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Congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21OHD-CAH) is an autosomal recessive disorder affecting steroidogenesis, due to mutations in CYP21A2 (6p21.3). 21OHD-CAH neonatal screening is based on 17-hydroxyprogesterone (17OHP) serum levels, showing high type I error rate and low sensitivity to mild CAH forms. Here, we used an epidemiological approach, which estimates the allelic frequency (q) of an autosomal recessive disorder using the proportion of homozygous patients, the mutational spectrum and the inbreeding coefficient in a sample of affected individuals. We applied this approach to 2 independent Italian cohorts of patients with both clinical and molecular diagnosis of 21OHD-CAH from mainland Italy (N = 240) and Sardinia (N = 53). We inferred q estimates of 2.87% and 1.83%, corresponding to a prevalence of 1/1214 and 1/2986, respectively. CYP21A2 mutational spectra were quite discrepant between the 2 cohorts, with V281L representing 74% of all the mutations detected in Sardinia vs 37% in mainland Italy. These findings provide an updated fine-grained picture of 21OHD-CAH genetic epidemiology in Italy and suggest the need for a screening approach suitable to the detection of the largest number of clinically significant forms of CAH.

Background 11ß-hydroxylase deficiency (11-OHD) is the second most common cause of congenital adrenal hyperplasia and it is caused by CYP11B1 gene mutations. It is characterized by genital ambiguity in affected girls and precocious pseudopuberty in both sexes. Hypertension can occur in about two thirds of patients. The non-classic form of 11OHD is manifested by signs of androgen excess during childhood. Objective and hypothesis To characterize the CYP11B1 gene alterations of these patients. Population and/or methods Herein we report 7 patients with classical and 4 with non-classical 11-OHD, selected for high hormonal levels of 11-deoxycortisol measured by ID-LC-MS/MS or for negative analysis of the CYP21A2 gene. The entire CYP11B1 gene was sequenced and four mutations were functionally characterized (paper submitted). Results 8 novel putative mutations were identified: 5 missense mutations (p.R143W, p.E310K, p.V316M, p.R332Q, p.Q337P), 2 splicing mutations (g.-1 IVS6 G>C, g.+148 IVS5 C>G) and one non sense mutation (p.R384X). Furthermore 5 already reported mutations were identified (p.L299P, p.A306V, p.T318R, p.Q356X, exon 7 g.ins4566-4567 GA). By in vitro studies, the p.E310K and the p.A306V resulted to be severe mutations causing classical 11-OHD; instead the p.R143W and the p.R332Q, with a higher residual activity, resulted milder mutations. In a pair of brothers with non classical 11-OHD due to the same CYP11B1 mutations, the boy showed an earlier manifestation of the symptoms, that may be due to an additional intron 2 splice site mutation in the CYP21A2 gene. Precise conclusions The detection of patients with non classic phenotypes underscore the importance to screen patients with a phenotype comparable to non classic 21OHD for mutations in the CYP11B1 gene in case of a negative analysis of the CYP21A2 gene. As CYP11B1 mutations are most often individual for a family, the in vitro analysis of novel mutations is essential for clinical and genetic counselling.

