The clinical management of children with achondroplasia in Italy: results of clinician and parent/caregiver surveys.

PURPOSE: This study aimed to assess the real-world management of achondroplasia in Italy. METHODS: Two online surveys addressed to (1) parents/caregivers of individuals with achondroplasia and (2) Italian clinicians managing individuals with achondroplasia were conducted to assess real-world perspectives on achondroplasia management. Both surveys collected data on either patient or clinician demographics, details on diagnoses and referrals, disease complications, and views/experiences with limb lengthening surgery. RESULTS: In total, 42 parents/caregivers and 19 clinicians (from 18 hospitals) completed the surveys. According to parents/caregivers, achondroplasia diagnosis was most commonly made in the third trimester of gestation (55% of respondents), with a genetic test performed to confirm the diagnosis in all but one case. In contrast, the clinicians indicated that, while achondroplasia was typically suspected during the prenatal period (78%), diagnosis was more frequently confirmed postnatally (72%). Parents/caregivers reported that the greatest impact of achondroplasia-related complications occurred in their children between the ages of 2-5 years. The most significant complications were otitis, sleep apnoea, stenosis of the foramen magnum or pressure on the spinal cord, and hearing difficulties. Lengthening surgery had been presented as a treatment option to 92% of responding parents/caregivers, with 76% of clinicians viewing surgery favourably. Typically, clinicians' reasons for suggesting limb lengthening surgery were to improve patient quality of life, increase patient autonomy and self-acceptance, improve trunk-limb disproportion, short stature and walking, and ensure that all possible treatment options had been presented to the parents/caregivers. CONCLUSION: This survey provides insight into the real-world management of individuals with achondroplasia in Italy.

Allergooncology: an expanding research area.

The relationship between allergic diseases and cancer is a very controversial topic, widely discussed in the last decades. Many studies have demonstrated inverse association between allergy and cancer, but others have reached neutral conclusions or have indicated a positive role of allergy in the development of cancer. However, either inhibiting or favoring, many cells and molecules relevant in the allergic process play a role in tumorigenesis. On the one hand, activated immune cells, like classically activated macrophages "M1", activated dendritic cells, IL-33 and amphiregulin stimulated Innate Lymphoid Cells (ILC2), Th1, IFN-γ producing T CD8+ and B lymphocytes have inhibitory effects on tumorigenesis and tumor progression. On the other hand, tolerogenic immune cells, like alternatively activated macrophages "M2" (M2a, M2b and M2c), tolerogenic dendritic cells, ILC3, T regulatory and B regulatory lymphocytes, while inhibiting allergic sensitization and response, appear to favour carcinogenesis. Furthermore, M2 subtypes macrophages (M2a, M2b), IL-25 stimulated ILC2 and Th2 lymphocytes have a role both in inducing allergic reactions and in favouring cancer progression. In addition, mast cells, pivotal cells in allergy, have a different effect of tumorigenesis based on their location - they can promote cancer progression or inhibit it. Finally, eosinophils have shown a prevalent tumoricidal function mediated by α-defensins, TNF-α, granzymes A and IL-18. Better understanding the role of various cells on carcinogenesis can help in developing new strategies (diagnostic, therapeutic and of follow up) against tumor.

Kounis Syndrome as First Manifestation of Allergic Sensitization.

Mast cells are abundant in the heart, among myocardial fibers, around coronary arteries, within arterial intima and intramural vessels, and in atherosclerotic plaques. Their mediators can be released during anaphylaxis and be responsible for acute coronary syndrome. This condition has been described as Kounis syndrome (KS). We report three cases of acute myocardial ischemia, which fulfill the definition for KS. In Cases 1 and 2, the association of intense chest pain with acute urticaria after an allergenic contact (wasp sting and betalactam antibiotic administration, respectively) was suspected to be an attack of angina related to an allergic reaction. No signs of an allergic reaction were observed in Case 3, but only the history of a wasp sting suggested its relationship to loss of consciousness and heart ischemia when hypersensitivity to venom was ascertained. These cases strongly recommend measurement of anaphylactic biomarkers, such as tryptase, during acute coronary syndromes to detect the possible involvement of an allergic reaction. Conversely, measurement of cardiac biomarkers during anaphylaxis, even without obvious signs of myocardial ischemia, might identify patients at risk of myocardial injury.

Say-Barber-Biesecker-Young-Simpson syndrome and Genitopatellar syndrome: Lumping or splitting?

