Extensive molecular analysis suggested the strong genetic heterogeneity of idiopathic chronic pancreatitis.

Rationale: Genetic features of Chronic Pancreatitis (CP) have been extensively investigated mainly testing genes associated to the trypsinogen activation pathway. However, different molecular pathways involving other genes may be implicated in CP pathogenesis. Objectives: 80 patients with Idiopathic CP were investigated using Next Generation Sequencing approach with a panel of 70 genes related to six different pancreatic pathways: premature activation of trypsinogen; modifier genes of Cystic Fibrosis phenotype; pancreatic secretion and ion homeostasis; Calcium signalling and zymogen granules exocytosis; autophagy; autoimmune pancreatitis related genes. Results: We detected mutations in 34 out of 70 genes examined; 64/80 patients (80.0%) were positive for mutations in one or more genes, 16/80 patients (20.0%) had no mutations. Mutations in CFTR were detected in 32/80 patients (40.0%) and 22 of them exhibited at least one mutation in genes of other pancreatic pathways. Of the remaining 48 patients, 13/80 (16.3%) had mutations in genes involved in premature activation of trypsinogen and 19/80 (23.8%) had mutations only in genes of the other pathways: 38/64 patients positive for mutations showed variants in two or more genes (59.3%). Conclusions: Our data, although to be extended with functional analysis of novel mutations, suggest a high rate of genetic heterogeneity in chronic pancreatitis and that trans-heterozygosity may predispose to the idiopathic CP phenotype.

CHARGE syndrome due to deletion of region upstream of CHD7 gene START codon.

BACKGROUND: CHARGE syndrome is an autosomal dominant disorder, characterized by ocular Coloboma, congenital Heart defects, choanal Atresia, Retardation, Genital anomalies and Ear anomalies. Over 90 % of typical CHARGE patients are mutated in the CHD7 gene, 65 %-70 % of the cases for all typical and suspected cases combined. The gene encoding for a protein involved in chromatin organization. The mutational spectrum include nonsense, frameshift, splice site, and missense mutations. Large deletions and genomic rearrangements are rare. CASE PRESENTATION: We report here on a 5.9 years old male of Moroccan origin displaying classic clinical features of CHARGE syndrome. Using CGH array and NGS analysis we detected a microdeletion (184 kb) involving the promoter region and exon 1 of CHD7 gene and the flanking RAB2 gene. CONCLUSION: The present observation suggests that deletion limited to the regulatory region of CHD7 is sufficient to cause the full blown CHARGE phenotype. Different size of deletions can result in different phenotypes, ranging from a milder to severe CHARGE syndrome; this is based on a combination of major and minor diagnostic characteristics, therefore to a more variable clinical features, likely due to the additive effect of other genetic imbalances. MLPA and CGH techniques should be considered in the diagnostic protocol of individuals with a clinical suspect of CHARGE syndrome.

Hypoplastic left heart syndrome and 21q22.3 deletion.

Hypoplastic left heart syndrome (HLHS) is a rare congenital heart defect (CHD), associated with extracardiac anomalies in the 15-28% of cases, in the setting of chromosomal anomalies, mendelian disorders, and organ defects. We report on a syndromic female newborn with HLHS and terminal 21q22.3 deletion (del 21q22.3), investigated by Fluorescence In Situ Hybridization (FISH) using a panel of 26 contiguous BAC probes. Although rare, del 21q22.3 has been described in two additional patients with HLHS. In order to investigate the frequency and role of this chromosomal imbalance in the pathogenesis of left-sided obstructive heart defects, we screened for del 21q22.3 a series of syndromic and non-syndromic children with HLHS, aortic coarctation and valvular aortic stenosis, consecutively admitted to our hospital in a three-year period. Although none of the 56 analyzed patients were hemizygous for this region, the present case report and published patients argue that del 21q22 should be added to the list of chromosomal imbalances associated with HLHS. Accordingly, the presence of a cardiac locus mapping in the critical region cannot be excluded.

A Novel Variant in RAD21 in Cornelia De Lange Syndrome Type 4: Case Report and Bioinformatic Analysis.