Background Non classical congenital adrenal hyperplasia (NC-CAH) due to 21-hydroxylase deficiency (21OHD) is one of the most common inherited defects of steroidogenesis. It is caused by mutations in the CYP21A2 gene that can be grouped in three categories according to the predicted level of enzymatic activity: A (complete loss of activity), B (severe) and C (mild). Objective and hypothesis The genetic characterization of suspected NC-CAH subjects with stimulated 17OHP values ranging from 800 and 15.000 ng/dl in order to: investigate the genotype and the contribution of the 1st and 2nd allele on the 17OHP levels; review the pathologic hormonal levels. Population and/or methods 287 subjects were investigated by complete sequencing of the CYP21A2 gene and by MLPA using the MRC-Holland P050B2 kit in order to identify all possible mutations including variation of the copy number. Results The 71.8% of the subjects showed both the alleles affected (37.4 % are compound heterozygous C/C, the 15.5 % are C/B and the 43.7 % are C/A), the 21.2 % resulted heterozygous and 7% normal. Among the group C/C the 50.6 % of the subjects are homozygous for V281L mutation. Taking into account the different mutations present in the 1st allele we found that the levels of 17OHP both basally and stimulated were progressively and significantly higher than the other group when the characterizing mutations were P482S, 3’UTR 13 G>A, P453S, V281L or P30L, respectively. Also the 2nd allele showed an influence on basal and stimulated 17OHP if the 1st allele is setted (i.e. V281L mutation). The V281L mutation is more frequent in the South Italian patients, instead the 3’UTR *13 G>A and the genic deletion are more frequent in North Italian patients. Precise conclusions Among affected subjects, the 94% showed 17OHP stimulated values >2000 ng/dl and only 1% values

All'esordio dell'apprendimento della matematica in prima elementare in italia si riscontra una contraddizionre lamapante. Da un lato l'allievo inizia la scuola elementare dopo aver frequentato la scuola dell'infanzia nella quale ha incontrato i numeri relativamente al campo di esperienza costitutito da spazio, ordine e misura. I bambini sanno contare, eseguire addizioni e sottrazioni, mettere in relazione giorno della settimana e numero del mese adottando strtegie risolutive complesse. I bambini incontrano nella loro esperienza quotidiana numeri con più cifre, e li usano mettendo in gioco strategie complesse. Gli allievi di 5 anni si orientano rispetto a questioni numeriche che coinvolgono numeri a più cifre. Dall'altro però, è prassi consolidata degli insegnanti che si inizi a fare matematica con numeri di una cifra legati a situazioni banali e poco sognificative rispetto all'esperienza vissuta del bambino. Si lavora in siutazioni ingnue che sottostimano le potenzialità dell'allievo, dimenticando gli indubbi vantaggi che hanno approcci più complessi e significativi nella costruzione del numero legati ai "campi di esperienza". Il capitolo del testo mostra i risultati di una ricecra e sperimentazione, condotta da un gruppo di insegnanti e ricercatori dell'RSDDM di Bologna sotto la guida di Bruno D'Amore. La ricerca ha coinvolto contemporaneamente classi della scuola dell'infanzia e della scuola elementare che hanno lavorato in situazioni di apprendimento che richiedevano approcci complessi e significativi nella costruzione del numero.

The screening of the usually tested CYP21 gene alterations (the large gene deletion/conversion and the P30L, IVS2-13A/C>G, Ex 3 D8nt, Ex6 cluster, I172N, V281L, 1766-1767insT, Q318X, R356Q, P453S) by means of Southern blotting and Allele-Specific PCR in an Italian population of 284 patients with 21-hydroxylase deficiency led us to characterize 461 of the total 506 different alleles (91.1%), while 45 (8,9 %) remained uncharacterized. Some incongruent genotype/phenotype correlation was also observed. In order to reduce the number of uncharacterized alleles and possibly to identify additional alterations in some patients’ alleles, we reanalysed the entire population (128 classic and 156 nonclassic forms selected for stimulated 17-HP level>10 ng/ml) by complete sequencing of the CYP21 gene, promoter included. Identified mutations were verified in the available parents to confirm allele segregation. Result a: of the 461 characterized alleles, 10 present an additional/different known mutation from that previously identified (2.1% false positive/negative results); 7 showed a conversion extending from the promoter to the P30L mutation (3 cases) or the IVS2 (4 cases). Result b: of the 34 uncharacterized alleles, 16 showed rare mutations (1 M283V, 1 R316X, 4 R341P, 4 R356Q, 1 R426H, 4 P482S, 1 R483P); 7 showed as far as we know unique mutations, 2 (W19X, L480Xfs) with an obvious implication in the phenotype, and 5 affecting residues L142, I171, R341, V358, L446 currently being studied; 11 remained uncharacterized. A total of 43 (23 of pseudogene origin) SNPs, 15 in the promoter, 3 in the 3’ UTR and 25 in introns were identified: further studies will verify their frequency in controls and the possible implication in the phenotypes. The high frequence of complex, rare or unique alleles in the Italian population underlines the importance to routinely analyse the CY21 gene by complete sequencing to avoid false/incomplete results, identify new mutations or sequence variations and to improve the genotype/phenotype correlation, genetic counselling and treatment.

