Clinical outcomes of screen-positive genome-wide cfDNA cases for trisomy 20: results from the global expanded NIPT Consortium.

Trisomy 20 has been shown to be one of the most frequent rare autosomal trisomies in patients that undergo genome-wide noninvasive prenatal testing. Here, we describe the clinical outcomes of cases that screened positive for trisomy 20 following prenatal genome-wide cell-free (cf.) DNA screening. These cases are part of a larger cohort of previously published cases. Members of the Global Expanded NIPT Consortium were invited to submit details on their cases with a single rare autosomal aneuploidy following genome-wide cfDNA screening for retrospective analysis. Clinical details including patient demographics, test indications, diagnostic testing, and obstetric pregnancy outcomes were collected. Genome-wide cfDNA screening was conducted following site-specific laboratory procedures. Cases which screened positive for trisomy 20 (n = 10) were reviewed. Clinical outcome information was available for 90% (9/10) of our screen-positive trisomy 20 cases; the case without diagnostic testing ended in a fetal demise. Of the nine cases with outcome information, one was found to have a mosaic partial duplication (duplication at 20p13), rather than a full trisomy 20. Only one case in the study cohort had placental testing; therefore, confined placental mosaicism could not be ruled out in most cases. Adverse pregnancy outcomes were seen in half of the cases, which could suggest the presence of underlying confined placental mosaicism or mosaic/full fetal trisomy 20. Based on our limited series, the likelihood of true fetal aneuploidy is low but pregnancies may be at increased risk for adverse obstetric outcomes and may benefit from additional surveillance.

Pulse-SILAC and Interactomics Reveal Distinct DDB1-CUL4-Associated Factors, Cellular Functions, and Protein Substrates.

Cullin-RING finger ligases represent the largest family of ubiquitin ligases. They are responsible for the ubiquitination of ∼20% of cellular proteins degraded through the proteasome, by catalyzing the transfer of E2-loaded ubiquitin to a substrate. Seven cullins are described in vertebrates. Among them, cullin 4 (CUL4) associates with DNA damage-binding protein 1 (DDB1) to form the CUL4-DDB1 ubiquitin ligase complex, which is involved in protein ubiquitination and in the regulation of many cellular processes. Substrate recognition adaptors named DDB1/CUL4-associated factors (DCAFs) mediate the specificity of CUL4-DDB1 and have a short structural motif of approximately forty amino acids terminating in tryptophan (W)-aspartic acid (D) dipeptide, called the WD40 domain. Using different approaches (bioinformatics/structural analyses), independent studies suggested that at least sixty WD40-containing proteins could act as adaptors for the DDB1/CUL4 complex. To better define this association and classification, the interaction of each DCAFs with DDB1 was determined, and new partners and potential substrates were identified. Using BioID and affinity purification-mass spectrometry approaches, we demonstrated that seven WD40 proteins can be considered DCAFs with a high confidence level. Identifying protein interactions does not always lead to identifying protein substrates for E3-ubiquitin ligases, so we measured changes in protein stability or degradation by pulse-stable isotope labeling with amino acids in cell culture to identify changes in protein degradation, following the expression of each DCAF. In conclusion, these results provide new insights into the roles of DCAFs in regulating the activity of the DDB1-CUL4 complex, in protein targeting, and characterized the cellular processes involved.

Patient attitudes and preferences about expanded noninvasive prenatal testing.

Introduction: Noninvasive prenatal testing (NIPT) using cell-free DNA (cfDNA) is typically carried out to screen for common fetal chromosomal anomalies, with the option to screen for a wider range of chromosomal changes (expanded NIPT) becoming increasingly available. However, little is known about pregnant patients' attitudes and preferences regarding expanded NIPT. Methods: To address this gap, we surveyed general-risk patients having first-tier cfDNA screening at a private prenatal clinic on their expectations for expanded NIPT. Patients were asked questions regarding their current pregnancy and previous pregnancy history, their opinions on fetal DNA screenings during pregnancy and incidental findings, information and opinions on financial resources for NIPT, as well as socio-cultural questions to determine patient demographics. Results: Of the 200 survey participants, the majority were educated, self-reported as white, had a higher than average income, and reported no aneuploidy risk factors. When asked what information they would like to receive from cfDNA screening, the vast majority of participants wanted all information available that could have an immediate impact on fetal health (88%) or an immediate impact on infant health from birth (82%). Many participants also wanted information that could have a future impact on the child's health or an immediate or future impact on the pregnant woman's own health. Most participants wanted information about the sex of fetus (86%) and common trisomies (71%), with almost half of participants desiring information about rare autosomal aneuploidies and/or all genetic information that may affect the baby. In addition, participants were found to be comfortable screening for conditions that are well-known, influence care during pregnancy, and are treatable. Finally, while most respondents either had insurance coverage for NIPT or were able to afford NIPT out of pocket, the majority of our participants felt that expanded NIPT should be either free for everyone or for those considered high risk. Discussion: Our findings suggest that with appropriate pre-test counseling, pregnant patients may choose NIPT for an expanding list of conditions.

