylmalonic acid (MMA) levels weekly for the first month and then monthly. MMA level remained always in the normal range.To date, placement of a subcutaneous catheter to minimize the pain related to parenteral vitamin B12 punctures has been described only in a patient with deficiency of the enzyme methylmalonyl-CoA mutase (MUT). No other experiences are described in the literature.Our case shows that OHCbl administration using a subcutaneous catheter is safe and effective even in patients with cblA deficiency. The use of subcutaneous devices may reduce difficulties in providing parenteral daily injections which is the main reason discouraging physicians and families to use such an invasive treatment. Moreover, our experience may be translated to other inherited metabolic disorders, such as cobalamin C (cblC) disease, which may require daily parenteral drug administration.

ObjectiveTo evaluate the incidence of congenital hypothyroidism (CH) with delayed TSH elevation among low-birth-weight (LBW) newborns in North-Eastern Italy and to verify if they need a second or third screening.DesignAnalysis of clinical and biochemical data of newborns affected by CH with delayed TSH elevation identified by neonatal screening.MethodsData of all newborns with birth weight (BW) 4levels. All their clinical signs at diagnosis were reported.Results57.5% of LBW newborns with delayed TSH increase at neonatal screening presented a CH with delayed TSH elevation and began a treatment withl-thyroxine. The incidence of this condition in North-Eastern Italy is therefore 1:908. The remaining infants presented a subclinical hypothyroidism (21.25%) or a complete normal serum thyroid function (21.25%). These data could be drawn only from a retesting strategy of neonatal screening.ConclusionsOur report describes the incidence of CH with delayed TSH rise in North-Eastern Italy and differentiates this clinical condition from other thyroid dysfunctions of preterm or LBW newborns. The second-screening strategy for CH in neonates with BW

Summary Objectives To establish the role of new TSH receptor (TSHr) variants (P27T, E34 K, R46P, D403N, W488R and M527T) recently identified in children with congenital hypothyroidism (CH) or subclinical hypothyroidism (SH) with a thyroid gland of normal size. Design and measurements TSHr variants were obtained by mutagenesis. Wild-type (wt) and TSHr mutants were expressed in COS cells and cAMP assay, 125I-TSH binding and microchip flow cytometry analyses were performed. Results D403N and M527T mutants showed a lower cAMP response to bovine TSH (bTSH) with respect to the wtTSHr. R46P and W488R mutants did not show any response to bTSH stimulation in terms of cAMP production. The E34 K mutant showed a significantly lower cAMP response to stimulation with bTSH, while P27T had a lower cAMP response only to the highest dose of bTSH used. P27T, E34 K, D403N and M527T mutants showed a lower TSH binding capacity with respect to the wtTSHr. R46P and W488R mutants did not show any TSH binding. Conclusions E34 K, D403N, M527T, R46P and W488R TSHr variants seem to cause a functional abnormality of the receptor which is responsible for the observed phenotype. The P27T TSHr variant does not seem to play a functional role in the pathogenesis of CH and should be considered as a polymorphism.

GH replacement therapy exhibits a wide spectrum of response in terms of growth. Nevertheless, standardized doses are still given in clinical practice. In order to optimize the therapy, it is necessary to identify its markers of responsiveness. Given the presence of GH receptors in the circulating lymphocytes, accessible by means of a simple blood withdrawal, blood becomes the tissue of choice as a source of RNA for in vivo gene expression analysis. Hence, the purpose of the present paper is to develop a method of preparation of RNA from lymphocytes suitable for microarray analysis, focusing on the reduction of the blood volume withdrawal in order to perform the analysis on pediatric subjects. After lymphocyte isolation and total RNA extraction from 6 ml of blood, we carried out an amplification procedure preserving the relative abundance of each transcript. Thereafter, we hybridized the labeled amplified RNA on an oligo chip (Human 30K A, MWG-Biotech), but the unsuccessful detection of a good signal to noise ratio indicates that labeled RNA is still insufficient. Therefore, we suggest performing pools of total RNA from different subjects with similar responsiveness to the therapy. It can be speculated that, upon comparison of the obtained data with those derived from pools of controls properly responding to the therapy, specific hallmarks of the condition of low responsiveness, devoid of inter-individual variability, will be evidenced.

