Genomic analyses elucidate S-locus evolution in response to intra-specific losses of distyly in Primula vulgaris.

Distyly, a floral dimorphism that promotes outcrossing, is controlled by a hemizygous genomic region known as the S-locus. Disruptions of genes within the S-locus are responsible for the loss of distyly and the emergence of homostyly, a floral monomorphism that favors selfing. Using whole-genome resequencing data of distylous and homostylous individuals from populations of Primula vulgaris and leveraging high-quality reference genomes of Primula we tested, for the first time, predictions about the evolutionary consequences of transitions to selfing on S-genes. Our results reveal a previously undetected structural rearrangement in CYPᵀ associated with the shift to homostyly and confirm previously reported, homostyle-specific, loss-of-function mutations in the exons of the S-gene CYPᵀ. We also discovered that the promoter and intronic regions of CYPᵀ in distylous and homostylous individuals are conserved, suggesting that down-regulation of CYPᵀ via mutations in its promoter and intronic regions is not a cause of the shift to homostyly. Furthermore, we found that hemizygosity is associated with reduced genetic diversity in S-genes compared with their paralogs outside the S-locus. Additionally, the shift to homostyly lowers genetic diversity in both the S-genes and their paralogs, as expected in primarily selfing plants. Finally, we tested, for the first time, long-standing theoretical models of changes in S-locus genotypes during early stages of the transition to homostyly, supporting the assumption that two copies of the S-locus might reduce homostyle fitness.

Halting targeted and collateral damage to red blood cells by the complement system.

The complement system is an important defense mechanism against pathogens; however, in certain pathologies, the system also attacks human cells, such as red blood cells (RBCs). In paroxysmal nocturnal hemoglobinuria (PNH), RBCs lack certain complement regulators which sensitize them to complement-mediated lysis, while in autoimmune hemolytic anemia (AIHA), antibodies against RBCs may initiate complement-mediated hemolysis. In recent years, complement inhibition has improved treatment prospects for these patients, with eculizumab now the standard of care for PNH patients. Current complement inhibitors are however not sufficient for all patients, and they come with high costs, patient burden, and increased infection risk. This review gives an overview of the underlying pathophysiology of complement-mediated hemolysis in PNH and AIHA, the role of therapeutic complement inhibition nowadays, and the high number of complement inhibitors currently under investigation, as for almost every complement protein, an inhibitor is being developed. The focus lies with novel therapeutics that inhibit complement activity specifically in the pathway that causes pathology or those that reduce costs or patient burden through novel administration routes.

The relevance of geriatric assessment for older patients receiving palliative chemotherapy.

OBJECTIVES: No tools accurately discriminate between older patients who are fit and those who are frail to tolerate systemic palliative treatment. This study evaluates whether domains of geriatric assessment (GA) are associated with increased risk of chemotherapy intolerance in patients who were considered fit to start palliative chemotherapy after clinical evaluation by their treating clinician. MATERIALS AND METHODS: This prospective multicenter study included patients ≥70 years who started first line palliative systemic treatment. Before treatment initiation, patients completed GA including Activities of Daily Life (ADL), Instrumental Activities of Daily Life (IADL), Mini-Mental State Examination (MMSE), Mini Nutritional Assessment (MNA), Geriatric Depression Scale (GDS-15) and the Timed Up and Go Test (TUGT). Primary endpoint was treatment modification, defined as inability to complete the first three sessions of systemic treatment as planned. Secondary endpoint was treatment related toxicity ≥ grade 3 (CTCAE Version 4). The association between GA and endpoints were assessed using univariable and multivariable logistic regression analysis. RESULTS: Ninety-nine patients with median age of 77 (+/- 8) years underwent GA. 48% of the patients required treatment modification and grade 3 toxicity occurred in 53% of patients. One or more geriatric impairments were present in 71% of patients and 32% of patients were frail in two or more domains. Only TUGT was associated with treatment modifications (OR 2.9 [95% CI 1.3-6.5]) and grade 3 toxicities (OR 2.8 [95% CI 1.2-6.3]). CONCLUSION: Frailty was common in older patients who were considered fit to receive palliative chemotherapy. Treatment modification was necessary in half of the patients. Only TUGT was significantly associated with treatment modifications and grade 3 chemotherapy toxicities.

