Study of LvsB in Dictyostelium discoideum provides insights into the Chediak-Higashi syndrome

The Chediak-Higashi Syndrome is a disorder affecting lysosome biogenesis. At the cellular level, the Chediak-Higashi syndrome is characterized by the presence of grossly enlarged lysosomes in every tissue. Impaired lysosomal function in CHS patients results in many physiological problems, including immunodeficiency, albinism and neurological problems. The Chediak-Higashi syndrome is caused by the loss of a BEACH protein of unknown function named Lyst. In this work, I have studied the function of the Dictyostelium LvsB protein, the ortholog of mammalian Lyst and a protein that is also important for lysosomal function. Using a knock-in approach we tagged LvsB with GFP and expressed it from its single chromosomal locus. GFP-LvsB was observed on endocytic and phagocytic compartments. Specific analysis of the endocytic compartments labeled by LvsB showed that they represented late lysosomes and postlysosomes. The analysis of LvsB-null cells revealed that loss of LvsB resulted in enlarged postlysosomes, in the abnormal localization of proton pumps on postlysosomes and their abnormal acidification. This work demonstrated that the abnormal postlysosomes in LvsB-null cells were produced by the inappropriate fusion of lysosomes with postlysosomal compartments. Furthermore, this work provided the first evidence that LvsB is a functional antagonist of the GTPase Rab14 in vesicle fusion events. In particular, we demonstrated that reduction of Rab14 activity suppressed the LvsB-null phenotype by reducing the enlarged post-lysosomes and the enhanced rate of heterotypic fusion. In contrast, expression of an active form of Rab14 enhanced the LvsB-null phenotype by causing an even more severe enlargement of endosome size. The results provided by this work support the model that LvsB and Lyst proteins act as negative regulators of fusion by limiting the heterotypic fusion of early with late compartments and antagonize Rab GTPases in membrane fusion. The LvsB localization studies and the functional assessment of the LvsB-null phenotype helped make unique contributions to the understanding of the molecular function of Lyst proteins.Microbiolog

The Epigenome View: An Effort towards Non-Invasive Prenatal Diagnosis

Epigenetic modifications have proven to play a significant role in cancer development, as well as fetal development. Taking advantage of the knowledge acquired during the last decade, great interest has been shown worldwide in deciphering the fetal epigenome towards the development of methylation-based non-invasive prenatal tests (NIPT). In this review, we highlight the different approaches implemented, such as sodium bisulfite conversion, restriction enzyme digestion and methylated DNA immunoprecipitation, for the identification of differentially methylated regions (DMRs) between free fetal DNA found in maternal blood and DNA from maternal blood cells. Furthermore, we evaluate the use of selected DMRs identified towards the development of NIPT for fetal chromosomal aneuploidies. In addition, we perform a comparison analysis, evaluate the performance of each assay and provide a comprehensive discussion on the potential use of different methylation-based technologies in retrieving the fetal methylome, with the aim of further expanding the development of NIPT assays

Development of a new methylation‐based fetal fraction estimation assay using multiplex ddPCR

Abstract Background Non‐invasive prenatal testing (NIPT) for fetal aneuploidies has rapidly been incorporated into clinical practice. Current NGS‐based methods can reliably detect fetal aneuploidies non‐invasively with fetal fraction of at least 4%. Inaccurate fetal fraction assessment can compromise the accuracy of the test as affected samples with low fetal fraction have an increased risk for misdiagnosis. Using a novel set of fetal‐specific differentially methylated regions (DMRs) and methylation sensitive restriction digestion (MSRD), we developed a multiplex ddPCR assay for accurate detection of fetal fraction in maternal plasma. Methods We initially performed MSRD followed by methylation DNA immunoprecipitation (MeDIP) and NGS on fetal and non‐pregnant female tissues to identify fetal‐specific DMRs. DMRs with the highest methylation difference between the two tissues were selected for fetal fraction estimation employing MSRD and multiplex ddPCR. Chromosome Y multiplex ddPCR assay (YMM) was used as a reference standard, to develop our fetal fraction estimation model in male pregnancy samples. Additional 123 samples were tested to examine whether the model is sex dependent and/or ploidy dependent. Results In all, 93 DMRs were identified of which seven were selected for fetal fraction estimation. Statistical analysis resulted in the final model which included four DMRs (FFMM). High correlation with YMM‐based fetal fractions was observed using 85 male pregnancies (r = 0.86 95% CI: 0.80–0.91). The model was confirmed using an independent set of 53 male pregnancies. Conclusion By employing a set of well‐characterized DMRs, we developed a SNP‐, sex‐ and ploidy‐independent methylation‐based multiplex ddPCR assay for accurate fetal fraction estimation

Targeted capture enrichment assay for non-invasive prenatal testing of large and small size sub-chromosomal deletions and duplications

<div><p>Noninvasive prenatal testing (NIPT) using whole genome and targeted sequencing has become increasingly accepted for clinical detection of Trisomy 21 and sex chromosome aneuploidies. Few studies have shown that sub-chromosomal deletions or duplications associated with genetic syndromes can also be detected in the fetus noninvasively. There are still limitations on these methodologies such as the detection of variants of unknown clinical significance, high number of false positives, and difficulties to detect small aberrations. We utilized a recently developed targeted sequencing approach for the development of a NIPT assay, for large and small size deletions/duplications, which overcomes these existing limitations. Artificial pregnancies with microdeletion/microduplication syndromes were created by spiking DNA from affected samples into cell free DNA (cfDNA) from non-pregnant samples. Unaffected spiked samples and normal pregnancies were used as controls. Target Capture Sequences (TACS) for seven syndromes were designed and utilized for targeted capture enrichment followed by sequencing. Data was analyzed using a statistical pipeline to identify deletions or duplications on targeted regions. Following the assay development a proof of concept study using 33 normal pregnancies, 21 artificial affected and 17 artificial unaffected pregnancies was carried out to test the sensitivity and specificity of the assay. All 21 abnormal spiked-in samples were correctly classified as subchromosomal aneuploidies while the 33 normal pregnancies or 17 normal spiked-in samples resulted in a false positive result. We have developed an NIPT assay for the detection of sub-chromosomal deletions and duplications using the targeted capture enrichment technology. This assay demonstrates high accuracy, high read depth of the genomic region of interest, and can identify deletions/duplications as small as 0.5 Mb. NIPT of fetal microdeletion/microduplication syndromes can be of enormous benefit in the management of pregnancies at risk both for prospective parents and health care providers.</p></div

Affected samples used to create artificial plasma pregnancies with deletion or duplication syndromes.

<p>Affected samples used to create artificial plasma pregnancies with deletion or duplication syndromes.</p