Decreased levels of ghrelin have been measured in growing children during puberty. No data are available for girls with central precocious puberty (CPP). Aims: To explore ghrelin changes before, during, and after GnRH analog treatment in girls with CPP. Subjects and methods: A sample of 20 Caucasian girls (8.08 ± 0.65 years of age) with CPP was recruited. Height and weight, bone age, LH, FSH, 17β estradiol (E2), and ghrelin were measured before starting treatment with GnRH analog, 18 months after therapy began and again 6 months after therapy discontinuation. Results: LH and E2 serum levels decreased significantly during treatment (2.45 ± 2.03 vs 0.67 ± 0.49 UI/l, P < 0.01 and 28.17 ± 9.7 vs 15 pmol/l, P < 0.01 respectively), returning to baseline levels after the discontinuation of therapy (4.75 ± 1.66 UI/l and 29.23 ± 6.99 pmol/l respectively). LH peaked following LHRH stimulation significantly (P < 0.01) decreased during treatment (24.45 ± 14.17 vs 1.3 ± 0.18 UI/l) and then increased after therapy discontinuation (12.58 ± 6.09, P < 0.01). Ghrelin decreased significantly (P < 0.05) during treatment (1849 ± 322 vs 1207 ± 637 pg/ml), and increased, though not significantly (P = 0.09) after therapy withdrawal (1567 ± 629 pg/ml). Conclusions: Contrary to what is expected in physiologic puberty, where ghrelin is progressively reduced, the prepubertal hormone milieau induced by GnRHa treatment in patients suffering from central precocious puberty (CPP) did not promote an increase in ghrelin circulating levels. Therefore, in CPP, ghrelin secretion seems to be independent from pubertal development per se. Concomitant estrogen suppression during treatment may play a potential role in the regulation of ghrelin secretion in these girls.

Mutations in COL1A1 and COL1A2 genes, encoding the alpha1 and alpha2 chain of type I collagen, respectively, are responsible for the vast majority of cases of osteogenesis imperfecta (OI) (95% of patients with a definite clinical diagnosis). We have investigated 22 OI patients, representing a heterogeneous phenotypic range, at the biochemical and molecular level. A causal mutation in either type I collagen gene was identified in 20 of them: no recurrent mutation was found in unrelated subjects; 15 out of 20 mutations had not been reported previously. In two patients, we could not find any causative mutation in either type I collagen gene, after extensive genomic DNA sequencing. Failure of COL1A1/COL1A2 mutation screening may be due, in a few cases, to further clinical heterogeneity, i.e. additional non-collagenous disease loci are presumably involved in OI types beyond the traditional Sillence's classification.

We report clinical findings, bone mineral density (BMD) and bone biopsy data in ten children with features of classic idiopathic juvenile osteoporosis (IJO). We also screened the patients for mutations in LRP5 and LRP6. We found low BMD in the lumbar spine, the hip and distal radius. In the spine and distal radius, the reduction in BMD was more marked in the trabecular compartment. Biopsy confirmed that the trabecular compartment is more severely involved with reduction in bone formation and increase in bone resorption. No mutations in LRP5 and LRP6 could be identified. IJO is likely to be a heterogeneous bone disorder, and next-generation genomic sequencing studies may help reveal causative genes.

Summary Objective TSH resistance ranges from overt nonautoimmune hypothyroidism to subclinical hypothyroidism, defined as mild hyperthyrotrophinaemia but a euthyroid state clinically. To date, 23 inactivating mutations of the TSH receptor (TSHR) gene have been proven responsible for the clinical condition, but an absence of mutations in the TSHR gene has been reported for several cases of TSH resistance as well. In this paper, we aimed to investigate the actual role of the TSHR gene in the development of both subclinical and congenital hypothyroidism. Patients 14 hypothyroid newborns, 116 subclinical hypothyroid subjects and 120 healthy controls. Measurements Through denaturing high performance liquid chromatography (DHPLC), we screened for mutations the TSHR gene (the proximal promoter, the exons and their flanking regions), and evaluated the association between serum TSH and functionally characterized alleles identified. Results In the hypothyroid patients, one patient was heterozygous for a new missense variation, E34K, whereas two others patients were either homozygous or heterozygous for the P162A substitution. In the subclinical hypothyroid subjects, we detected only heterozygous substitutions: they are mostly new (123–124insTGCA, P27T, R46P, 555–561delTATTCTT, D403N, W488R, M527T), while six correspond to already published mutations (P162A, R109Q, L252P and three C41S). We only focused on those mutations that had been functionally characterized in vitro, and in whom serum TSH was available from family members. Conclusions A single grossly mutated allele (such as C41S or 555–561del) invariably leads to a condition of subclinical hypothyroidism, whereas in case of heterozygous carriers of mutations partially affecting the receptor function (such as P162A or L252P), a remarkable variable expressivity was detected among individuals belonging to different generations.