The Say-Barber-Biesecker-Young-Simpson variant of Ohdo syndrome (SBBYSS) and Genitopatellar syndrome (GTPTS) are 2 rare but clinically well-described diseases caused by de novo heterozygous sequence variants in the KAT6B gene. Both phenotypes are characterized by significant global developmental delay/intellectual disability, hypotonia, genital abnormalities, and patellar hypoplasia/agenesis. In addition, congenital heart defects, dental abnormalities, hearing loss, and thyroid anomalies are common to both phenotypes. This broad clinical overlap led some authors to propose the concept of KAT6B spectrum disorders. On the other hand, some clinical features could help to differentiate the 2 disorders. Furthermore, it is possible to establish a genotype-phenotype correlation when considering the position of the sequence variant along the gene, supporting the notion of the 2 disorders as really distinct entities.

Diagnostic application of a capture based NGS test for the concurrent detection of variants in sequence and copy number as well as LOH.

Whole exome sequencing (WES) has made the identification of causative SNVs/InDels associated with rare Mendelian conditions increasingly accessible. Incorporation of softwares allowing CNVs detection into the WES bioinformatics pipelines may increase the diagnostic yield. However, no standard protocols for this analysis are so far available and CNVs in non-coding regions are totally missed by WES, in spite of their possible role in the regulation of the flanking genes expression. So, in a number of cases the diagnostic workflow contemplates an initial investigation by genomic arrays followed, in the negative cases, by WES. The opposite workflow may also be applied, according to the familial segregation of the disease. We show preliminary results for a diagnostic application of a single next generation sequencing panel permitting the concurrent detection of LOH and variations in sequences and copy number. This approach allowed us to highlight compound heterozygosity for a CNV and a sequence variant in a number of cases, the duplication of a non-coding region responsible for sex reversal, and a whole-chromosome isodisomy causing reduction to homozygosity for a WFS1 variant. Moreover, the panel enabled us to detect deletions, duplications, and amplifications with sensitivity comparable to that of the most widely used array-CGH platforms.

SNORD116 deletions cause Prader-Willi syndrome with a mild phenotype and macrocephaly.

Prader-Willi syndrome is a complex condition caused by lack of expression of imprinted genes in the paternally derived region of chromosome 15 (15q11q13). A small number of patients with Prader-Willi phenotype have been discovered to have narrow deletions, not encompassing the whole critical region, but only the SNORD116 cluster, which includes genes codifying for small nucleolar RNAs. This kind of deletion usually is not detected by the classic DNA methylation analysis test. We present the case of a male patient with a mild Prader-Willi phenotype and a small deletion including SNORD116, diagnosed by methylation-sensitive multiplex ligation-dependent probe amplification (MLPA. The patient showed neonatal hypotonia, hyperphagia, obesity, central hypogonadism, hypothyroidism, strabismus. Stature and intellectual development are within the normal range. The presence of macrocephaly, observed in other cases of SNORD116 deletions as well, is uncommon for the classic phenotype of the syndrome.

Insights into genotype-phenotype correlations from CREBBP point mutation screening in a cohort of 46 Rubinstein-Taybi syndrome patients.

The genetic basis of Rubinstein-Taybi syndrome (RSTS), a rare, sporadic, clinically heterogeneous disorder characterized by cognitive impairment and a wide spectrum of multiple congenital anomalies, is primarily due to private mutations in CREBBP (approximately 55% of cases) or EP300 (approximately 8% of cases). Herein, we report the clinical and the genetic data taken from a cohort of 46 RSTS patients, all carriers of CREBBP point mutations. Molecular analysis revealed 45 different gene alterations including 31 inactivating (21 frameshift and 10 nonsense), 10 missense and 4 splicing mutations. Bioinformatic tools and transcript analyses were used to predict the functional effects of missense and splicing alterations. Of the 45 mutations, 42 are unreported and 3 were described previously. Recurrent mutations maybe a key tool in addressing genotype-phenotype correlations in patients sharing the same defects (at the genomic or transcript level) and specific clinical signs, demonstrated here in two cases. The clinical data of our cohort evidenced frequent signs such as arched eyebrows, epicanthus, synophrys and/or frontal hypertrichosis and broad phalanges that, previously overlooked in RSTS diagnosis, now could be considered. Some suggested correlations between organ-specific anomalies and affected CREB-binding protein domains broaden the RSTS clinical spectrum and perhaps will enhance patient follow-up and clinical care.

Clinical and molecular characterization of Rubinstein-Taybi syndrome patients carrying distinct novel mutations of the EP300 gene.

