First report of whole CFTR gene duplication in a healthy newborn carrying R74W and V855I variants on the same allele.

Cystic fibrosis (CF) is the most common severe autosomal recessive genetic disorder among Caucasians. The improvement of genetic techniques has allowed the identification of an increasing number of genetic variants, including large rearrangements such as duplications. We report the first case of a whole CFTR gene duplication in a healthy newborn, who had normal sweat test, also carrying R74W and V855I variants on the same allele. Familial segregation analysis and the observed frequencies of all the CFTR gene variants, revealed that R74W and V855I were probably both present in a cis arrangement on the allele also containing the duplication (i.e., in a double complex allele). Since R74W is a "variant of varying clinical consequence" its arrangement in trans with one pathogenic variant may not be sufficient to cause a classic CF disease phenotype. Moreover, its duplication could even be an advantage that could compensate for the effect of the alteration.

TNFRSF13B gene mutation in familial acute myeloid leukemia: A new piece in the complex scenario of hereditary predisposition?

TNFRSF13B mutations are widely associated with common variable immunodeficiency. TNFRSF13B was recently counted among relevant genes associated with childhood-onset of hematological malignancies; nonetheless, its role in acute myeloid leukemia (AML) remains unexplored. We report the study of a family with two cases of AML, sharing a germline TNFRSF13B mutation favoring the formation of a more stable complex with its ligand TNFSF13: a positive regulator of AML-initiating cells. Our data turn the spotlight onto the TNFRSF13B role in AML onset, inserting a new fragment into the complex scenario of a hereditary predisposition to myeloid neoplasms.

Whole-Exome and Transcriptome Sequencing Expands the Genotype of Majewski Osteodysplastic Primordial Dwarfism Type II.

Microcephalic Osteodysplastic Primordial Dwarfism type II (MOPDII) represents the most common form of primordial dwarfism. MOPD clinical features include severe prenatal and postnatal growth retardation, postnatal severe microcephaly, hypotonia, and an increased risk for cerebrovascular disease and insulin resistance. Autosomal recessive biallelic loss-of-function genomic variants in the centrosomal pericentrin (PCNT) gene on chromosome 21q22 cause MOPDII. Over the past decade, exome sequencing (ES) and massive RNA sequencing have been effectively employed for both the discovery of novel disease genes and to expand the genotypes of well-known diseases. In this paper we report the results both the RNA sequencing and ES of three patients affected by MOPDII with the aim of exploring whether differentially expressed genes and previously uncharacterized gene variants, in addition to PCNT pathogenic variants, could be associated with the complex phenotype of this disease. We discovered a downregulation of key factors involved in growth, such as IGF1R, IGF2R, and RAF1, in all three investigated patients. Moreover, ES identified a shortlist of genes associated with deleterious, rare variants in MOPDII patients. Our results suggest that Next Generation Sequencing (NGS) technologies can be successfully applied for the molecular characterization of the complex genotypic background of MOPDII.

Prenatal diagnosis of Pfeiffer syndrome and role of three-dimensional ultrasound: case report and review of literature.

Purpose/aim of the study: We report a rare case of autosomal dominant genetic syndrome "Pfeiffer", which is part of the group of acrocephalosyndactyly, with an annual incidence <1/100,000. Three forms are known. Type I is the less common form and it is characterized by moderate-severe mediofacial hypoplasia usually with normal cognitive development. Conversely, types 2 and 3 are more common and they are associated with more severe signs and complications with a more unfavorable prognosis. The type 3 form due to the presence of a cloverleaf skull distinguishes type 2.Materials and methods: Thirty-eight-year-old primigravida was referred to our center, at 28 weeks of gestation due to borderline ventriculomegaly, macrocrania, and a short femur. First trimester screening for chromosomopathies and CF-DNA was low risk; II trimester screening ultrasound showed the presence of "short femur" and macrocrania.Result: Our ultrasound evaluation, assisted by 3D ultrasound, showed cloverleaf skull, turricephaly, moderate ventriculomegaly (13 mm), hypertelorism and exophthalmos, low ear implantation, mild rhizomelia. Ultrasound depicts Pfeiffer syndrome or other acrocephalosyndactyly syndromes (Apert syndromes, Saethre-Chotzen) or other syndromic forms of craniosynostosis like Crouzon syndrome. The NGS panel for molecular analysis of genes involved in skeletal dysplasias showed the mutation of the FGFR2 gene, de novo.Conclusions: Using three-dimensional (3D) ultrasound, it is easier to distinguish rare syndromes characterized by facial dysmorphisms such as exophthalmos, mediofacial hypoplasia, and craniosynostosis.