A collaboration among 157 newborn screening programs in 47 countries has lead to the creation of a database of 705,333 discrete analyte concentrations from 11,462 cases affected with 57 metabolic disorders, and from 631 heterozygotes for 12 conditions. This evidence was first applied to establish disease ranges for amino acids and acylcarnitines, and clinically validate 114 cutoff target ranges. Objective: To improve quality and performance with an evidence-based approach, multivariate pattern recognition software has been developed to aid in the interpretation of complex analyte profiles. The software generates tools that convert multiple clinically significant results into a single numerical score based on overlap between normal and disease ranges, penetration within the disease range, differences between specific conditions, and weighted correction factors. Design: Eighty-five on-line tools target either a single condition or the differential diagnosis between two or more conditions. Scores are expressed as a numerical value and as the percentile rank among all cases with the condition chosen as primary target, and are compared to interpretation guidelines. Tools are updated automatically after any new data submission (2009- 2011: 5.2 new cases added per day on average). Main outcome measures: Retrospective evaluation of past cases suggest that these tools could have avoided at least half of 277 false positive outcomes caused by carrier status for fatty acid oxidation disorders, and could have prevented 88% of false negative events caused by cutoff 7 values set inappropriately. In Minnesota, their prospective application has been a major contributing factor to the sustained achievement of a false positive rate below 0.1% and a positive predictive value above 60%. Conclusions: Application of this computational approach to raw data could make cutoff values for single analytes effectively obsolete. This paradigm is not limited to newborn screening and is applicable to the interpretation of diverse multi-analyte profiles utilized in laboratory medicine. Abstract word

Background: Lysinuric protein intolerance (LPI; MIM# 222700) is a rare metabolic disorder caused by a defective cationic amino acids (CAA) membrane transport leading to decreased circulating plasma CAA levels and resulting in dysfunction of the urea cycle. Short stature is commonly observed in children with LPI and has been associated with protein malnutrition. A correlation between LPI and growth hormone deficiency (GHD) has also been postulated because of the known interaction between the AA arginine, ornithine, and lysine and growth hormone (GH) secretion. Our report describes a case of GHD in an LPI patient, who has not presented a significant increase in growth velocity with recombinant-human GH (rhGH) therapy, suggesting some possible pathogenic mechanisms of growth failure.

Background: Congenital hypothyroidism is often secondary to thyroid dysgenesis, including thyroid agenesis, hypoplasia, ectopic thyroid tissue or cysts. Loss of function mutations in TSHR, PAX8, NKX2.1, NKX2.5 and FOXE1 genes are responsible for some forms of inherited congenital hypothyroidism, with or without hypoplastic thyroid. The aim of this study was to analyse the PAX8 gene sequence in several members of the same family in order to understand whether the variable phenotypic expression, ranging from congenital hypothyroidism with thyroid hypoplasia to mild subclinical hypothyroidism, could be associated to the genetic variant in the PAX8 gene, detected in the proband. Methods: We screened a hypothyroid child with thyroid hypoplasia for mutations in PAX8, TSHR, NKX2.1, NKX2.5 and FOXE1 genes. We studied the inheritance of the new variant R133W detected in the PAX8 gene in the proband’s family, and we looked for the same substitution in 115 Caucasian European subjects and in 26 hypothyroid children. Functional studies were performed to assess the in vitro effect of the newly identified PAX8 gene variant. Results: A new heterozygous nucleotide substitution was detected in the PAX8 DNA-binding motif (c.397C/T, R133W) in the proband, affected by congenital hypothyroidism with thyroid hypoplasia, in his older sister, displaying a subclinical hypothyroidism associated with thyroid hypoplasia and thyroid nodules, in his father, affected by hypothyroidism with thyroid hypoplasia and thyroid nodules, and his first cousin as well, who revealed only a subclinical hypothyroidism. Functional studies of R133W-PAX8 in the HEK293 cells showed activation of the TG promoter comparable to the wild-type PAX8. Conclusions: In vitro data do not prove that R133W-PAX8 is directly involved in the development of the thyroid phenotypes reported for family members carrying the substitution. However, it is reasonable to conceive that, in the cases of transcriptions factors, such as Pax8, which establish several interactions in different protein complexes, genetic variants could have an impact in vivo.