Cornelia de Lange Syndrome (CdLS) is a rare genetic disorder that affects many organs. The diagnosis of this condition is primarily clinical and it can be confirmed by molecular analysis of the genes known to cause this disease, although about 30% of CdLS patients are without a genetic diagnosis. Here we report clinical and genetic findings of a patient with CdLS type 4, a syndrome of which the clinical features of only 30 patients have been previously described in the literature. The index patient presented with clinical characteristics previously associated with CdLS type 4 (short nose, thick eyebrow, global development delay, synophrys, microcephaly, weight < 2DS, small hands, height < 2DS). She also presented cardiac anomalies, cleft palate and laryngomalacia, which was never described before. The index patient was diagnosed with a novel de novo RAD21 variant (c.1722_1723delTG, p.Gly575SerfsTer2): segregation analysis, bioinformatic analysis, population data and in silico structural modelling indicate the pathogenicity of the novel variant. This report summarizes previously reported clinical manifestations of CdLS type 4 but also highlights new clinical symptoms, which will aid correct counselling of future CdLS type 4 cases.

Identification of a Novel Mutation in TNFAIP3 in a Family With Poly-Autoimmunity.

Haploinsufficiency of A20 (HA20) is an inflammatory disease caused by mutations in the TNFAIP3 gene classically presenting with Behcet's-like disease. A20 acts as an inhibitor of inflammation through its effect on NF-kB pathway. Here we describe four consanguineous patients (three sisters and their mother) with a predominantly autoimmune phenotype, including thyroiditis, type I diabetes, hemolytic anemia and chronic polyarthritis. All patients had recurrent oral ulcers, with only 1 patient presenting also recurrent fever episodes, as a classical autoinflammatory feature. Next generation sequencing identified a novel heterozygous frameshift mutation (p.His577Alafs*95) that causes a premature stop codon in the zinc finger domain of A20, leading to a putative haploinsufficiency of the protein. Functional analyses confirmed the pathogenicity of the mutation. The variant was associated with decreased levels of A20 in blood cells. Accordingly, ex-vivo lipopolysaccharide (LPS)-stimulated patients' peripheral blood mononuclear cells (PBMCs) showed higher levels of p65 NF-kB phosphorylation, as well as increased production of the proinflammatory cytokines IL-1ß, IL-6 and TNF-α. Moreover, in agreement with recent observations, demonstrating a role for A20 in inhibiting STAT1 and IFNγ pathways, markedly higher circulating levels of the two IFNγ-inducible chemokines CXCL9 and CXCL10 were detected in all patients. Supporting the findings of a hyperactivation of IFNγ signaling pathway in HA20 patients, patients' monocytes showed higher levels of STAT1 without stimulation, as well as higher phosphorylated (active) STAT1 levels following IFNγ stimulation. In conclusion, our study show that in the clinical spectrum of HA20 autoimmune features may predominate over autoinflammatory features and demonstrate, from a molecular point of view, the involvement of A20 in modulating not only the NF-kB, but also the IFNγ pathway.

Clinical hallmarks and genetic polymorphisms in the CFTR gene contribute to the disclosure of the A1006E mutation.

Since the identification of the Cystic Fibrosis transmembrane conductance regulator (CFTR) gene in 1989, many genetic mutations have been found in cystic fibrosis (CF) patients. Dysfunctions of the CFTR gene are responsible for the highly variable clinical presentation ranging from severe CF, disseminated bronchiectasis, idiopathic chronic pancreatitis and congenital bilateral absence of vas deferens (CBAVD). Linkage disequilibrium studies have shown that some mutations are stringently coupled with polymorphisms in a genetic complex called haplotype. From a familial study of a patient with CBAVD, carrier of the A1006E mutation, we have observed its strict association with the polymorphism 5T-TG11. In order to speed up the genetic diagnosis and to correlate the clinical setting to this genetic feature, we have directly investigated the exon 17a, where the A1006E mutation is located, of five cystic fibrosis patients belonging to two unrelated families. All patients had the 5T-TG11 tract, F508del and one unknown mutation. One more family with two affected individuals carrying the Q220X/A1006E mutations was investigated for the poly-T polymorphism. All the members were found to have the A1006E mutation and the 5T-TG11 in the same DNA strand, demonstrating that this strategy is a reliable and inexpensive method for genotyping the CFTR gene. A detailed description of the clinical presentation and follow-up are provided in order to highlight common phenotypic features useful to improve the management of cystic fibrosis patients.

NK cell effector functions in a Chédiak-Higashi patient undergoing cord blood transplantation: Effects of in vitro treatment with IL-2.