Rubinstein-Taybi syndrome (RSTS) is a rare congenital neurodevelopmental disorder characterized by postnatal growth deficiency, skeletal abnormalities, dysmorphic features and cognitive deficit. Mutations in two genes, CREBBP and EP300, encoding two homologous transcriptional co-activators, have been identified in ˜55% and ˜3-5% of affected individuals, respectively. To date, only eight EP300-mutated RSTS patients have been described and 12 additional mutations are reported in the database LOVD. In this study, EP300 analysis was performed on 33 CREBBP-negative RSTS patients leading to the identification of six unreported germline EP300 alterations comprising one deletion and five point mutations. All six patients showed a convincing, albeit mild, RSTS phenotype with minor skeletal anomalies, slight cognitive impairment and few major malformations. Beyond the expansion of the RSTS-EP300-mutated cohort, this study indicates that EP300-related RSTS cases occur more frequently than previously thought (˜8% vs 3-5%); furthermore, the characterization of novel EP300 mutations in RSTS patients will enhance the clinical practice and genotype-phenotype correlations.

Histone acetylation deficits in lymphoblastoid cell lines from patients with Rubinstein-Taybi syndrome.

BACKGROUND: Rubinstein-Taybi syndrome (RSTS) is a congenital neurodevelopmental disorder defined by postnatal growth deficiency, characteristic skeletal abnormalities and mental retardation and caused by mutations in the genes encoding for the transcriptional co-activators with intrinsic lysine acetyltransferase (KAT) activity CBP and p300. Previous studies have shown that neuronal histone acetylation is reduced in mouse models of RSTS. METHODS: The authors identified different mutations at the CREBBP locus and generated lymphoblastoid cell lines derived from nine patients with RSTS carrying distinct CREBBP mutations that illustrate different grades of the clinical severity in the spectrum of the syndrome. They next assessed whether histone acetylation levels were altered in these cell lines. RESULTS: The comparison of CREBBP-mutated RSTS cell lines with cell lines derived from patients with an unrelated mental retardation syndrome or healthy controls revealed significant deficits in histone acetylation, affecting primarily histone H2B and histone H2A. The most severe defects were observed in the lines carrying the whole deletion of the CREBBP gene and the truncating mutation, both leading to a haploinsufficiency state. Interestingly, this deficit was rescued by treatment with an inhibitor of histone deacetylases (HDACi). CONCLUSIONS: The authors' results extend to humans the seminal observations in RSTS mouse models and point to histone acetylation defects, mainly involving H2B and H2A, as relevant molecular markers of the disease.

Interleukin (IL)-6 and receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) are increased in the serum of a patient with primary pachydermoperiostosis.

Primary pachydermoperiostosis (PDP) is a rare syndrome, characterized by digital clubbing, periostosis, and pachydermia. We have evaluated biochemical bone turnover markers, including components of interleukin-6 (IL-6) and osteoprotegerin/receptor activator of nuclear factor (NF)-kappaB ligand (OPG/RANKL) systems, in an 18-year-old man affected by primary PDP. The acute phase of the disease was characterized in our patient by high serum levels of IL-6 and RANKL. The observed high serum levels of these parameters are associated with increased values in markers of bone resorption (degradation products of C-terminal telopeptides of type-I collagen and urinary hydroxyproline/creatinine ratio) and reduced serum levels of bone alkaline phosphatase, a marker of bone formation. Serum levels of osteotrophic hormones were in the normal range. Our data suggest that, despite the radiographic findings, the acute phase of primary PDP is characterized by increased bone resorption, probably mediated by IL-6 and RANKL.

Assessing the memorization of TV commercials with the use of high resolution EEG: a pilot study.

The present work intends to evaluate the functional characteristics of the cerebral network during the successful memory encoding of TV commercials. We estimated the functional networks in the frequency domain from a set of high-resolution EEG data. High resolution EEG recordings were performed in a group of healthy subjects and the cortical activity during the observation of TV commercials was evaluated in several regions of interest coincident with the Brodmann areas (BAs). Summarizing the main results of the present study, a sign of the memorization of a particular set of TV commercials have been found in a group of investigated subjects with the aid of advanced modern tools for the acquisition and the processing of EEG data. The cerebral processes involved during the observation of TV commercials that were remembered successively by the population examined (RMB dataset) are generated by the posterior parietal cortices and the prefrontal areas, rather bilaterally and are irrespective of the frequency bands analyzed. Such results are compatible with previously results obtained from EEG recordings with superficial electrodes as well as with the brain activations observed with the use of MEG and fMRI devices.

Clinical score of 62 Italian patients with Cornelia de Lange syndrome and correlations with the presence and type of NIPBL mutation.