Multisite assessment of the impact of cell-free DNA-based screening for rare autosomal aneuploidies on pregnancy management and outcomes.

Cell-free (cf) DNA screening is a noninvasive prenatal screening approach that is typically used to screen for common fetal trisomies, with optional screening for sex chromosomal aneuploidies and fetal sex. Genome-wide cfDNA screening can screen for a wide variety of additional anomalies, including rare autosomal aneuploidies (RAAs) and copy number variants. Here, we describe a multi-cohort, global retrospective study that looked at the clinical outcomes of cases with a high-risk cfDNA screening result for a RAA. Our study cohort included a total of 109 cases from five different sites, with diagnostic outcome information available for 68% (74/109) of patients. Based on confirmatory diagnostic testing, we found a concordance rate of 20.3% for presence of a RAA (15/74) in our study population. Pregnancy outcome was also available for 77% (84/109) of cases in our cohort. Many of the patients experienced adverse pregnancy outcomes, including intrauterine fetal demise, fetal growth restriction, and preterm birth. These adverse outcomes were observed both in patients with fetal or placental confirmation of the presence of a RAA, as well as patients that did not undergo fetal and/or placental diagnostic testing. In addition, we have proposed some suggestions for pregnancy management and counseling considerations for situations where a RAA is noted on a cfDNA screen. In conclusion, our study has shown that genome-wide cfDNA screening for the presence of rare autosomal aneuploidies can be beneficial for both patients and their healthcare practitioners. This can provide a possible explanation for an adverse pregnancy outcome or result in a change in pregnancy management, such as increased monitoring for adverse outcomes.

UBB pseudogene 4 encodes functional ubiquitin variants.

Pseudogenes are mutated copies of protein-coding genes that cannot be translated into proteins, but a small subset of pseudogenes has been detected at the protein level. Although ubiquitin pseudogenes represent one of the most abundant pseudogene families in many organisms, little is known about their expression and signaling potential. By re-analyzing public RNA-sequencing and proteomics datasets, we here provide evidence for the expression of several ubiquitin pseudogenes including UBB pseudogene 4 (UBBP4), which encodes UbKEKS (Q2K, K33E, Q49K, N60S). The functional consequences of UbKEKS conjugation appear to differ from canonical ubiquitylation. Quantitative proteomics shows that UbKEKS modifies specific proteins including lamins. Knockout of UBBP4 results in slower cell division, and accumulation of lamin A within the nucleolus. Our work suggests that a subset of proteins reported as ubiquitin targets may instead be modified by ubiquitin variants that are the products of wrongly annotated pseudogenes and induce different functional effects.

Protein interaction network of alternatively spliced NudCD1 isoforms.

NudCD1, also known as CML66 or OVA66, is a protein initially identified as overexpressed in patients with chronic myelogenous leukemia. The mRNA of NudCD1 is expressed in heart and testis of normal tissues, and is overexpressed in several cancers. Previous studies have shown that the expression level of the protein correlates with tumoral phenotype, possibly interacting upstream of the Insulin Growth Factor - 1 Receptor (IGF-1R). The gene encoding the NudCD1 protein consists of 12 exons that can be alternative spliced, leading to the expression of three different isoforms. These isoforms possess a common region of 492 amino acids in their C-terminus region and have an isoform specific N-terminus. To determine the distinct function of each isoforms, we have localised the isoforms within the cells using immunofluorescence microscopy and used a quantitative proteomics approach (SILAC) to identify specific protein interaction partners for each isoforms. Localization studies showed a different subcellular distribution for the different isoforms, with the first isoform being nuclear, while the other two isoforms have distinct cytoplasmic and nuclear location. We found that the different NudCD1 isoforms have unique interacting partners, with the first isoform binding to a putative RNA helicase named DHX15 involved in mRNA splicing.

Subcellular proteomics analysis of different stages of colorectal cancer cell lines.