Hair analysis is often used in forensic toxicology to study, retrospectively, chronic exposure of individuals to drugs, and consequently newborn hair may become an ideal sample to study intrauterine exposure to xenobiotics as well as to endogenous compounds. As a tool to investigate a supposed maternal thyroxine (T4) supply to the congenital hypothyroid fetus, we devised to use the analysis of T4 extracted from newborn hair. In the present paper, the analytical method based on T4 extraction from hair followed by a radioimmunoassay is described. To verify the nature of the T4-like immunoreactive material present in newborn hair, it was further studied by HPLC fractionation with radioimmunoassay of the eluted fractions. On the basis of a clear correspondence between retention times of T4 standard and T4-immunoreactive compound extracted from hair, we assigned this immunoreactive material to T4. Then, we determined T4 hair concentrations in 19 control newborns at birth and 12 congenital hypothyroid infants at 22 days of life. Values obtained from hypothyroid infants (31.47+/-8.8 pg/mg(hair), mean+/-S.D.) were not significantly lower than those obtained from healthy newborns at birth (36.10+/-13.2 pg/mg(hair)). Such results are in agreement with the hypothesis of a maternal supply of thyroxine to the fetus through placental crossing.

The human HADH gene encodes the short-chain-L-3-hydroxyacyl-CoA dehydrogenase, the enzyme which catalyzes the third step of the β-oxidation of the fatty acids in the mitochondrial matrix. Loss-of-function mutations in the HADH gene lead to short-chain-L-3-hydroxyacyl-CoA dehydrogenase deficiency, an autosomal recessive genetic defect of unknown prevalence with a wide spectrum of phenotypic variability. As in other metabolic diseases, the diagnostic relevance of the biochemical evaluations, plasma acylcarnitines, and urinary organic acids, are crucially dependent on the clinical conditions of the patient during specimen collection.This paper describes the eighth patient carrying a HADH gene mutation, a new homozygous deletion c.565delG leading to an early stop codon (p.V116Wfs124X), in an infant with hyperinsulininemic hypoglycemia, displaying abnormal patterns of plasma acylcarnitines and urinary organic acids. We conclude that, when the residual catalytic activity of the mutated enzyme is seriously reduced, the biochemical hallmarks of the disease, namely plasma 3-hydroxybutyrylcarnitine and urinary 3-hydroxyglutaric acid, are invariably present.

In this prospective study, we analyzed 30 male infants with increased neonatal 17-hydroxyprogesterone (17OH-P) (patients) and for comparison 52 age-matched healthy babies (control subjects) with the aim of investigating the hypothalamic-pituitary-testis axis in the first 6 months of life. Although T, FSH, and LH levels were not significantly different in patients and control subjects, inhibin B was higher in patients than in control subjects. Therefore we suggest a clinical follow-up of these babies during childhood and puberty to verify the evolution of their condition.

Objective To investigate the prepubertal serum inhibin B levels in monorchid boys with compensatory testicular hypertrophy (CTH) and in surgically treated cryptorchid boys with normal testicular volume. Design Prospective study. Setting Pediatric Division, University of Verona, Italy. Patient(s) Thirty-two prepubertal boys: 11 monorchids with CTH and 21 cryptorchids. For comparison we analyzed 15 healthy boys. Main Outcome Measure(s) All patients underwent a GnRH agonist test. Inhibin B measurement was performed at basal time. Result(s) There was a significant difference in mean testicular volume between monorchid and cryptorchid or healthy children, with boys with CTH evidencing larger testicle volume. There was no significant difference in inhibin B levels between boys with CTH and cryptorchid, but the levels were significantly lower in both groups than in the control group. Boys with CTH had significantly higher serum levels of basal FSH than children with cryptorchid. The FSH peak response to GnRH agonist stimulation was significantly higher in boys with CTH than in those with cryptorchid. Conclusion(s) Monorchid infants with CTH showed low inhibin B and high FSH levels. Our finding may confirm the hypothesis that CTH is unable to prevent testicular insufficiency in adulthood. We suggest an early hormonal evaluation of boys with CTH at puberty, together with early sperm analysis.

After a couple of decades from the institution of nationwide congenital hypothyroidism (CH) newborn screening program, the first generation properly treated is now displaying normal reproductive rate and the causative molecular defects are spreading from one generation to the next. In the present study we propose a method of detection of mutations in the thyrotropin receptor (TSHR) and in the paired box 8 (PAX8) genes that have been proved to be responsible for some forms of CH. The method, carried out by means of denaturing high-performance liquid chromatography (DHPLC) followed by direct sequencing, takes advantage of the CH newborn screening procedure, because genomic DNA for the analysis is extracted from the same blood spot collected for recall confirmation. Among 16 hypothyroid newborns with thyroid hypoplasia born between January 1999 and April 2005 in northeastern Italy, three heterozygous causative mutations in the TSHR gene were evidenced, whereas the analysis of the PAX8 gene revealed an unknown heterozygous substitution that could interfere with the start of transcription.