Alkali-activated materials are a promising type of binder candidate as a substitute to Portland cement. Fly ashes can be used as binder precursors giving higher environmental benefits. In the present research, fly ashes (Type F) containing different amounts of unburned carbonaceous matter have been used to formulate mortars. Serious problems concerning the workability in the fresh state have been found when high carbon content are reached. An attempt to avoid the preliminary treatments used to eliminate the unburned matter is carried out by exploiting different mix-design receipts obtained by changing the water/binder ratio, the ratio of the alkaline activators and using different types of superplasticizer additives. Data so far collected underline that a high amount of unburned carbonaceous matter can not only compromise the mechanical properties of the materials, but also the rheological ones and underline the necessity to develop ad hoc additives for this type of binders.

Congenital adrenal hyperplasia includes autosomal recessive conditions that affect the adrenal cortex steroidogenic enzymes (cholesterol side-chain cleavage enzyme; 3β-hydroxysteroid dehydrogenase; 17α-hydroxylase/17,20 lyase; P450 oxidoreductase; 21-hydroxylase; and 11β-hydroxylase) and proteins (steroidogenic acute regulatory protein). These are located within the three major pathways of the steroidogenic apparatus involved in the production of mineralocorticoids, glucocorticoids, and androgens. Many countries have introduced newborn screening program (NSP) based on 17-OH-progesterone (17-OHP) immunoassays on dried blood spots, which enable faster diagnosis and treatment of the most severe forms of 21-hydroxylase deficiency (21-OHD). However, in several others, the use of this diagnostic tool has not yet been implemented and clinical diagnosis remains challenging, especially for males. Furthermore, less severe classic forms of 21-OHD and other rarer types of CAHs are not identified by NSP. The aim of this mini review is to highlight both the main clinical characteristics and therapeutic options of these conditions, which may be useful for a differential diagnosis in the neonatal period, while contributing to the biochemical evolution taking place in the steroidogenic field. Currently, chromatographic techniques coupled with tandem mass spectrometry are gaining attention due to an increase in the reliability of the test results of NPS for detecting 21-OHD. Furthermore, the possibility of identifying CAH patients that are not affected by 21-OHD but presenting elevated levels of 17-OHP by NSP and the opportunity to include the recently investigated 11-oxygenated androgens in the steroid profiles are promising tools for a more precise diagnosis and monitoring of some of these conditions.

We report the case of 2 sisters (46,XX) born from consanguineous Moroccan parents. Both sisters had normal female genitalia, but within 2 weeks after birth, they presented with a severe salt-wasting crisis. Hormonal investigations suggested the diagnosis of congenital adrenal hyperplasia, which was confirmed by subsequent molecular analysis to be caused by 3β-hydroxysteroid dehydrogenase type 2 deficiency. Here, we discuss the main features like onset, possible complications, genetics, and replacement therapy of this rare disease.

