Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by antibody deficiency, poor humoral response to antigens, and recurrent infections. To investigate the molecular ...cause of CVID, we carried out exome sequence analysis of a family diagnosed with CVID and identified a heterozygous frameshift mutation, c.2564delA (p.Lys855Serfs∗7), in NFKB2 affecting the C terminus of NF-κB2 (also known as p100/p52 or p100/p49). Subsequent screening of NFKB2 in 33 unrelated CVID-affected individuals uncovered a second heterozygous nonsense mutation, c.2557C>T (p.Arg853∗), in one simplex case. Affected individuals in both families presented with an unusual combination of childhood-onset hypogammaglobulinemia with recurrent infections, autoimmune features, and adrenal insufficiency. NF-κB2 is the principal protein involved in the noncanonical NF-κB pathway, is evolutionarily conserved, and functions in peripheral lymphoid organ development, B cell development, and antibody production. In addition, Nfkb2 mouse models demonstrate a CVID-like phenotype with hypogammaglobulinemia and poor humoral response to antigens. Immunoblot analysis and immunofluorescence microscopy of transformed B cells from affected individuals show that the NFKB2 mutations affect phosphorylation and proteasomal processing of p100 and, ultimately, p52 nuclear translocation. These findings describe germline mutations in NFKB2 and establish the noncanonical NF-κB signaling pathway as a genetic etiology for this primary immunodeficiency syndrome.
Studies of homotypic vacuole-vacuole fusion in the yeast Saccharomyces cerevisiae have been instrumental in determining the cellular machinery required for eukaryotic membrane fusion and have ...implicated the vacuolar H+-ATPase (V-ATPase). The V-ATPase is a multisubunit, rotary proton pump whose precise role in homotypic fusion is controversial. Models formulated from in vitro studies suggest that it is the proteolipid proton-translocating pore of the V-ATPase that functions in fusion, with further studies in worms, flies, zebrafish, and mice appearing to support this model. We present two in vivo assays and use a mutant V-ATPase subunit to establish that it is the H+-translocation/vacuole acidification function, rather than the physical presence of the V-ATPase, that promotes homotypic vacuole fusion in yeast. Furthermore, we show that acidification of the yeast vacuole in the absence of the V-ATPase rescues vacuole-fusion defects. Our results clarify the in vivo requirements of acidification for membrane fusion.
•Development of two in vivo assays for membrane fusion•Yeast vacuole-vacuole fusion requires an acidified vacuole•The V-ATPase complex per se is not required for vacuole-vacuole fusion in vivo
The vacuolar H+-ATPase (V-ATPase) complex has been proposed to play a mechanistic role in eukaryotic membrane fusion. Coonrod et al. show that H+-translocation function, rather than the physical presence of the V-ATPase, promotes vacuole membrane fusion in yeast. These results clarify the in vivo requirement of acidification for membrane fusion.
To the Editor: There is a great diversity of RAG1- and RAG2-dependent primary immunodeficiencies ranging from severe combined immunodeficiency (SCID) to various late-onset combined ...immunodeficiencies, as complete lack of RAG activity leads to absence of mature B and T cells (T-B- SCID), whereas hypomorphic RAG mutations allow limited generation of T and or B cells, resulting in various clinical phenotypes distinct from SCID.1,2 Currently, there are no peripheral blood-based functional assays for the evaluation of RAG activity. ...the pathogenicity of RAG variants is determined using prediction programs and in vitro V(D)J recombination assays.3 Critical to this evaluation is the population frequency of the variants. The number of individuals who are homozygous or compound heterozygous for decreased-function RAG variants is about 9.68 x 10-7 or approximately 1 in 1,000,000. Because this estimation is substantially lower than the frequency of RAG1/2 mutations observed by newborn screening (1 in 336,000),E4 we conclude that the ExAC database currently underestimates the frequency of RAG1/2 deficiency diseases, and the number of individuals who are homozygous or compound heterozygous for decreased-function RAG variants is estimated to be between 1 in 181,000 and 1 in 336,000.
