Tracheal agenesis versus tracheal atresia: anatomical conditions, pathomechanisms and causes with a possible link to a novel MAPK11 variant in one case.

BACKGROUND: In this study we aimed to describe the morphological and pathogenetic differences between tracheal agenesis and tracheal atresia, which are not clearly distinguished from each other in the literature, and to contribute thereby to the understanding and management of these conditions. Both tracheal agenesis and tracheal atresia represent rare disorders of still unknown aetiology that cannot be detected by prenatal ultrasound. If the affected foetuses survive until birth these conditions result in respiratory failure and in futile attempts to rescue the infant's life. RESULTS: Autopsies and genetic analyses, including singleton or trio exome sequencing, were performed on five neonates/foetuses with tracheal agenesis and three foetuses with tracheal atresia. Tracheal agenesis was characterized by absence of the sublaryngeal trachea and presence of a bronchooesophageal fistula and by pulmonary isomerism and occurred as an isolated malformation complex or as part of a VACTERL association. Special findings were an additional so-called 'pig bronchus' and a first case of tracheal agenesis with sirenomelia. Tracheal atresia presenting with partial obliteration of its lumen and persistence of a fibromuscular streak resulted in CHAOS. This condition was associated with normal lung lobulation and single, non-VACTERL type malformations. Trio ES revealed a novel variant of MAPK11 in one tracheal agenesis case. Its involvement in tracheooesophageal malformation is herein discussed, but remains hypothetical. CONCLUSION: Tracheal agenesis and tracheal atresia represent different disease entities in terms of morphology, pathogenesis and accompanying anomalies due to a primary developmental and secondary disruptive possibly vascular disturbance, respectively.

Large neutral amino acid levels tune perinatal neuronal excitability and survival.

Little is known about the critical metabolic changes that neural cells have to undergo during development and how temporary shifts in this program can influence brain circuitries and behavior. Inspired by the discovery that mutations in SLC7A5, a transporter of metabolically essential large neutral amino acids (LNAAs), lead to autism, we employed metabolomic profiling to study the metabolic states of the cerebral cortex across different developmental stages. We found that the forebrain undergoes significant metabolic remodeling throughout development, with certain groups of metabolites showing stage-specific changes, but what are the consequences of perturbing this metabolic program? By manipulating Slc7a5 expression in neural cells, we found that the metabolism of LNAAs and lipids are interconnected in the cortex. Deletion of Slc7a5 in neurons affects the postnatal metabolic state, leading to a shift in lipid metabolism. Additionally, it causes stage- and cell-type-specific alterations in neuronal activity patterns, resulting in a long-term circuit dysfunction.

Singleton exome sequencing of 90 fetuses with ultrasound anomalies revealing novel disease-causing variants and genotype-phenotype correlations.

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.

Distal Arthrogryposis with Impaired Proprioception and Touch: Description of an Early Phenotype in a Boy with Compound Heterozygosity of PIEZO2 Mutations and Review of the Literature.

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.

Utilization of Benchtop Next Generation Sequencing Platforms Ion Torrent PGM and MiSeq in Noninvasive Prenatal Testing for Chromosome 21 Trisomy and Testing of Impact of In Silico and Physical Size Selection on Its Analytical Performance.

OBJECTIVES: The 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. METHODS: Samples 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. RESULTS: Using 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. CONCLUSIONS: Noninvasive 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.

Molecular genetic methods in the diagnosis of myelodysplastic syndromes. A review.

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 array-based 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.

Identification of a novel mutations BRCA1*c.80 + 3del4 and BRCA2*c.6589delA in Slovak HBOC families.

Mutations in the BRCA1 and BRCA2 genes account for the majority of hereditary breast ovarian cancer (HBOC) cases. However, after BRCA1 and BRCA2 screening still the most HBOC cases remain negative for any mutational event. Accordingly, in these cases raises the relevance to analyze the unusual BRCA1/2 variants of uncertain clinical significance. Complex RNA/cDNA analysis may constitute the solution and help to interpret the HBOC syndrome in the family. In our study we analyzed the novel, to our knowledge, not yet published mutations identified in Slovak HBOC families, c.80 + 3del4 (IVS2 + 3delAGTC) in BRCA1 gene and mutation c.6589delA (6817delA) in BRCA2 gene. To determine the effect of the BRCA1 mutation, we applied different approaches: segregation analysis of mutation with disease, presence in the set of unaffected controls and finally RNA/cDNA BRCA1 analysis. Novel BRCA2 mutation was determined performing direct sequencing analysis. In conclusion, considering the results from all used techniques we approved the mentioned mutations as seriously pathogenic and disease causing with clear effect on the onset of HBOC syndrome.