Cornelia de Lange syndrome (CdLS) is a rare multisystem disorder characterized by facial dysmorphisms, upper limb abnormalities, growth and cognitive retardation. About half of all patients with CdLS carry mutations in the NIPBL gene. The first Italian CdLS cohort involving 62 patients (including 4 related members) was screened for NIPBL mutations after a clinical evaluation using a quantitative score that integrates auxological, malformation and neurodevelopmental parameters. The patients were classified as having an overall 'severe', 'moderate' or 'mild' phenotype. NIPBL screening showed 26 mutations so classified: truncating (13), splice-site (8), missense (3), in-frame deletion (1) and regulatory (1). The truncating mutations were most frequently found in the patients with a high clinical score, whereas most of the splice-site and all missense mutations clustered in the low-medium score groups. The NIPBL-negative group included patients covering the entire clinical spectrum. The prevalence of a severe phenotype in the mutated group and a mild phenotype in the non-mutated group was statistically significant. In terms of the isolated clinical signs, the statistically significant differences between the mutation-positive and mutation-negative individuals were pre- and post-natal growth deficits, limb reduction, and delayed speech development. The proposed score seems to be a valuable means of prioritizing the patients with CdLS to undergo an NIPBL mutation test.

WNT4 deficiency--a clinical phenotype distinct from the classic Mayer-Rokitansky-Kuster-Hauser syndrome: a case report.

The pathways leading to female sexual determination in mammals are incompletely defined. Loss-of-function mutations in the WNT4 gene appear to cause developmental abnormalities of sexual differentiation in women and mice. We recruited six patients with different degrees of Müllerian abnormalities, with or without renal aberrations and a normal female 46,XX karyotype. A clear androgen excess was found only in one patient. This 19-year-old woman was affected by primary amenorrhoea, absence of Müllerian ducts derivatives, clinical (acne and hirsutism) and biochemical (repeatedly high levels of testosterone) signs of androgen excess. Direct sequencing of her WNT4 gene followed by functional studies in human ovarian cells (OVCAR3) was performed. This patient carried the novel R83C loss-of-function dominant negative mutation in her WNT4, confirming the role of WNT4 in the development and maintenance of the female phenotype in women. Our study can also help refine the phenotype of WNT4 deficiency in humans. In fact, it appears that at least in this limited casuistic small group of patients, the absence of a uterus (and not other Müllerian abnormalities) and the androgen excess are the pathognomonic signs of WNT4 defects, suggesting that this might be a clinical entity distinct from the classic Mayer-Rokitansky-Kuster-Hauser syndrome.

Mutation analysis of the NSD1 gene in a group of 59 patients with congenital overgrowth.

Sotos syndrome is characterized by pre- and post-natal overgrowth, typical craniofacial features, advanced bone age, and developmental delay. Some degree of phenotypic overlap exists with other overgrowth syndromes, in particular with Weaver syndrome. Sotos syndrome is caused by haploinsufficiency of the NSD1 (nuclear receptor SET domain containing gene 1) gene. Microdeletions involving the gene are the major cause of the syndrome in Japanese patients, whereas intragenic mutations are more frequent in non-Japanese patients. NSD1 aberrations have also been described in some patients diagnosed as Weaver syndrome. Some authors have suggested a certain degree of genotype-phenotype correlation, with a milder degree of overgrowth, a more severe mental retardation, and a higher frequency of congenital anomalies in microdeleted patients. Data on larger series are needed to confirm this suggestion. We report here on microdeletion and mutation analysis of NSD1 in 59 patients with congenital overgrowth. Fourteen novel mutations, two previously described and one microdeletion were identified. All patients with a NSD1 mutation had been clinically classified as "classical Sotos," although their phenotype analysis demonstrated that some major criteria, such as overgrowth and macrocephaly, could be absent. All patients with confirmed mutations shared the typical Sotos facial gestalt. A high frequency of congenital heart defects was present in patients with intragenic mutations, supporting the relevance of the NSD1 gene in the pathogenesis of this particular defect.

Prenatal diagnosis of severe structural congenital malformations in Europe.