Subclinical hypothyroidism is defined as a serum TSH level above the statistically set reference range, associated to normal free thyroid hormone concentrations. Genetic and environmental factors contribute to the inter- and intra-individual biological variations of TSH levels, sometimes leading to uncertainty of treatment in the clinical practice, especially when moderate elevations above the upper limit of the reference range are considered (5< TSH

The aim of this work was to perform genetic analysis on 18 different blood-spot samples collected from neonates detected as hyperphenylalaninemic by Northeastern Italian screening program. DNA was extracted from blood-spots. Exons/introns of PAH gene were amplified by polymerase chain reaction (PCR), and PCR products were purified and sequenced with both forward and reverse primers. The most frequent mutations were IVS12nt1ga (16.7%) and R408W, P281L and L48S (all together 11.1%). As expected, compound heterozygosity was the usual finding; homozygosity was found only in two patients with R158Q and IVS2nt5gc mutations. The V230I mutation was reported for the first time in Italy. We found six previously described polymorphisms (V245V, IVS4nt47ct, IVS2nt19tc, IVS3nt-22ct, IVS5nt-54ag, and E280Q280). To our knowledge, four genotypes were not previously described: R158Q/V230I present in one patient with classical PKU; and L48S/R408Q, A403V/IVS2nt-13tg, and G272X/V230I present in patients showing HPA phenotype. Most of the mutations were located in the exons 12 and 7 and in exon/intron 2 (83.3% detection of total mutations in PKU or HPA patients of Northeastern Italy). From a practical viewpoint, the genetic analysis of blood-spots collected on Guthrie cards for neonatal screening for PKU could be a simple method to establish the genotype of neonates. Consequently, the genotype/phenotype correlation could lead to a more accurate diagnosis and prognosis for families.

Aims: To evaluate the incidence of congenital adrenal hyperplasia (CAH) in the Northern Italian population and the efficiency of the North-Eastern Italy screening program. To adjust cut-off levels for 17-hydroxyprogesterone (17-OHP) in relation to gestational age and birth weight, comparing the benefits in terms of reduction of recall rates with the two approaches and ultimately choosing the better of the two. Subjects and Methods: Since September 2001, blood samples from neonates born in North-Eastern Italy have been screened with a fluoroimmunoassay method for 17-OHP determination (DELFIA). A preliminary cut-off level of ≧30 nmol/l was set both for term and preterm newborns. The values of 17-OHP were analysed usingstatistical methods in relation to gestational age and birth weight in order to modify the cut-off on the basis of our data. Results: After 33 months of screening we screened 128,282 newborns and detected 6 affected babies. During the first 8 months of screening among the recalled babies, 89.6 and 78.1% were preterm and low-birth-weight newborns, respectively, with a recall rate of 2.59% for premature neonates and of 4.94% for babies with birth weights Conclusion: After 33 months of screening for CAH in North-Eastern Italy, we report an incidence of 1:21,380. In 5 out of 6 affected babies, the diagnosis was established only after a positive screening test, which prevented a severe salt-wasting crisis in these babies. The cut-off level related to gestational age led to a significant reduction in the number of false-positives among preterm babies.We therefore intend to continue with the screening program for CAH in North-Eastern Italy, keeping a gestational-age-related cut-off in the hope that our data may encourage a national screening program for CAH.

Purpose: To improve quality of newborn screening by tandem mass spectrometry with a novel approach made possible by the collaboration of 154 laboratories in 49 countries. Methods: A database of 767,464 results from 12,721 cases affected with 60 conditions was used to build multivariate pattern recognition software that generates tools integrating multiple clinically significant results into a single score. This score is determined by the overlap between normal and disease ranges, penetration within the disease range, differences between conditions, and weighted correction factors. Results: Ninety tools target either a single condition or the differential diagnosis between multiple conditions. Scores are expressed as the percentile rank among all cases with the same condition and are compared to interpretation guidelines. Retrospective evaluation of past cases suggests that these tools could have avoided at least half of 279 false-positive outcomes caused by carrier status for fatty-acid oxidation disorders and could have prevented 88% of known false-negative events. Conclusion: Application of this computational approach to raw data is independent from single analyte cutoff values. In Minnesota, the tools have been a major contributing factor to the sustained achievement of a false-positive rate below 0.1% and a positive predictive value above 60%. © 2012 American College of Medical Genetics and Genomics.