Use of the MLPA Assay in the Molecular Diagnosis of Gene Copy Number Alterations in Human Genetic Diseases

Multiplex Ligation-dependent Probe Amplification (MLPA) assay is a recently developed technique able to evidence variations in the copy number of several human genes. Due to this ability, MLPA can be used in the molecular diagnosis of several genetic diseases whose pathogenesis is related to the presence of deletions or duplications of specific genes. Moreover, MLPA assay can also be used in the molecular diagnosis of genetic diseases characterized by the presence of abnormal DNA methylation. Due to the large number of genes that can be analyzed by a single technique, MLPA assay represents the gold standard for molecular analysis of all pathologies derived from the presence of gene copy number variation. In this review, the main applications of the MLPA technique for the molecular diagnosis of human diseases are described

Testis transcriptome analysis in male infertility: new insight on the pathogenesis of oligo-azoospermia in cases with and without AZFc microdeletion

<p>Abstract</p> <p>Background</p> <p>About 10% of cases of male infertility are due to the presence of microdeletions within the long arm of the Y chromosome (Yq). Despite the large literature covering this critical issue, very little is known about the pathogenic mechanism leading to spermatogenesis disruption in patients carrying these microdeletions. In order to identify the presence of specific molecular pathways leading to spermatogenic damage, testicular gene expression profiling was carried out by employing a microarray assay in 16 patients carrying an AZFc microdeletion or affected by idiopathic infertility. Hierarchical clustering was performed pooling the data set from 26 experiments (16 patients, 10 replicates).</p> <p>Results</p> <p>An intriguing and unexpected finding is that all the samples showing the AZFc deletion cluster together irrespectively of their testicular phenotypes. This cluster, including also four patients affected by idiopathic infertility, showed a downregulation of several genes related to spermatogenesis that are mainly involved in testicular mRNA storage. Interestingly, the four idiopathic patients present in the cluster showed no testicular expression of <it>DAZ </it>despite the absence of AZFc deletion in the peripheral blood.</p> <p>Conclusions</p> <p>Our expression profiles analysis indicates that several forms of infertility can be triggered by a common pathogenic mechanism that is likely related to alterations in testicular mRNA storage. Our data suggest that a lack of testicular DAZ gene expression may be the trigger of such mechanism. Furthermore, the presence of AZFc deletions in mosaic or the loss of function of AZFc genes in absence of Yq deletion can perhaps explain these findings. Finally, based on our data, it is intriguing to hypothesize that <it>DAZ </it>gene dysfunctions can account for a larger number of previously thought "idiopathic" infertility cases and investigation of such testicular gene dysfunction can be important to reveal the molecular determinant of infertility than are undetected when only testing Yq deletions in peripheral blood.</p

Isolation of osteogenic progenitors from human amniotic fluid using a single step culture protocol

<p>Abstract</p> <p>Background</p> <p>Stem cells isolated from amniotic fluid are known to be able to differentiate into different cells types, being thus considered as a potential tool for cellular therapy of different human diseases. In the present study, we report a novel single step protocol for the osteoblastic differentiation of human amniotic fluid cells.</p> <p>Results</p> <p>The described protocol is able to provide osteoblastic cells producing nodules of calcium mineralization within 18 days from withdrawal of amniotic fluid samples. These cells display a complete expression of osteogenic markers (COL1, ONC, OPN, OCN, OPG, BSP, Runx2) within 30 days from withdrawal. In order to test the ability of these cells to proliferate on surfaces commonly used in oral osteointegrated implantology, we carried out cultures onto different test disks, namely smooth copper, machined titanium and Sandblasted and Acid Etching titanium (SLA titanium). Electron microscopy analysis evidenced the best cell growth on this latter surface.</p> <p>Conclusion</p> <p>The described protocol provides an efficient and time-saving tool for the production of osteogenic cells from amniotic fluid that in the future could be used in oral osteointegrated implantology.</p

PLCB1 (phospholipase C, beta 1 (phosphoinositide-specific))

Review on PLCB1 (phospholipase C, beta 1 (phosphoinositide-specific)), with data on DNA, on the protein encoded, and where the gene is implicated

Identification of the second CFTR mutation in patients with congenital bilateral absence of vas deferens undergoing ART protocols.

Congenital bilateral absence of vas deferens (CBAVD) is a manifestation of the mildest form of cystic fibrosis (CF) and is characterized by obstructive azoospermia in otherwise healthy patients. Owing to the availability of assisted reproductive technology, CBAVD patients can father children. These fathers are at risk of transmitting a mutated allele of the CF transmembrane conductance regulator (CFTR) gene, responsible for CF, to their offspring. The identification of mutations in both CFTR alleles in CBAVD patients is a crucial requirement for calculating the risk of producing a child with full-blown CF if the female partner is a healthy CF carrier. However, in the majority of CBAVD patients, conventional mutation screening is not able to detect mutations in both CFTR alleles, and this difficulty hampers the execution of correct genetic counselling. To obtain information about the most represented CFTR mutations in CBAVD patients, we analysed 23 CBAVD patients, 15 of whom had a single CFTR mutation after screening for 36 mutations and the 5T allele. The search for the second CFTR mutation in these cases was performed by using a triplex approach: (i) first, a reverse dot-blot analysis was performed to detect mutations with regional impact; (ii) next, multiple ligation-dependent probe amplification assays were conducted to search for large rearrangements; and (iii) finally, denaturing high-performance liquid chromatography was used to search for point mutations in the entire coding region. Using these approaches, the second CFTR mutation was detected in six patients, which increased the final detection rate to 60.8%

A case of triploidy detected by crosstrimester test

A 40-year-old woman presented in her second pregnancy, naturally conceived. Maternal serum screening and ultrasound examination raised concerns regarding aneuploidy. After genetic counselling an amniocentesis was performed, showing a 69,XXX karyotype. Here we report a case of digynic triploidy, which resulted from fertilization of a diploid ovum by a single sperm

Testis Transcriptome Modulation in Klinefelter Patients with Hypospermatogenesis

The main genetic cause of male infertility is represented by the Klinefelter Syndrome (KS), a condition accounting for 3% of all cases of infertility and up to15% of cases of azoospermia. KS is generally characterized by azoospermia; approximately 10% of cases have severe oligozoospermia. Among these, the 30–40% of patients show hypospermatogenesis. The mechanisms leading to adult testis dysfunctions are not completely understood. A microarray transcriptome analysis was performed on testis biopsies obtained from three KS patients with hypospermatogenesis and three control subjects. KS testis showed a differential up- and down-regulation of 303 and 747 transcripts, respectively, as compared to controls. The majority of down-regulated transcripts were involved in spermiogenesis failure and testis morphological defects, whereas up-regulated genes were responsible for testis apoptotic processes. Functional analysis of the transcriptionally altered genes indicated a deregulation in cell death, germ cell function and morphology as well as blood-testis-barrier maintenance and Leydig cells activity. These data support a complex scenario in which spermatogenic impairment is the result of functional and morphological alterations in both germinal and somatic components of KS testis. These findings could represent the basis for evaluating new markers of KS spermatogenesis and potential targets of therapeutic intervention to preserve residual spermatogenesis