OBJECTIVES: To assess at a population-based level the frequency with which severe structural congenital malformations are detected prenatally in Europe and the gestational age at detection, and to describe regional variation in these indicators. METHODS: In the period 1995-1999, data were obtained from 17 European population-based registries of congenital malformations (EUROCAT). Included were all live births, fetal deaths and terminations of pregnancy diagnosed with one or more of the following malformations: anencephalus, encephalocele, spina bifida, hydrocephalus, transposition of great arteries, hypoplastic left heart, limb reduction defect, bilateral renal agenesis, diaphragmatic hernia, omphalocele and gastroschisis. RESULTS: The 17 registries reported 4366 cases diagnosed with the 11 severe structural malformations and of these 2300 were live births (53%), 181 were fetal deaths (4%) and 1863 were terminations of pregnancy (43%); in 22 cases pregnancy outcome was unknown. The overall prenatal detection rate was 64% (range, 25-88% across regions). The proportion of terminations of pregnancy varied between regions from 15% to 59% of all cases. Gestational age at discovery for prenatally diagnosed cases was less than 24 weeks for 68% (range, 36-88%) of cases. There was a significant relationship between high prenatal detection rate and early diagnosis (P < 0.0001). For individual malformations, the prenatal detection rate was highest for anencephalus (469/498, 94%) and lowest for transposition of the great arteries (89/324, 27%). Termination of pregnancy was performed in more than half of the prenatally diagnosed cases, except for those with transposition of the great arteries, diaphragmatic hernia and gastroschisis, in which 30-40% of the pregnancies with a prenatal diagnosis were terminated. CONCLUSION: European countries currently vary widely in the provision and uptake of prenatal screening and its quality, as well as the "culture" in terms of decision to continue the pregnancy. This inevitably contributes to variation between countries in perinatal and infant mortality and in childhood prevalence and cost to health services of congenital anomalies.

Deletion of the SHOX gene in patients with short stature of unknown cause.

A fluorescence in situ hybridization (FISH) study was performed in 56 patients with short stature of unknown cause in order to establish the role of deletion of the SHOX gene in this population. FISH analysis was carried out on metaphase spreads and interphase lymphocytes from blood smears using a probe specific for the SHOX gene. Deletion of SHOX was found in four patients (7.1%). No skeletal abnormalities were detected in these patients either at the physical examination or at X-rays of the upper and lower limbs. Present results indicate that SHOX plays an important role also in short stature of unknown cause, and FISH analysis appears as an easy, appropriate, and inexpensive method for the detection of SHOX deletion.

Synthesis and stability of phytochelatins induced by cadmium and lead in the marine diatom Phaeodactylum tricornutum.

The synthesis of phytochelatins (PC), intracellular metal-binding polypeptides characterized by a repeating sequence of gamma-glutamic acid- cysteine (gamma-Glu-Cys) pairs, has been studied in laboratory cultures of the marine diatom Phaeodactylum tricornutum exposed to Cd, Pb or Zn. Cd and Pb were able to induce PC of different degree of polymerization. The accumulation of the peptides follows a direct relationship with the metal exposure. No PC induction was observed in Zn-treated cultures, although the intracellular concentration of Zn increased during exposure. Both in short-term (7 h exposure, 10 microM Cd or Pb) and 3-day experiments (metal concentration less than 0.5 microM), the major fraction of total PC gamma-Glu-Cys subunits synthesized was polymerized as PC2 when cells were exposed to Pb, but as PC4 when cells were exposed to Cd. In short-term experiments about 50% of the gamma-Glu-Cys residues of the cellular pool of glutathione was quickly and almost quantitatively converted into PC. Recovery experiments, in which metal-stressed cells are suspended in a metal-free medium, showed a decrease of the PC pool and a concomitant increase of glutathione, suggesting a mechanism of degradation and release of metal-phytochelatin complexes.

Evaluation of benzene exposure in children living in Campania (Italy) by urinary trans,trans-muconic acid assay.

The urinary benzene metabolite trans,trans-muconic acid (MA) was determined in 144 children living in Campania (Italy): 92 from Naples (1,300,000 inhabitants), designated as an urban source, and compared to 52 from Pollica (300 inhabitants), considered a rural, background exposure for benzene. The children participating in the study were tested by an anonymous questionnaire about the possible sources of exposure to benzene. Quantifiable levels of MA were found in 63% of the urine samples analyzed. Setting the value of nondetectable urinary samples at 7 microg/L MA, a value that is one-half of the instrument detection limit of 14 microg/L, the mean urinary concentration levels were 98.7+/-81.0 microg/L and 48.4+/-71.7 microg/L in Naples and Pollica, respectively; adjustment of these values to creatinine clearance resulted in MA levels of 141.2+/-145.4 microg/L in Naples and 109.8+/-133.2 microg/L in Pollica. Passive smoke exposure did not significantly affect urinary MA levels, but proximity of the home to traffic increased urine MA content. Data show that MA can be utilized as a biomarker for exposure; however, a clear-cut association to benzene requires personal monitoring and control of dietary sorbic acid.