Objective: We aimed at defining the most effective routine immunoassay- or liquid chromatography-tandem mass spectrometry (LC–MS/MS)-determined steroid biomarkers for identifying non-classic adrenal hyperplasia due to 21-hydroxylase deficiency (21-NCAH) in a PCOS-like population before genotyping. Methods: Seventy PCOS-like patients in reproductive age with immunoassay-determined follicular 17OH-progesterone (17OHP) ≥ 2.00 ng/mL underwent CYP21A2 gene analysis and 1–24ACTH test. Serum steroids were measured by immunoassays at baseline and 60 min after ACTH stimulation; basal steroid profile was measured by LC–MS/MS. Results: Genotyping revealed 23 21-NCAH, 15 single allele heterozygous CYP21A2 mutations (21-HTZ) and 32 PCOS patients displaying similar clinical and metabolic features. Immunoassays revealed higher baseline 17OHP and testosterone, and after ACTH stimulation, higher 17OHP (17OHP60) and lower cortisol, whereas LC–MS/MS revealed higher 17OHP (17OHPLC-MS/MS), progesterone and 21-deoxycortisol and lower corticosterone in 21-NCAH compared with both 21-HTZ and PCOS patients. Steroid thresholds best discriminating 21-NCAH from 21-HTZ and PCOS were estimated, and their diagnostic accuracy in identifying 21-NCAH from PCOS was established by ROC analysis. The highest accuracy was observed for 21-deoxycortisol ≥ 0.087 ng/mL, showing 100% sensitivity, while the combination of 17OHPLC-MS/MS ≥ 1.79 ng/mL and corticosterone ≤ 8.76 ng/mL, as well as the combination of ACTH-stimulated 17OHP ≥ 6.77 ng/mL and cortisol ≤ 240 ng/mL by immunoassay, showed 100% specificity. Conclusions: LC–MS/MS measurement of basal follicular 21-deoxycortisol, 17OHP and corticosterone seems the most convenient method for diagnosing 21-NCAH in a population of PCOS with a positive first level screening, providing high accuracy and reducing the need for ACTH stimulation test.

Thyroid-stimulating hormone receptor (TSHR) loss-of-function (LOF) mutations lead to a wide spectrum of phenotypes, ranging from severe congenital hypothyroidism (CH) to mild euthyroid hyperthyrotropinemia. The degree of TSH resistance depends on the severity of the impairment of the receptor function caused by the mutation and on the number of mutated alleles In this review data about genotype-phenotype correlation and criteria for clinical work-up will be presented and discussed. Complete TSH resistance due to biallelic LOF TSHR mutations must be suspected in all patients with severe not syndromic CH and severe thyroid hypoplasia diagnosed at birth by neonatal screening. Partial forms of TSH resistance show a more heterogeneous hormonal and clinical pattern . In these cases TSH serum levels are above the upper limit of normal range for the age but with a very variable pattern, free thyroxine (T4) concentrations are within the normal range and thyroid size can be normal or hypoplastic at ultrasound scan. An early substitutive treatment with L-T4 must be mandatory in all patients with severe CH due to complete uncompensated TSH resistance diagnosed at birth by neonatal screening. The usefulness of substitutive treatment appears much more controversial in patients with subclinical hypothyroidism due to partial TSH resistance in whom the increased TSH concentration should be able to compensate the mild functional impairment of the mutant receptor. Together with standard criteria we recommend also an accurate clinical work-up to select patients who are candidates for a LOF TSHR mutation. Conflict of interest:None declared.

Purpose: 11β-Hydroxylase deficiency (11OHD) represents the second most common cause of congenital adrenal hyperplasia. It is caused by mutations in the CYP11B1 gene localized about 40 kb from the CYP11B2 gene with which it shares a homology of 95 %. The asymmetric recombination of these two genes is involved both in 11OHD and in glucocorticoid-remediable aldosteronism (GRA). Our objective was to set up an easy and rapid method to detect these hybrid genes and other kinds of deletions, to improve the molecular diagnosis of 11OHD. Methods: A set of 8 specific probes for both the CYP11B1 and the CYP11B2 genes to be used for multiplex ligation-dependent probe amplification (MLPA) analysis was designed to detect rearrangements of these genes. Results: The method developed was tested on 15 healthy controls and was proved to be specific and reliable; it led us to identify a novel chimeric CYP11B2/CYP11B1 gene in one patient that carried the known A306V mutation on the other allele. Specific amplification and sequencing of the hybrid gene confirmed the breakpoint localization in the second intron. Conclusions: The MLPA kit developed enables the detection of deletions, duplications or chimeric genes and represents an optimal supplement to DNA sequence analysis in patients with 11OHD. In addition, it can also be used to show the presence of the opposite chimaera associated with GRA.

