ObjectivesThe aims of this study were to test the utility of benchtop NGS platforms for NIPT for trisomy 21 using previously published z score calculation methods and to optimize the sample preparation and data analysis with use of in silico and physical size selection methods.MethodsSamples from 130 pregnant women were analyzed by whole genome sequencing on benchtop NGS systems Ion Torrent PGM and MiSeq. The targeted yield of 3 million raw reads on each platform was used for z score calculation. The impact of in silico and physical size selection on analytical performance of the test was studied.ResultsUsing a z score value of 3 as the cut-off, 98.11% - 100% (104-106/106) specificity and 100% (24/24) sensitivity and 99.06% - 100% (105-106/106) specificity and 100% (24/24) sensitivity were observed for Ion Torrent PGM and MiSeq, respectively. After in silico based size selection both platforms reached 100% specificity and sensitivity. Following the physical size selection z scores of tested trisomic samples increased significantly—p = 0.0141 and p = 0.025 for Ion Torrent PGM and MiSeq, respectively.ConclusionsNoninvasive prenatal testing for chromosome 21 trisomy with the utilization of benchtop NGS systems led to results equivalent to previously published studies performed on high-to-ultrahigh throughput NGS systems. The in silico size selection led to higher specificity of the test. Physical size selection performed on isolated DNA led to significant increase in z scores. The observed results could represent a basis for increasing of cost effectiveness of the test and thus help with its penetration worldwide.

BACKGROUND:In the workup of patients with suspected hereditary nonpolyposis colorectal cancer (HNPCC), detection of loss of heterozygosity (LOH) could help pinpoint the mismatch-repair (MMR) gene carrying the germline mutation, but analysis of microsatellite markers has proved unreliable for this purpose. We developed a simple, low-cost method based on singlenucleotide polymorphism (SNP) genotyping and capillary electrophoresis for the assessment of LOH at 2 MMR loci simultaneously.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a dominantly-inherited cancer predisposition syndrome, in which the susceptibility to cancer of the colon, endometrium and ovary is linked to germline mutations in DNA mismatch repair (MMR) genes. We have recently initiated a cancer prevention program in suspected HNPCC families in the Slovak Republic. The first ten families fulfilling Amsterdam criteria or Bethesda guidelines were screened for germline mutations in MLH1 and MSH2, two MMR genes most frequently mutated in HNPCC families. Six mutations were identified, five of which have not been reported previously. Two of the three new mutations in MLH1 (c.380+2T>A; c.307-2A>C) were absent from 100 chromosomes of healthy controls and probably cause a splicing defect, while the third was a 1 bp deletion (c.1261delA). In the MSH2 gene, one new nonsense (c.1030C>T [p.Q344X]) and one missense (c.524T>C [p.L175P]) mutation were identified. This latter variant was not found in 104 alleles of healthy control individuals. Moreover, a previously-reported pathogenic mutation (c.677G>T [p.R226L]) was found in one kindred. The clinical data and the genotypic and phenotypic evaluation of the tumors indicate that all the new alterations are pathogenic HNPCC mutations.

Background. Myelodysplastic syndromes (MDS) represent a heterogeneous group of premalignant hematologic disorders characterized by ineffective hematopoiesis, peripheral blood cytopenias and increased risk of progression to acute leukemia. Cytogenetic analysis still plays a central role in the diagnosis of MDS, as clonal chromosomal abnormalities are observed in 30-50% of MDS patients. Despite their technical limitations, standard karyotyping and fluorescence in situ hybridization (FISH) are routinely used for identifying recurrent chromosomal rearrangements. However, using this approach means that submicroscopic and not targeted chromosomal aberrations, as well as somatic mutations and epigenetic changes remain largely undetected. Methods and Results. Introduction of methods for the analysis of copy-number variations (CNV), including arraybased technologies and Multiplex ligation-dependent probe amplification (MLPA) has provided novel insights into the molecular pathogenesis of MDS and considerably extended possibilities for genetic laboratory testing. Several novel molecular markers have been discovered and used for diagnosis and prognostic evaluation of patients with MDS. At present, mutational analysis is not routinely performed, as the clinical significance of somatic mutations in MDS has only begun to emerge. However, recently introduced Next-generation sequencing (NGS) technologies could help to elucidate the relationship between chromosomal and molecular aberrations in MDS and lead to further improvement in its diagnosis. Conclusion. This review focuses on the advantages, limitations, clinical applications and future perspectives of three molecular methods (array-based analysis, MLPA and NGS) currently used in genetic testing and/ or translational research of MDS. In conclusion, a brief summary for clinicians from the routine diagnostic point of view is given.

