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.

Oral Signs and HLA-DQB1∗02 Haplotypes in the Celiac Paediatric Patient: A Preliminary Study.

Celiac disease (CD) diagnosis can be extremely challenging in the case of atypical patterns. In this context, oral signs seem to play a decisive role in arousing suspicion of these forms of the disease. At the same time, the different expressions of the HLA-DQB1∗02 allele apparently seem to facilitate the interpretation of signs and highlighted symptoms. The aim of this work was to verify whether it is possible to identify a correlation between the development of oral signs and different DQ2 haplotypes in celiac pediatric patients. 44 celiac patients with a medium age of 9.9 were studied. Oral examinations were performed in order to identify recurrent aphthous stomatitis (RAS) and dental enamel defects (DED). The diagnosis of DED resulted as being related to allele expression (P value = 0.042) while it was impossible to find a similar correlation with RAS. When both oral signs were considered, there was an increase in correlation with HLA-DQB1∗02 expression (P value = 0.018). The obtained results identified both the fundamental role that dentists can play in early diagnosis of CD, as well as the possible role of HLA haplotype analysis in arousing suspicion of atypical forms of the disease.

Refining the phenotype associated with MEF2C haploinsufficiency.

Recently, submicroscopic deletions of the 5q14.3 region have been described in patients with severe mental retardation (MR), stereotypic movements, epilepsy and cerebral malformations. Further delineation of a critical region of overlap in these patients pointed to MEF2C as the responsible gene. This finding was further reinforced by the identification of a nonsense mutation in a patient with a similar phenotype. In brain, MEF2C is essential for early neurogenesis, neuronal migration and differentiation. Here we present two additional patients with severe MR, autism spectrum disorder and epilepsy, carrying a very small deletion encompassing the MEF2C gene. This finding strengthens the role of this gene in severe MR, and enables further delineation of the clinical phenotype.

Periventricular heterotopia, mental retardation, and epilepsy associated with 5q14.3-q15 deletion.

BACKGROUND: Periventricular heterotopia (PH) is an etiologically heterogeneous disorder characterized by nodules of neurons ectopically placed along the lateral ventricles. Most affected patients have seizures and their cognitive level varies from normal to severely impaired. At present, two genes have been identified to cause PH when mutated. Mutations in FLNA (Xq28) and ARFGEF2 (20q13) are responsible for X-linked bilateral PH and a rare autosomal recessive form of PH with microcephaly. Chromosomal rearrangements involving the 1p36, 5p15, and 7q11 regions have also been reported in association with PH but the genes implicated remain unknown. Fourteen additional distinct anatomoclinical PH syndromes have been described, but no genetic insights into their causes have been gleaned. METHODS: We report the clinical and imaging features of three unrelated patients with epilepsy, mental retardation, and bilateral PH in the walls of the temporal horns of the lateral ventricles, associated with a de novo deletion of the 5q14.3-15 region. We used microarray-based comparative genomic hybridization to define the boundaries of the deletions. RESULTS: The three patients shared a common deleted region spanning 5.8 Mb and containing 14 candidate genes. CONCLUSION: We identified a new syndrome featuring bilateral periventricular heterotopia (PH), mental retardation, and epilepsy, mapping to chromosome 5q14.3-q15. This observation reinforces the extreme clinical and genetic heterogeneity of PH. Array comparative genomic hybridization is a powerful diagnostic tool for characterizing causative chromosomal rearrangements of limited size, identifying potential candidate genes for, and improving genetic counseling in, malformations of cortical development.

Clinical and molecular characteristics of 1qter microdeletion syndrome: delineating a critical region for corpus callosum agenesis/hypogenesis.

BACKGROUND: Patients with a microscopically visible deletion of the distal part of the long arm of chromosome 1 have a recognisable phenotype, including mental retardation, microcephaly, growth retardation, a distinct facial appearance and various midline defects including corpus callosum abnormalities, cardiac, gastro-oesophageal and urogenital defects, as well as various central nervous system anomalies. Patients with a submicroscopic, subtelomeric 1qter deletion have a similar phenotype, suggesting that the main phenotype of these patients is caused by haploinsufficiency of genes in this region. OBJECTIVE: To describe the clinical presentation of 13 new patients with a submicroscopic deletion of 1q43q44, of which nine were interstitial, and to report on the molecular characterisation of the deletion size. RESULTS AND CONCLUSIONS: The clinical presentation of these patients has clear similarities with previously reported cases with a terminal 1q deletion. Corpus callosum abnormalities were present in 10 of our patients. The AKT3 gene has been reported as an important candidate gene causing this abnormality. However, through detailed molecular analysis of the deletion sizes in our patient cohort, we were able to delineate the critical region for corpus callosum abnormalities to a 360 kb genomic segment which contains four possible candidate genes, but excluding the AKT3 gene.