In 1992, Raymond et al. published a compilation of the 41 yeast vacuolar protein sorting (vps) mutant groups and described a large class of mutants (class E vps mutants) that accumulated an ...exaggerated prevacuolar endosome-like compartment. Further analysis revealed that this "class E compartment" contained soluble vacuolar hydrolases, vacuolar membrane proteins, and Golgi membrane proteins unable to recycle back to the Golgi complex, yet these class E vps mutants had what seemed to be normal vacuoles. The 13 class E VPS genes were later shown to encode the proteins that make up the complexes required for formation of intralumenal vesicles in late endosomal compartments called multivesicular bodies, and for the sorting of ubiquitinated cargo proteins into these internal vesicles for eventual delivery to the vacuole or lysosome.
Next-generation sequencing is being implemented in the clinical laboratory environment for the purposes of candidate causal variant discovery in patients affected with a variety of genetic disorders. ...The successful implementation of this technology for diagnosing genetic disorders requires a rapid, user-friendly method to annotate variants and generate short lists of clinically relevant variants of interest. This report describes Omicia's Opal platform, a new software tool designed for variant discovery and interpretation in a clinical laboratory environment. The software allows clinical scientists to process, analyze, interpret and report on personal genome files.
To demonstrate the software, the authors describe the interactive use of the system for the rapid discovery of disease-causing variants using three cases.
Here, the authors show the features of the Opal system and their use in uncovering variants of clinical significance.
Mitochondrial division requires coordinated interactions among Fis1p, Mdv1p, and the Dnm1p GTPase, which assemble into fission complexes on the outer mitochondrial membrane. The integral outer ...membrane protein Fis1p contains a cytoplasmic domain consisting of a tetratricopeptide repeat (TPR)-like fold and a short NH₂-terminal helix. Although it is known that the cytoplasmic domain is necessary for assembly of Mdv1p and Dnm1p into fission complexes, the molecular details of this assembly are not clear. In this study, we provide new evidence that the Fis1p-Mdv1p interaction is direct. Furthermore, we show that conditional mutations in the Fis1p TPR-like domain cause fission complex assembly defects that are suppressed by mutations in the Mdv1p-predicted coiled coil. We also define separable functions for the Fis1p NH₂-terminal arm and TPR-like fold. These studies suggest that the concave binding surface of the Fis1p TPR-like fold interacts with Mdv1p during mitochondrial fission and that Mdv1p facilitates Dnm1p recruitment into functional fission complexes.
Breast cancer, particularly the estrogen receptor positive (ER+) subtype, remains a leading cause of cancer-related death among women. Endocrine therapy is the most effective treatment for ER+ breast ...cancer; however, the development of resistance presents a significant challenge. This study explored the role of the breast cancer antiestrogen resistance 4 (BCAR4) gene as a potential driver of resistance and a pivotal biomarker in breast cancer.
The researchers undertook a comprehensive analysis of 1743 patients spanning 6 independent cohorts. They examined the association of BCAR4 expression with patient outcomes across all breast cancer types and the PAM50 molecular subtypes. The relationship between elevated BCAR4 expression and resistance to endocrine therapy including AIs, the prevailing standard-of-care for endocrine therapy, was also investigated.
This meta-analysis corroborated the link between BCAR4 expression and adverse outcomes as well as resistance to endocrine therapy in breast cancer. Notably, BCAR4 expression is clinically significant in luminal A and B subtypes. Additionally, an association between BCAR4 expression and resistance to AI treatment was discerned.
This study expands on previous findings by demonstrating that BCAR4 expression is associated with resistance to newer therapies. The identification of patients with intrinsic resistance to hormone therapy is crucial to avoid ineffective treatment strategies. These findings contribute to our understanding of endocrine therapy resistance in breast cancer and could potentially guide the development of more effective treatment strategies.
Estrogen receptor positive (ER+) breast cancer is a leading cause of cancer-related deaths among women due to treatment resistance. This study explored the role of the breast cancer antiestrogen resistance 4 (BCAR4) gene as a potential driver of resistance and a pivotal biomarker in breast cancer. Through an extensive study of 1743 patients across 6 cohorts, high BCAR4 expression was found to correlate with poor outcome, particularly in luminal A and B subtypes, with resistance to endocrine therapy including aromatase inhibitor (AI) treatments. These insights enrich our understanding of endocrine therapy resistance and may lead to improved therapeutic strategies.