Genetic Pattern, Orthodontic and Surgical Management of Multiple Supplementary Impacted Teeth in a Rare, Cleidocranial Dysplasia Patient: A Case Report.

Background: Cleidocranial dysplasia (CCD) is a rare, autosomal dominant skeletal dysplasia with a prevalence of one per million births. The main causes of CCD are mutations in the core-binding factor alpha-1 (CBFA1) or runt-related transcription factor-2 (RUNX2), located at the 6p21 chromosomal region. RUNX2 plays important roles in osteoblast differentiation, chondrocyte proliferation and differentiation, and tooth formation. The disease is characterized by clavicular aplasia or hypoplasia, Wormian bones, delayed closure of cranial suture, brachycephalic head, maxillary deficiency, retention of primary teeth, inclusion of permanent teeth, and multiple supernumerary teeth. Materials and Methods: A 22-year-old girl suffering from cleidocranial dysplasia with short stature, narrow shoulders, craniofacial manifestations (short face, broad forehead, etc.) and dental anomalies (different lower dental elements under eruption, supernumerary and impacted multiple teeth, etc.) was examined at our service (Complex Operative Unit of Odontostomatology of Policlinico of Bari). RX Orthopantomography (OPG) and cone beam computed tomography (CBCT) were requested to better assess the position of the supernumerary teeth and their relationships with others and to evaluate the bone tissue. Results: Under eruption was probably caused by dental interferences with supernumerary teeth; hence, extractions of supernumerary upper canines and lower premolars were performed under general anaesthesia. Surgery outcome was excellent with good tissue healing and improvements in the therapeutic possibilities with future orthodontics. Conclusions: The objective of this article is to give an update about radiological, clinical, and molecular features of CCD and to alert the health team about the importance of establishing an early diagnosis and an appropriate treatment in these patients to prevent impacted teeth complications and to offer them a better quality of life.

Cytogenetic and Array-CGH Characterization of a Simple Case of Reciprocal t(3;10) Translocation Reveals a Hidden Deletion at 5q12.

Chromosome deletions, including band 5q12, have rarely been reported and have been associated with a wide range of clinical manifestations, such as postnatal growth retardation, intellectual disability, hyperactivity, nonspecific ocular defects, facial dysmorphism, and epilepsy. In this study, we describe for the first time a child with growth retardation in which we identified a balanced t(3;10) translocation by conventional cytogenetic analysis in addition to an 8.6 Mb 5q12 deletion through array-CGH. Our results show that the phenotypic abnormalities of a case that had been interpreted as "balanced" by conventional cytogenetics are mainly due to a cryptic deletion, highlighting the need for molecular investigation in subjects with an abnormal phenotype before assuming the cause is an apparently simple cytogenetic rearrangement. Finally, we identify PDE4D and PIK3R1 genes as the two major candidates responsible for the clinical features expressed in our patient.

Functional evidence of mTORß splice variant involvement in the pathogenesis of congenital heart defects.

mTOR dysregulation has been described in pathological conditions, such as cardiovascular and overgrowth disorders. Here we report on the first case of a patient with a complex congenital heart disease and an interstitial duplication in the short arm of chromosome 1, encompassing part of the mTOR gene. Our results suggest that an intragenic mTOR microduplication might play a role in the pathogenesis of non-syndromic congenital heart defects (CHDs) due to an upregulation of mTOR/Rictor and consequently an increased phosphorylation of PI3K/AKT and MEK/ERK signaling pathways in patient-derived amniocytes. This is the first report which shows a causative role of intragenic mTOR microduplication in the etiology of an isolated complex CHD.

Bilateral Phyllodes Giant Tumor. A Case Report Analyzed by Array-CGH.

The breast phyllodes tumor is a biphasic tumor that accounts for less than of 1% of all breast neoplasms. It is classified as benign, borderline, or malignant, and can mimic benign masses. Some recurrent alterations have been identified. However, a precise molecular classification of these tumors has not yet been established. Herein, we describe a case of a 43-year-old woman that was admitted to the emergency room for a significant bleeding from the breast skin. A voluminous ulcerative mass of the left breast and multiple nodules with micro-calcifications on the right side were detected at a physical examination. A left total mastectomy and a nodulectomy of the right breast was performed. The histological diagnosis of the surgical specimens reported a bilateral giant phyllodes tumor, showing malignant features on the left and borderline characteristics associated with a fibroadenoma on the right. A further molecular analysis was carried out by an array-Comparative Genomic Hybridization (CGH) to characterize copy-number alterations. Many losses were detected in the malignant mass, involving several tumor suppressor genes. These findings could explain the malignant growth and the metastatic risk. In our study, genomic profiling by an array-CGH revealed a greater chromosomal instability in the borderline mass (40 total defects) than in the malignant (19 total defects) giant phyllodes tumor, reflecting the tumor heterogeneity. Should our results be confirmed with more sensitive and specific molecular tests (DNA sequencing and FISH analysis), they could allow a better selection of patients with adverse pathological features, thus optimizing and improving patient's management.