Cryptic deletions are a common finding in "balanced" reciprocal and complex chromosome rearrangements: a study of 59 patients.

Using array comparative genome hybridisation (CGH) 41 de novo reciprocal translocations and 18 de novo complex chromosome rearrangements (CCRs) were screened. All cases had been interpreted as "balanced" by conventional cytogenetics. In all, 27 cases of reciprocal translocations were detected in patients with an abnormal phenotype, and after array CGH analysis, 11 were found to be unbalanced. Thus 40% (11 of 27) of patients with a "chromosomal phenotype" and an apparently balanced translocation were in fact unbalanced, and 18% (5 of 27) of the reciprocal translocations were instead complex rearrangements with >3 breakpoints. Fourteen fetuses with de novo, apparently balanced translocations, all but two with normal ultrasound findings, were also analysed and all were found to be normal using array CGH. Thirteen CCRs were detected in patients with abnormal phenotypes, two in women who had experienced repeated spontaneous abortions and three in fetuses. Sixteen patients were found to have unbalanced mutations, with up to 4 deletions. These results suggest that genome-wide array CGH may be advisable in all carriers of "balanced" CCRs. The parental origin of the deletions was investigated in 5 reciprocal translocations and 11 CCRs; all were found to be paternal. Using customized platforms in seven cases of CCRs, the deletion breakpoints were narrowed down to regions of a few hundred base pairs in length. No susceptibility motifs were associated with the imbalances. These results show that the phenotypic abnormalities of apparently balanced de novo CCRs are mainly due to cryptic deletions and that spermatogenesis is more prone to generate multiple chaotic chromosome imbalances and reciprocal translocations than oogenesis.

Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute.

There is great interest in mesenchymal stromal cells (MSCs) for cell-therapy and tissue engineering approaches. MSCs are currently expanded in vitro in the presence of fetal calf serum (FCS); however, FCS raises concerns when used in clinical grade preparations. The aim of this study was to evaluate whether MSCs expanded in medium supplemented with platelet-lysate (PL), already shown to promote MSC growth, are endowed with biological properties appropriate for cell-therapy approaches. We confirm previously published data showing that MSCs expanded in either FCS or PL display comparable morphology, phenotype, and differentiation capacity, while PL-MSCs were superior in terms of clonogenic efficiency and proliferative capacity. We further extended these data by investigating the immune-regulatory effect of MSCs on the alloantigen-specific immune response in mixed lymphocyte culture (MLC). We found that MSCs-PL are comparable to MSCs-FCS in their capacity to: (i) decrease alloantigen-induced cytotoxic activity; (ii) favor differentiation of CD4+ T-cell subsets expressing a Treg phenotype; (iii) increase early secretion of IL-10 in MLC supernatant, as well as induce a striking augmentation of IL-6 production. As compared with MSCs-PL, MSCs-FCS were more efficient in suppressing alloantigen-induced lymphocyte subset proliferation and reducing early IFNgamma-secretion. Resistance to spontaneous transformation into tumor cells of expanded MSCs was demonstrated by molecular karyotyping and maintenance of normal morphology/phenotype after prolonged in vitro culture. Our data support the immunological functional plasticity of MSCs and suggest that MSCs-PL can be used as an alternative to MSCs-FCS, although these latter cells might be more suitable for preventing/treating alloreactivity-related immune complications.

[Energy expenditure at rest and body composition in obese subjects before and after weight loss]. / Spesa energetica a riposo e composizione corporea in obesi prima e dopo dimagramento.

In 7 obese subjects (3 males and 4 females), before (T0) and after (T60) weight loss, and in 10 nonobese subjects (4 males and 6 females) as controls, resting energy expenditure (REE) and body composition were evaluated. In obese subjects fat-free mass (FFM) and fat mass (FM) were significantly reduced after weight loss. REE, expressed in absolute value, was significantly higher in obese subjects at T0 with respect to controls (1683 +/- 77.3 vs. 1425 +/- 70.5 Kcal/day; p less than 0.05); in obese group REE was significantly lower at T60 (1563 +/- 63.5 Kcal/day) than at T0 (p less than 0.01). The decrease in REE observed in the obese group after weight loss was related only to the loss of FFM (r = 0.88; p less than 0.01). REE, expressed in relation to FFM, was not different between control and obese subjects at T0 (28.64 +/- 0.42 vs 26.40 +/- 1.08 Kcal/Kg-FFM/day; pns); however, this value was significantly lower in the obese group at T60 (25.99 +/- 1.07 Kcal/Kg-FFM/day) with respect to controls (p less than 0.05). It is concluded that a reduction in REE, due at least in part to the loss of FFM, may contribute to the difficulty in the maintenance of body weight often observed in obese subjects after a period on hypocaloric diet.