NK cell cytotoxicity in Chédiak-Higashi syndrome (CHS) is strongly impaired as lytic granules are not released upon NK-target cell contact, contributing to several defects typical of this severe immunodeficiency. Correction of NK cell defects in CHS should improve the outcome of hematopoietic stem-cell transplantation, proposed as therapy. We investigated NK cell functions in a CHS patient before and after cord-blood transplantation, and the ability of in vitro IL-2 treatment to restore them. Before the transplant, the strong defect in NK cell-mediated natural and antibody-dependent cytotoxicity, as well as in IFN-γ production, could be restored up to normal levels by in vitro IL-2 treatment. This cytokine also caused the appearance of smaller lysosomal granules and their orientation towards the NK-target cell contact area, thus suggesting that IL-2 had a more general capacity to restore NK cell effector functions. Moreover after the transplant, although the successful engraftment, NK cell cytotoxicity resulted still partially impaired at one year, almost normal at ten years and, anyhow, fully recovered by in vitro IL-2 treatment. Taken together, our results indicate that IL-2 had a wide capacity to restore NK cell effector functions, being able to reverse the altered cytotoxic activity, lytic granule pattern, and cytokine production observed in the CHS patient.

Relationship between CFTR and CTRC variants and the clinical phenotype in late-onset cystic fibrosis disease with chronic pancreatitis.

Cystic fibrosis (CF), the most common autosomal recessive disease in whites, is caused by mutations in the CF transmembrane conductance regulator (CFTR). So far, >1900 mutations have been described, most of which are nonsense, missense, and frameshift, and can lead to severe phenotypes, reducing the level of function of the CFTR protein. Synonymous variations are usually considered silent without pathogenic effects. However, synonymous mutations exhibiting exon skipping as a consequence of aberrant splicing of pre-mRNA differ. Herein, we describe the effect of the aberrant splicing of the c.273G>C (G91G) synonymous variation found in a 9-year-old white (ΔF508) patient affected by CF and pancreatitis associated with a variant in chymotrypsin C (CTRC). Magnetic resonance imaging showed an atrophic pancreatic gland with substitution of the pancreatic parenchyma with three cysts. Genetic examination revealed compound heterozygosity for the c.1521_1523delCTT (ΔF508) pathogenic variant and the c.273G>C (G91G) variant in CFTR. Sweat test results confirmed the diagnosis of CF. We have thus identified a synonymous variation (G91G) causing the skipping of exon 3 in a CF patient carrying the ΔF508 mutation. However, the clinical phenotype with pancreatic symptoms encouraged us to investigate a panel of pancreas-related genes, which resulted in finding a known sequence variation inside CTRC. We further discuss the role of these variants and their possible interactions in determining the current phenotype.

Kabuki syndrome: clinical and molecular diagnosis in the first year of life.

OBJECTIVE: To review the clinical and molecular genetic characteristics of 16 patients presenting a suspected diagnosis of Kabuki syndrome (KS) in the first year of life, to evaluate the clinical handles leading to a prompt diagnosis of KS in newborns. Clinical diagnosis of KS can be challenging during the first year of life, as many diagnostic features become evident only in subsequent years. METHODS: All patients were clinically investigated by trained clinical geneticists. A literature review was performed using the Pubmed online database and diagnostic criteria suggested by DYSCERNE-Kabuki Syndrome Guidelines (2010) were used (a European Network of Centres of Expertise for Dysmorphology, funded by the European Commission Executive Agency for Health and Consumers (DG Sanco), Project 2006122). Molecular analysis of the known causative genes of KS, KMT2D/MLL2 and KDM6A, was performed through MiSeq-targeted sequencing platform. All mutations identified were validated by Sanger sequencing protocols. RESULTS: Mutations in KMT2D gene were identified in 10/16 (62%) of the patients, whereas none of the patients had KDM6A mutations. Facial dysmorphisms (94%), feeding difficulties (100%) and hypotonia (100%) suggested the clinical diagnosis of KS. No significative differences in terms of facial features were noticed between mutation positive and negative patients of the cohort. Brachydactyly, joint laxity and nail dysplasia were present in about 80% of the patients. Other congenital anomalies were most commonly present in the mutated group of patients, including left-sided cardiac abnormalities, skeletal, renal and anorectal malformations and hypertricosis. CONCLUSIONS: We present an overview of patients with KS diagnosed during the first year of life. Early diagnosis is serviceable in terms of clinical management and for targeted genetic counselling.

Epilepsy in patients with duplications of chromosome 14 harboring FOXG1.