Novel exostosin-2 missense variants in a family with autosomal recessive exostosin-2-related syndrome: further evidences on the phenotype.

Biallelic exostosin-2 (EXT2) pathogenic variants have been described as the cause of the Seizures-Scoliosis-Macrocephaly syndrome (OMIM 616682) characterized by intellectual disability, facial dysmorphisms and seizures. More recently, it has been proposed to rename this disorder with the acronym AREXT2 (autosomal recessive EXT2-related syndrome). Here, we report the third family affected by AREXT2 syndrome, harboring compound missense variants in EXT2, p.Asp227Asn, and p.Tyr608Cys. In addition, our patients developed multiple exostoses, which were not observed in the previously described families. AREXT2 syndrome can be considered as a multiorgan Congenital Disorder of Glycosylation caused by a significant, but non-lethal, decrease in EXT2 expression, thereby affecting the synthesis of the heparan sulfate proteoglycans, which is relevant in many physiological processes. Our finding expands the clinical and molecular spectrum of the AREXT2 syndrome and suggests a possible genotype/phenotype correlation in the development of the exostoses.

Risk of Fetal Loss in Pregnancies Undergoing Midtrimester Amniocentesis after Inconclusive Chorionic Villus Sampling.

OBJECTIVE: To estimate the procedure-related risk of miscarriage in pregnancies undergoing amniocentesis (AC) following inconclusive results for a chorionic villus sampling (CVS). METHODS: This was a multicentric retrospective cohort study of patients in which both CVS at 11-13 weeks' gestation and AC at 16-22 weeks were performed between January 1st, 2008, and July 31st, 2017. The primary outcome measure was pregnancy loss prior to 24 weeks gestation; the secondary one was intrauterine demise after 24 weeks. RESULTS: A total of 287 patients underwent transabdominal CVS and AC. Nine patients were lost at follow-up; therefore, the analysis was conducted on a population of 278 patients (275 singletons and 3 dichorionic twin pregnancies). AC was performed because of placental mosaicism (93.6%), failure of direct/semidirect preparation of trophoblastic cells (3.2%), or targeted genetic testing after the diagnosis of an anomaly in the second trimester (3.2%). In continuing pregnancies, there were no fetal losses prior to 24 weeks' gestation. Two intrauterine demises (including 1 fetus with multiple anomalies and growth restriction) in the third trimester were recorded. CONCLUSION: Patients undergoing midtrimester AC because of an inconclusive result of CVS can be reasonably reassured that in general the risk of miscarriage and fetal loss following the procedure is very small.

Genomic changes of chromosomes 8p23.1 and 1q21: Novel mutations in malignant mesothelioma.

INTRODUCTION: Malignant mesothelioma is an aggressive malignancy of the thoracic cavity caused by prior asbestos exposure. In the peritoneum the mesothelioma is an extremely rare condition. In the present preliminary study, high-resolution array-comparative genomic hybridization (a-CGH) was performed to identify genetic imbalances in a series of malignant peritoneal mesothelioma cases. MATERIALS AND METHODS: Between 1990 and 2008, among the cases recorded in the Apulia Mesothelioma Register, we found 22 peritoneal mesothelioma cases. CGH-array was performed on samples from all patients. RESULTS: The CGH-array analysis revealed multiple chromosomal imbalances. Interestingly, deletion at 8p23.1 was observed in 12 cases. Furthermore, another novel deletion at 1q21 was present in 11. Often, 1q21 and 8p23.1 losses were present in the same patient (7 cases). Losses of BAP1 and CDKN2A loci were not detected. DISCUSSION: The region at 8p23.1 contains the beta-defensin gene cluster (DEF) and 1q21 contains ubiquitin conjugating enzyme E2 (UBE2Q1). We hypotesized that the loss of function of ubiquitination, as well as of the defensins, could play an important role in the initial development and subsequent progression of mesothelioma.

Clinical biomarkers for cancer recognition and prevention: A novel approach with optical measurements.