Congenital adrenal hyperplasia (CAH) is most commonly due to 21-hydroxylase deficiency and presents with a wide spectrum of clinical manifestations, from prenatal virilization and salt-wasting in the neonatal period to precocious pubarche and late-onset hyperandrogenic symptoms during adulthood. A limited number of mutations account for the majority of all mutated alleles, but a growing number of rare mutations are responsible for the disease in some patients. By sequence analysis of the CYP21A2 gene, we identified two novel (I171N and L446P) and two rare (R341P and R426H) mutations in seven Italian patients with CAH. One of the patients was diagnosed with mild non-classical CAH and was found to be a compound heterozygote (I171N/V281L), while all other patients showed severe phenotypes with latent or manifest salt-wasting. The residual activities measured after expression of the four mutant enzymes in COS-1 cells were all below 1% towards both natural substrates (17-OH-progesterone and progesterone) compared with the wild-type protein. All four mutations are, thus, associated with severe enzyme deficiency and are predicted to cause classic CAH if found in trans with other mutations causing severe enzyme deficiency.

In a retrospective study we evaluated long-term growth, pubertal developmental patterns to final height (FH), and medication in 55 patients (35 females) affected by 21-hydroxylase deficiency. The patients were classified into 3 groups according to predicted mutation severity: group A (11 women and 9 men), homozygous or compound heterozygous for null or In2 splice mutations [residual enzymatic activity (RA), 30%) or compound heterozygous with any of the group A, B, or C mutations. Three patients showed unclassifiable genotypes. FH was similar in the female groups, whereas male patients in group B were shorter than males in groups A and C. Fifty-five percent of patients in group A, 33% in group B, and 40% in group C reached an FH within 0.5 sd of target height. Four of the 7 patients diagnosed via neonatal screening achieved an FH equal to or above the target height. In the entire group, early diagnosis (

Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine disease. Steroid 11b-hydroxylase deficiency (11b-OHD) is the second most common form of CAH. The aim of the study was to study the functional consequences of three novel and one previously described CYP11B1 gene mutations (p.(Arg143Trp), p.(Ala306Val), p.(Glu310Lys) and p.(Arg332Gln)) detected in patients suffering from classical and non-classical 11b-OHD. Functional analyses were performed by using a HEK293 cell in vitro expression system comparing wild type (WT) with mutant 11b-hydroxylase activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of the protein. Two mutations (p.(Ala306Val) and p.(Glu310Lys)) detected in patients with classical 11b-OHD showed a nearly complete loss of 11b-hydroxylase activity. The mutations p.(Arg143Trp) and p.(Arg332Gln) detected in patients with non-classical 11b-OHD showed a partial functional impairment with approximately 8% and 6% of WT activity, respectively. Functional mutation analysis allows the classification of novel CYP11B1 mutations as causes of classical and non-classical 11b-OHD. The detection of patients with non-classical phenotypes underscores the importance to screen patients with a phenotype comparable to non-classical 21-hydroxylase deficiency for mutations in the CYP11B1 gene in case of a negative analysis of the CYP21A2 gene. As CYP11B1 mutations are most often individual for a family, the in vitro analysis of novel mutations is essential for clinical and genetic counselling. Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine disease. Steroid 11β-hydroxylase deficiency (11β-OHD) is the second most common form of CAH. The aim of the study was to study the functional consequences of three novel and one previously described CYP11B1 gene mutations (p.(Arg143Trp), p.(Ala306Val), p.(Glu310Lys) and p.(Arg332Gln)) detected in patients suffering from classical and non-classical 11β-OHD. Functional analyses were performed by using a HEK293 cell in vitro expression system comparing wild type (WT) with mutant 11β-hydroxylase activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of the protein. Two mutations (p.(Ala306Val) and p.(Glu310Lys)) detected in patients with classical 11β-OHD showed a nearly complete loss of 11β-hydroxylase activity. The mutations p.(Arg143Trp) and p.(Arg332Gln) detected in patients with non-classical 11β-OHD showed a partial functional impairment with approximately 8% and 6% of WT activity, respectively. Functional mutation analysis allows the classification of novel CYP11B1 mutations as causes of classical and non-classical 11β-OHD. The detection of patients with non-classical phenotypes underscores the importance to screen patients with a phenotype comparable to non-classical 21-hydroxylase deficiency for mutations in the CYP11B1 gene in case of a negative analysis of the CYP21A2 gene. As CYP11B1 mutations are most often individual for a family, the in vitro analysis of novel mutations is essential for clinical and genetic counselling. © 2014 Macmillan Publishers Limited All rights reserved.