Heteroaryldihydropyrimidines (HAPs) are compounds that inhibit hepatitis B virus (HBV) replication by modulating viral capsid assembly. While their biophysical effects on capsid assembly
have been ...previously studied, the effect of HAP treatment on capsid protein (Cp) in individual HBV-infected cells remains unknown. We report here that the HAP Bay 38-7690 promotes aggregation of recombinant Cp
and causes a time- and dose-dependent decrease of Cp in infected cells, consistent with previously studied HAPs. Interestingly, immunofluorescence analysis showed Cp aggregating in nuclear foci of Bay 38-7690-treated infected cells in a time- and dose-dependent manner. We found these foci to be associated with promyelocytic leukemia (PML) nuclear bodies (NBs), which are structures that affect many cellular functions, including DNA damage response, transcription, apoptosis, and antiviral responses. Cp aggregation is not an artifact of the cell system used, as it is observed in HBV-expressing HepAD38 cells, in HepG2 cells transfected with an HBV-expressing plasmid, and in HepG2-NTCP cells infected with HBV. Use of a Cp overexpression vector without HBV sequences shows that aggregation is independent of viral replication, and use of an HBV-expressing plasmid harboring a HAP resistance mutation in Cp abrogated the aggregation, demonstrating that the effect is due to direct compound-Cp interactions. These studies provide novel insight into the effects of HAP-based treatment at a single-cell level.
Despite the availability of effective vaccines and treatments, HBV remains a significant global health concern, with more than 240 million individuals chronically infected. Current treatments are highly effective at controlling viral replication and disease progression but rarely cure infections. Therefore, much emphasis is being placed on finding therapeutics with new drug targets, such as viral gene expression, covalently closed circular DNA formation and stability, capsid formation, and host immune modulators, with the ultimate goal of an HBV cure. Understanding the mechanisms by which novel antiviral agents act will be imperative for the development of curative HBV therapies.
Purpose
Combined immunodeficiency (CID) presents a unique challenge to clinicians. Two patients presented with the prior clinical diagnosis of common variable immunodeficiency (CVID) disorder marked ...by an early age of presentation, opportunistic infections, and persistent lymphopenia. Due to the presence of atypical clinical features, next generation sequencing was applied documenting RAG deficiency in both patients.
Methods
Two different genetic analysis techniques were applied in these patients including whole exome sequencing in one patient and the use of a gene panel designed to target genes known to cause primary immunodeficiency disorders (PIDD) in a second patient. Sanger dideoxy sequencing was used to confirm
RAG1
mutations in both patients.
Results
Two young adults with a history of recurrent bacterial sinopulmonary infections, viral infections, and autoimmune disease as well as progressive hypogammaglobulinemia, abnormal antibody responses, lymphopenia and a prior diagnosis of CVID disorder were evaluated. Compound heterozygous mutations in
RAG1
(1) c256_257delAA, p86VfsX32 and (2) c1835A>G, pH612R were documented in one patient. Compound heterozygous mutations in
RAG1
(1) c.1566G>T, p.W522C and (2) c.2689C>T, p. R897X) were documented in a second patient post-mortem following a fatal opportunistic infection.
Conclusion
Astute clinical judgment in the evaluation of patients with PIDD is necessary. Atypical clinical findings such as early onset, granulomatous disease, or opportunistic infections should support the consideration of atypical forms of late onset CID secondary to RAG deficiency. Next generation sequencing approaches provide powerful tools in the investigation of these patients and may expedite definitive treatments.
Advances in sequencing technology with the commercialization of next-generation sequencing (NGS) has substantially increased the feasibility of sequencing human genomes and exomes. Next-generation ...sequencing has been successfully applied to the discovery of disease-causing genes in rare, inherited disorders. By necessity, the advent of NGS has fostered the concurrent development of bioinformatics approaches to expeditiously analyze the large data sets generated. Next-generation sequencing has been used for important discoveries in the research setting and is now being implemented into the clinical diagnostic arena.
To review the current literature on technical and bioinformatics approaches for exome and genome sequencing and highlight examples of successful disease gene discovery in inherited disorders. To discuss the challenges for implementing NGS in the clinical research and diagnostic arenas.
Literature review and authors' experience.
Next-generation sequencing approaches are powerful and require an investment in infrastructure and personnel expertise for effective use; however, the potential for improvement of patient care through faster and more accurate molecular diagnoses is high.
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