Cancer is the most important cause of death worldwide, and early cancer detection is the most fundamental factor for efficacy of treatment, prognosis, and increasing survival rate. Over the years great effort has been devoted to discovering and testing new biomarkers that can improve its diagnosis, especially at an early stage. Here we report the potential usefulness of new, easily applicable, non-invasive and relatively low-cost clinical biomarkers, based on abnormalities of oral mucosa spectral reflectance and fractal geometry of the vascular networks in several different tissues, for identification of hereditary non-polyposis colorectal cancer carriers as well for detection of other tumors, even at an early stage. In the near future the methodology/technology of these procedures should be improved, thus making possible their applicability worldwide as screening tools for early recognition and prevention of cancer.

Peritoneal Mesothelioma with Residential Asbestos Exposure. Report of a Case with Long Survival (Seventeen Years) Analyzed by Cgh-Array.

Malignant mesothelioma is a rare and aggressive tumor with limited therapeutic options. We report a case of a malignant peritoneal mesothelioma (MPM) epithelioid type, with environmental asbestos exposure, in a 36-year-old man, with a long survival (17 years). The patient received standard treatment which included cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). METHODS AND RESULTS: Molecular analysis with comparative genomic hybridization (CGH)-array was performed on paraffin-embedded tumoral samples. Multiple chromosomal imbalances were detected. The gains were prevalent. Losses at 1q21, 2q11.1→q13, 8p23.1, 9p12→p11, 9q21.33→q33.1, 9q12→q21.33, and 17p12→p11.2 are observed. Chromosome band 3p21 (BAP1), 9p21 (CDKN2A) and 22q12 (NF2) are not affected. Conclusions: the defects observed in this case are uncommon in malignant peritoneal mesothelioma. Some chromosomal aberrations that appear to be random here, might actually be relevant events explaining the response to therapy, the long survival and, finally, may be considered useful prognostic factors in peritoneal malignant mesothelioma (PMM).

Increased Wnt and Notch signaling: a clue to the renal disease in Schimke immuno-osseous dysplasia?

BACKGROUND: Schimke immuno-osseous dysplasia (SIOD) is a multisystemic disorder caused by biallelic mutations in the SWI/SNF-related matrix-associated actin-dependent regulator of chromatin, subfamily A-like 1 (SMARCAL1) gene. Changes in gene expression underlie the arteriosclerosis and T-cell immunodeficiency of SIOD; therefore, we hypothesized that SMARCAL1 deficiency causes the focal segmental glomerulosclerosis (FSGS) of SIOD by altering renal gene expression. We tested this hypothesis by gene expression analysis of an SIOD patient kidney and verified these findings through immunofluorescent analysis in additional SIOD patients and a genetic interaction analysis in Drosophila. RESULTS: We found increased expression of components and targets of the Wnt and Notch signaling pathways in the SIOD patient kidney, increased levels of unphosphorylated ß-catenin and Notch1 intracellular domain in the glomeruli of most SIOD patient kidneys, and genetic interaction between the Drosophila SMARCAL1 homologue Marcal1 and genes of the Wnt and Notch signaling pathways. CONCLUSIONS: We conclude that increased Wnt and Notch activity result from SMARCAL1 deficiency and, as established causes of FSGS, contribute to the renal disease of most SIOD patients. This further clarifies the pathogenesis of SIOD and will hopefully direct potential therapeutic approaches for SIOD patients.

Pulmonary alveolar microlithiasis: review of the 1022 cases reported worldwide.

Pulmonary alveolar microlithiasis (PAM) is a rare disease characterised by the widespread intra-alveolar accumulation of minute calculi called microliths. It is caused by mutation of the SLC34A2 gene encoding the type IIb sodium phosphate cotransporter in alveolar type II cells. The present study explores the epidemiological, familial, genetic, clinical, diagnostic, radiological and therapeutic aspects with the aim of contributing to a better understanding of this uncommon disease.We searched articles on PAM published up to December 2014 and 544 papers were found, accounting for 1022 cases.PAM is present in all continents and in many nations, in particular in Turkey, China, Japan, India, Italy and the USA. Familiality is frequent. The clinical course is not uniform and the causes of this clinical variability seem to be largely nongenetic. The optimal diagnostic procedure is the association of chest high-resolution computed tomography (HRCT) with bronchoalveolar lavage, but a chest radiograph may suffice in families in which a case has already been diagnosed. Moreover, chest radiography and HRCT allow the classification of the evolutionary phase of the disease and its severity. At present lung transplantation is the only effective therapy. However, better knowledge of the gene responsible offers hope for new therapies.

Lack of IL7Rα expression in T cells is a hallmark of T-cell immunodeficiency in Schimke immuno-osseous dysplasia (SIOD).