Background Congenital adrenal hyperplasia is mainly caused by the deficiency of the 21-hydroxylase enzyme coded by the CYP21A2 gene. However, some alleles in the nonclassical form (NC-CAH) remain without identified mutations, suggesting the involvement of regulatory regions. Aim Our objective was to study an allele carrying the variant *13 G>A in the 3’UTR of the CYP21A2 gene identified in some patients with a mild form of NC-CAH in order to verify the possible implication of this variation with the phenotype observed. Subjects and Methods Among all the subjects in whom the CYP21A2 gene was analysed, 14 patients and 7 relatives heterozygous or homozygous for the *13 G>A substitution in 3’UTR were selected. Sequencing of DNA, genotyping, multiplex ligation-dependent probe amplification (MLPA), in-vitro studies and bioinformatic analysis were performed. Results The haplotype of the *13 G>A allele was identical in all the subjects with a monomodular structure composed by one C4A gene and one CYP21A2 gene without a second module with the CYP21A1P pseudogene. No other concomitant mutations were found in the region extending from 3 Kb in the promoter and encompassing the polyadenylation signal. Both bioinformatic analysis and in vitro studies predicted an alteration of the RNA folding and expression, but no miRNA target sequences were found in this region. Conclusions The identification of a substitution in the 3’UTR of the gene associated with a mild form of NC-CAH suggests the importance of analysing the CYP21A2 untranslated regions to better characterize and treat this subgroup of patients.

Background Congenital adrenal hyperplasia is caused mostly by 21-hydroxylase deficiency (>90%) and 11ß-hydroxylase deficiency (5–8%). Both enzymes are required for cortisol synthesis and the non classical (NC) phenotype of both deficiencies is characterized by hyper-androgenic manifestations in childhood/adolescence. Objectives To complete the characterization of a NC case in which the identified CYP21A2 mutations do not mach completely the phenotype by analysis of the CYP11B1 gene. Population and/or methods Case: female that present: precocious puberty at 5,5 yrs; at 6,7 yrs advanced bone age (10 yrs), hypertrophic clitoris and the following hormonal data: 17-OHP 8200/14600 ng/dl, ACTH 66 pg/ml; T 0.98 ng/ml; Cortisol 190/170 ng/ml; D4-A 315/377 ng/dl. A therapy with hydrocortisone (5 mg x3/die) has been started. A first clinical diagnosis of NC-21OHD was made. Genetic analysis of CYP21A2 and CYP11B1 genes was performed as previously described. Results The sequencing analysis of CYP21A2 gene revealed the presence of 3 mutations: Q318X (null), A391T (mild) and *13 G>A 3’UTR (very mild) all carried by the maternal allele. Complete sequencing of the paternal CYP21A2 gene do not show any alterations. The MLPA analysis of the family revealed a normal arrangement of the locus, therefore all the 3 maternal mutations lie on a unique gene. In order to identify other possible molecular causes, we performed the CYP11B1 gene analysis even without a complete hormonal profile, as the patient was in therapy. This analysis revealed the presence of 2 mutations: R43Q and A386V, both present on the paternal allele and individually reported as mild. Conclusions The patient, with the standard clinical NC-CAH manifestations, resulted to be heterozygous for both the deficiencies. We suggest therefore the possibility of a cumulative effect on the phenotype of the two mutants as previously reported in other conditions for enzymes catalyzing consecutive reactions on the same pathway. This is the first report of a di-genic inheritance in CAH.