BACKGROUND: Dup(14q12) harboring FOXG1 has been recently reported in individuals with developmental delay of variable severity, delayed/absent speech, and epilepsy/infantile spasms. FOXG1 was described as a dosage-sensitive gene encoding G1, a forkhead protein that is a brain-specific transcription factor with a role in brain development. PATIENTS: We extensively reviewed all published cases with dup(14) harboring FOXG1 and highlighted those epileptological features that are more commonly found among such cases. We also describe one new patient, detailing his peculiar clinical and neurophysiological findings. RESULTS: To date, 15 patients with dup(14) including FOXG1 have been reported; within those patients, nine also presented with epilepsy. At onset, the more frequent seizure type in the report and also in our patient is the epileptic spasm. Focal seizures might also be present. Outcomes in patients with epilepsy associated with dup(14) should be considered separately regarding seizures and cognitive and motor development. In the majority of patients (seven of 10, including ours), seizures tend to disappear and motor skills improve; however, instead stagnation of cognitive development is evident in all of them, associated with severe speech difficulties. CONCLUSIONS: There are some common features that should be considered: seizures with onset during the first year of life, particularly clusters of spasms and focal seizures with hypsarrhythmic electroencephalograph pattern; different degrees of cognitive impairment possibly associated with behavior disturbances and severe speech disabilities; and dysmorphic features in the absence of significant microcephaly.

Telomere shortening and telomere position effect in mild ring 17 syndrome.

BACKGROUND: Ring chromosome 17 syndrome is a rare disease that arises from the breakage and reunion of the short and long arms of chromosome 17. Usually this abnormality results in deletion of genetic material, which explains the clinical features of the syndrome. Moreover, similar phenotypic features have been observed in cases with complete or partial loss of the telomeric repeats and conservation of the euchromatic regions. We studied two different cases of ring 17 syndrome, firstly, to clarify, by analyzing gene expression analysis using real-time qPCR, the role of the telomere absence in relationship with the clinical symptoms, and secondly, to look for a new model of the mechanism of ring chromosome transmission in a rare case of familial mosaicism, through cytomolecular and quantitative fluorescence in-situ hybridization (Q-FISH) investigations. RESULTS: The results for the first case showed that the expression levels of genes selected, which were located close to the p and q ends of chromosome 17, were significantly downregulated in comparison with controls. Moreover, for the second case, we demonstrated that the telomeres were conserved, but were significantly shorter than those of age-matched controls; data from segregation analysis showed that the ring chromosome was transmitted only to the affected subjects of the family. CONCLUSIONS: Subtelomeric gene regulation is responsible for the phenotypic aspects of ring 17 syndrome; telomere shortening influences the phenotypic spectrum of this disease and strongly contributes to the familial transmission of the mosaic ring. Together, these results provide new insights into the genotype-phenotype relationships in mild ring 17 syndrome.

Complex chromosome rearrangements related 15q14 microdeletion plays a relevant role in phenotype expression and delineates a novel recurrent syndrome.

Complex chromosome rearrangements are constitutional structural rearrangements involving three or more chromosomes or having more than two breakpoints. These are rarely seen in the general population but their frequency should be much higher due to balanced states with no phenotypic presentation. These abnormalities preferentially occur de novo during spermatogenesis and are transmitted in families through oogenesis.Here, we report a de novo complex chromosome rearrangement that interests eight chromosomes in eighteen-year-old boy with an abnormal phenotype consisting in moderate developmental delay, cleft palate, and facial dysmorphisms.Standard G-banding revealed four apparently balanced translocations [corrected] involving the chromosomes 1;13, 3;19, 9;15 and 14;18 that appeared to be reciprocal. Array-based comparative genomic hybridization analysis showed no imbalances at all the breakpoints observed except for an interstitial microdeletion on chromosome 15. This deletion is 1.6 Mb in size and is located at chromosome band 15q14, distal to the Prader-Willi/Angelman region. Comparing the features of our patient with published reports of patients with 15q14 deletion this finding corresponds to the smallest genomic region of overlap. The deleted segment at 15q14 was investigated for gene content.

Characterization of a novel isolated deletion of the exon 3 within the CFTR gene: Relevance for phenotypic expression and genetic counseling.

OBJECTIVES: To characterize a novel deletion of exon 3 of CFTR gene and to evaluate the implications in Cystic Fibrosis (CF) care and genetic counseling. DESIGN AND METHODS: We performed a wide mutational analysis of CFTR gene, using reverse dot blot, Multiplex Ligation-dependent Probe Amplification (MLPA) assay and Real Time Quantitative PCR, in a carrier male and two CF patients with the F508del mutation. RESULTS: We found a novel isolate 538bp deletion of exon 3, described as 328del538, giving rise to a nonsense codon 60bp at the 3' end of the new coding sequence or, alternatively, a novel splice site at the breakpoints. CONCLUSIONS: The 328del538 is a rare lesion with the characteristics of a complete, but moderate, phenotypic expression. Its finding underlines the importance of improving the detection of mutations using different methods.