The recessive PIEZO2-associated disease, distal arthrogryposis with impaired proprioception and touch (DAIPT), is characterized by hypotonia, perinatal respiratory distress, significantly delayed motor milestones, and progressive symptoms of distal arthrogryposis and scoliosis. Here, we describe the youngest patient with DAIPT to date, who, at the age of 3.5 years, did not show a single clinical sign of distal arthrogryposis or contractures, but had a history of bilateral clubfoot operations. On the contrary, he presented with some features, not described thus far, such as syringohydromyelia, a small cyst of the spinal cord, moderate microcephaly with premature closure of anterior fontanelle, and spontaneous unilateral patella dislocation at the age of 32 months. Using whole exome sequencing, we identified 2 new different loss-of-function mutations in the PIEZO2 gene in our patient. We also review the phenotypes of all 16 previously published patients with DAIPT, summarize the distinctive clinical features of this rare genetic disorder, and recommend that DAIPT be included in the differential diagnosis of floppy infant. PIEZO2 is a unique ion channel that converts mechanical impulses into cellular signals and is involved in various mechanotransduction pathways. In addition to DAIPT, mutations in PIEZO2 have been described to cause 3 more distinct phenotypes of distal arthrogryposis, which are dominant and associated with gain-of-function mutations. On the contrary, recessive DAIPT is associated with loss-of-function PIEZO2 mutations.

Exome sequencing has been increasingly implemented in prenatal genetic testing for fetuses with morphological abnormalities but normal rapid aneuploidy detection and microarray analysis. We present a retrospective study of 90 fetuses with different abnormal ultrasound findings, in which we employed the singleton exome sequencing (sES; 75 fetuses) or to a lesser extent (15 fetuses) a multigene panel analysis of 6713 genes as a primary tool for the detection of monogenic diseases. The detection rate of pathogenic or likely pathogenic variants in this study was 34.4%. The highest diagnostic rate of 56% was in fetuses with multiple anomalies, followed by cases with skeletal or renal abnormalities (diagnostic rate of 50%, respectively). We report 20 novel disease-causing variants in different known disease-associated genes and new genotype–phenotype associations for the genes KMT2D, MN1, CDK10, and EXOC3L2. Based on our data, we postulate that sES of fetal index cases with a concurrent sampling of parental probes for targeted testing of the origin of detected fetal variants could be a suitable tool to obtain reliable and rapid prenatal results, particularly in situations where a trio analysis is not possible.

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Surprisingly little is known about the critical metabolic changes that neural cells have to undergo during development and how even mild, temporary shifts in this program can influence brain circuitries and behavior. Inspired by the discovery that mutations in SLC7A5, a transporter of metabolically-relevant large neutral amino acids, lead to a form of autism spectrum disorder, we employed metabolomic profiling to study the metabolic states of the cerebral cortex across different stages of life. We found that the cerebral cortex undergoes significant metabolic remodeling throughout development, with certain groups of metabolites showing stage-specific changes. But what are the consequences of interfering with this metabolic program? By manipulating Slc7a5 expression in neural cells, we found that the metabolism of large neutral amino acids and lipids in the cerebral cortex are highly interconnected. Deletion of Slc7a5 in neurons perturbs specifically the postnatal metabolic state leading to a shift in lipid metabolism and a stage- and cell-type-specific alteration in neuronal activity patterns, resulting in a long-term cortical circuit dysfunction.