Mutations in TSH receptor (TSHR) are notoriously associated with congenital hypothyroidism as well as with non-autoimmune subclinical hypothyroidism, a mild form of TSH resistance that is not as well characterized by diagnostic procedures.The genetic analysis of the TSHR gene was performed to determine the prevalence of TSHR gene mutations in non-autoimmune subclinical hypothyroidism during the pediatric age. The new mutations were studied for genotypic-phenotypic correlation.Thirty-eight children (ages 0.5-18.0 yr) affected by non-autoimmune subclinical hypothyroidism diagnosed in our center (follow-up from 1 to 11.5 yr) and normal at neonatal screening were enrolled in the genetic study. In 11 cases, the relatives were included in the genetic analysis.Eleven different mutations of the TSHR gene were identified in 11 of the 38 patients. Two are new: the nonsense mutation C31X and the missense mutation P68S, which shows a decrease in TSH binding capacity but not in biological activity. In all cases the carrier parent was identified.To date, this study demonstrates the highest prevalence (29%) of TSHR gene mutations in children and adolescents with non-autoimmune subclinical hypothyroidism not selected by neonatal screening. Functional studies show that some mutations cause a slight inactivation of the TSHR. This reveals a possible limit of the in vitro study or of the knowledge of mechanisms involving TSHR, or that other candidate genes must be considered.

The development of a testis requires the proper spatiotemporal expression of the SR Y gene and other genes that act in a dosage-sensitive manner. Mutations in the SRY gene account for only 10-15% of patients with 46,XY gonadal disorder of sex development (DSD). To enable the diagnostics of deletions and duplications of genes known to be involved in different forms of DSD, we developed a synthetic probe set for multiplex ligation-dependent probe amplification (MLPA) analysis. Here, we report the results from the analysis of 22 patients with 46,XY gonadal DSD. The analysis with the DSD probe set has led to the identification of two copy number variations, an 800-kb NROB1 (DAX1) locus duplication on Xp21 in a patient with isolated partial gonadal dysgenesis and a duplication of the SRD5A2 gene that represents a rare normal variant. The described MLPA kit represents an optimal complement to DNA sequence analysis in patients with DSD, enabling screening for deletions and duplications of several genes simultaneously. Furthermore, the second identification of an NROB1 locus duplication in a patient with isolated gonadal dysgenesis, without dysmorphic features and/or mental retardation, highlights the importance of evaluating NROB1 duplication in patients with gonadal dysgenesis.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) is one of the most common autosomal recessive metabolic disease and occurs worldwide. Ethnic-specific differences in the distribution of CYP21A2 gene mutations have been described so far. In this report we focused our attention to the Mediterranean area and compared the types and the relative frequencies of molecular defects in 233 unrelated patients of Italian origin (as defined by review of pedigrees extending to two or three generations) with the reported frequencies in other “Mediterranean” Countries. In particular, the distribution of mutations were available for samples of Croatian, French, Hellenic, Lebanese, Slovenian, Spanish, Tunisian, and Turkish populations. The predominance of the prevalent allelic mutations and genotypes in some of these populations was significant and it appeared that ethnic- or even intra-ethnic specific mutations were present. A large deletion is prevalent in the Slovenjans; a Q318X mutation is prevalent in the Tunisians; a R356W is prevalent in the Croatians; the D8bp mutation was rather frequent in the Lebanese population and exclusively in the Christian Maronite group. Intra-National differences were also found when the comparison was extended to groups coming from different areas of the same Nation such as North-West vs. Central Spain or North-Central vs. South Italy. The more frequent genotype/phenotype discrepancies will be discussed.