Schimke immuno-osseous dysplasia (SIOD) is an autosomal recessive, fatal childhood disorder associated with skeletal dysplasia, renal dysfunction, and T-cell immunodeficiency. This disease is linked to biallelic loss-of-function mutations of the SMARCAL1 gene. Although recurrent infection, due to T-cell deficiency, is a leading cause of morbidity and mortality, the etiology of the T-cell immunodeficiency is unclear. Here, we demonstrate that the T cells of SIOD patients have undetectable levels of protein and mRNA for the IL-7 receptor alpha chain (IL7Rα) and are unresponsive to stimulation with IL-7, indicating a loss of functional receptor. No pathogenic mutations were detected in the exons of IL7R in these patients; however, CpG sites in the IL7R promoter were hypermethylated in SIOD T cells. We propose therefore that the lack of IL7Rα expression, associated with hypermethylation of the IL7R promoter, in T cells and possibly their earlier progenitors, restricts T-cell development in SIOD patients.

High Wilms' tumour gene (WT1) expression and low mitotic count are independent predictors of survival in diffuse peritoneal mesothelioma.

AIMS: To evaluate the use of the Wilms' tumour gene (WT1) marker and histomorphological parameters as indicators of prognosis in malignant peritoneal mesothelioma (MPM). METHODS AND RESULTS: Histological samples of 31 MPM were stained immunohistochemically for the WT1 protein. The results were quantified by recording the number of stained nuclei, and then correlated with patient survival. Statistical correlation was evaluated for tumour histotype, mitotic count (MC), nuclear grade (NG), necrosis, lymphoid response (grade of inflammation) and desmoplasia with regard to survival. High-grade histology (solid epithelioid, pure sarcomatoid or biphasic tumours), high NG, MC more than five per 10 per high-power field (HPF), necrosis and desmoplasia were associated with a significantly worse prognosis. Patients with MPM with low WT1 expression (≤25% of positive cells) survived for a significantly shorter time compared to those with high WT1 expression (>25% of positive cells) (P = 0.0001). The 50% survival time of subjects with low WT1 expression was 2.9 months [95% confidence interval (CI): 2.05-3.71] versus 31.5 months (95% CI: 20.4-42.5) for those with high WT1 expression. On multivariate analysis, WT1 and MC were found to be associated independently with survival (P = 0.002; P = 0.005, respectively). CONCLUSIONS: Our study suggests that low WT1 expression and high MC may be indicative of an unfavourable prognosis in patients with advanced malignant peritoneal mesothelioma.

Rfx6 directs islet formation and insulin production in mice and humans.

Insulin from the beta-cells of the pancreatic islets of Langerhans controls energy homeostasis in vertebrates, and its deficiency causes diabetes mellitus. During embryonic development, the transcription factor neurogenin 3 (Neurog3) initiates the differentiation of the beta-cells and other islet cell types from pancreatic endoderm, but the genetic program that subsequently completes this differentiation remains incompletely understood. Here we show that the transcription factor Rfx6 directs islet cell differentiation downstream of Neurog3. Mice lacking Rfx6 failed to generate any of the normal islet cell types except for pancreatic-polypeptide-producing cells. In human infants with a similar autosomal recessive syndrome of neonatal diabetes, genetic mapping and subsequent sequencing identified mutations in the human RFX6 gene. These studies demonstrate a unique position for Rfx6 in the hierarchy of factors that coordinate pancreatic islet development in both mice and humans. Rfx6 could prove useful in efforts to generate beta-cells for patients with diabetes.

Characterization of a complex chromosome aberration in two cases of peritoneal mesothelioma arising primarily in the hernial sac.

Malignant mesotheliomas of the hernial sac are uncommon and only a few cases have been diagnosed incidentally during herniorrhaphy procedures. The prognosis is poor and patient management is difficult because current treatment modalities do little to prolong survival. Molecular markers could be useful to identify potential therapeutic targets. Using microarray-comparative genomic hybridization (aCGH), two cases of peritoneal mesothelioma that were found incidentally at the time of hernia repair, were investigated. A high number of genetic aberrations was detected in both cases. The gains were prevalent. The tumors showed identical lost regions at 2q13, 6q25.3, 6q26, 6q26-->q27, 9q31.1-->9q31.3, 10p15.3, 11q13.2, 13q14.2, 19q13.42-->q43, and gains at 1p36.33, 3q29, 5p15.33, 7p22.3, 10p15.1-->10p14, 11q13.2, 12q24.23, 12q24.33, 16p13.3, 17p13.3, 18p11.31, 19q13.43, 21q21.1-->q21.2, 22q11.1-->q11.22, Xp21.2, Xq28. Survival was longer in the patient with a lower total number of genetic defects. aCGH provides a high-resolution map of copy number changes that may be critical to mesothelioma progression.