Studying cell differentiation and transformation allows a better understanding of the mechanisms involved in the initiation and the evolution of cancer. The role of proteins which participate in these processes is dependent on their location within the cell. Determining the subcellular localization of proteins or the changes in localization is, therefore, paramount in elucidating their role. Using quantitative mass spectrometry, we characterized the protein expression and subcellular localization of nearly 5000 proteins from seven different colorectal cancer (CRC) cell lines, as well as normal colon fibroblasts and intestinal epithelial cells. This cellular characterization allowed the identification of colon cancer-associated proteins with differential expression patterns as well as deregulated protein networks and pathways. Indeed, our results demonstrate differential expression of proteins involved in cell adhesion, cytoskeleton, and transcription in colon cancer cells compared to normal colon-derived cells. Pathway analyses identified different cellular functions, including endocytosis and eIF2 signaling, whose deregulation correlates with mutations found in the different CRC phenotypes. Our results provide an unbiased, quantitative and high-throughput approach to measure changes in protein expression and subcellular protein locations in different CRC cell lines.

Comprehensive Characterization of Minichromosome Maintenance Complex (MCM) Protein Interactions Using Affinity and Proximity Purifications Coupled to Mass Spectrometry.

The extensive identification of protein-protein interactions under different conditions is an important challenge to understand the cellular functions of proteins. Here we use and compare different approaches including affinity purification and purification by proximity coupled to mass spectrometry to identify protein complexes. We explore the complete interactome of the minichromosome maintenance (MCM) complex by using both approaches for all of the different MCM proteins. Overall, our analysis identified unique and shared interaction partners and proteins enriched for distinct biological processes including DNA replication, DNA repair, and cell cycle regulation. Furthermore, we mapped the changes in protein interactions of the MCM complex in response to DNA damage, identifying a new role for this complex in DNA repair. In summary, we demonstrate the complementarity of these approaches for the characterization of protein interactions within the MCM complex.

Active Yeast Telomerase Shares Subunits with Ribonucleoproteins RNase P and RNase MRP.

Telomerase is the ribonucleoprotein enzyme that replenishes telomeric DNA and maintains genome integrity. Minimally, telomerase activity requires a templating RNA and a catalytic protein. Additional proteins are required for activity on telomeres in vivo. Here, we report that the Pop1, Pop6, and Pop7 proteins, known components of RNase P and RNase MRP, bind to yeast telomerase RNA and are essential constituents of the telomerase holoenzyme. Pop1/Pop6/Pop7 binding is specific and involves an RNA domain highly similar to a protein-binding domain in the RNAs of RNase P/MRP. The results also show that Pop1/Pop6/Pop7 function to maintain the essential components Est1 and Est2 on the RNA in vivo. Consistently, addition of Pop1 allows for telomerase activity reconstitution with wild-type telomerase RNA in vitro. Thus, the same chaperoning module has allowed the evolution of functionally and, remarkably, structurally distinct RNPs, telomerase, and RNases P/MRP from unrelated progenitor RNAs.

Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage.

The minichromosome maintenance complex (MCM) proteins are required for processive DNA replication and are a target of S-phase checkpoints. The eukaryotic MCM complex consists of six proteins (MCM2-7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex. Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork. The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair. Here, we employed a combination of stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein-tagged fusion proteins to identify proteins interacting with the MCM complex, and quantify changes in interactions in response to DNA damage. Interestingly, the MCM complex showed very dynamic changes in interaction with proteins such as Importin7, the histone chaperone ASF1, and the Chromodomain helicase DNA binding protein 3 (CHD3) following DNA damage. These changes in interactions were accompanied by an increase in phosphorylation and ubiquitination on specific sites on the MCM proteins and an increase in the co-localization of the MCM complex with γ-H2AX, confirming the recruitment of these proteins to sites of DNA damage. In summary, our data indicate that the MCM proteins is involved in chromatin remodeling in response to DNA damage.

Proteomics methods for subcellular proteome analysis.

The elucidation of the subcellular distribution of proteins under different conditions is a major challenge in cell biology. This challenge is further complicated by the multicompartmental and dynamic nature of protein localization. To address this issue, quantitative proteomics workflows have been developed to reliably identify the protein complement of whole organelles, as well as for protein assignment to subcellular location and relative protein quantification based on different cell culture conditions. Here, we review quantitative MS-based approaches that combine cellular fractionation with proteomic analysis. The application of these methods to the characterization of organellar composition and to the determination of the dynamic nature of protein complexes is improving our understanding of protein functions and dynamics.