Monoclonal gammopathy of renal significance (MGRS) histopathologic classification, diagnostic workup, and therapeutic options.

Monoclonal gammopathy of renal significance (MGRS) includes all kidney disorders caused by a monoclonal protein (M-protein) secreted by a small plasma cell clone or other B-cell clones in patients who do not meet the diagnostic criteria for multiple myeloma or other B-cell malignancies. The underlying disorder in patients with MGRS is generally consistent with monoclonal gammopathy of undetermined significance (MGUS). MGRS-associated kidney disorders are various and the list is still expanding. The kidney disorders can manifest as glomerular diseases, tubulopathies, and vascular involvement with varying clinical presentations. Diagnosis is often challenging because of the wide spectrum of MGRS, and it is difficult to establish a pathogenic link between the presence of the M-protein or serum free light chains and kidney diseases; further complicating accurate diagnosis is the high incidence of MGUS and/or kidney disorders, independent of MGRS, in elderly patients. However, MGRS can significantly impair kidney function. Because treatment can stop and also reverse kidney disease, early recognition is of great importance. A combined haematologic and nephrologic approach is crucial to establish the causative role of the M-protein in the pathogenesis of kidney disease. Clone-directed therapy, which may include autologous stem cell transplantation in eligible patients, often results in improved outcomes. In this review, we discuss the histopathologic classification of MGRS lesions, provide a renal and haematologic diagnostic workup, discuss treatment options for MGRS, and introduce a Benelux MGRS Working Group.

Guideline for diagnosis and treatment of Waldenström's macroglobulinaemia.

On behalf of the lymphoma and multiple myeloma working parties of the Dutch/Belgian Haemato-Oncology Foundation for Adults in The Netherlands (HOVON), we present a guideline for diagnosis and management of Waldenström's macroglobulinemia (WM). Considering the indolent behaviour and heterogeneous clinical presentation of WM, it is crucial to determine the right indications for treatment, as well as to individualise therapeutic options. There are significant differences from the approach to multiple myeloma or the treatment of other indolent non-Hodkgin lymphomas, and these results cannot always be extrapolated. There is a lack of large clinical trials due to the low incidence of WM. Based on the available data, we provide a practical diagnostic classification, as well as recommendations for first-line therapy and options for treating relapsed disease. Some typical clinical features of WM, such as autoimmune phenomena and 'IgM flare' after rituximab treatment, are highlighted. A more elaborate version of this guideline was published in the 'Nederlands Tijdschrift voor Hematologie' (Dutch Journal for Hematology) September 2012.

A survey on diagnostic methods and treatment strategies used in patients with Waldenström's macroglobulinaemia in The Netherlands.

BACKGROUND: Waldenström's macroglobulinaemia (WM) is defined as a lymphoplasmacytic lymphoma primarily located in the bone marrow, accompanied by an immunoglobulin M (IgM) monoclonal protein in the serum. The symptoms are highly variable, which can sometimes lead to a diagnostic delay. Currently, there is a wide range of therapeutic options used for the management of WM but no approved therapeutic agents are available specifically for this disease. METHODS: An online survey was prepared and sent out to haematologists and haemato-oncologists in The Netherlands, together with an invitational letter to participate. Information was gathered about the preferred methods of diagnosing and treating patients with WM in general, and about the last WM patient diagnosed in their department. RESULTS: 83 (31.8%) responses were obtained, out of which 68 (81.9%) contained responses to all three parts of the survey. The respondents most commonly used either rituximab-CVP or chlorambucil as first-line treatment, whereas rituximab in combination with purine analogues was the most frequently applied second-line treatment. The prevention of an IgM 'flare' was managed by the respondents in various ways, and rituximab maintenance treatment was not commonly used. CONCLUSION: This survey indicates that in general the diagnostic methods and treatment options for WM are well known to a representative number of Dutch haematologists. The areas of uncertainty are knowledge about asymptomatic vs symptomatic disease, risk of hyperviscosity in relation to IgM level, and the occurrence and prevention of an IgM 'flare'. These issues should be addressed in clinical research and guidelines to improve care for WM patients in The Netherlands.

[Diagnostic image (196). A newborn with fever and a swollen finger]. / Diagnose in beeld (196). Een pasgeborene met koorts en met een dik vingertje.

A 5-day-old girl was presented with monoarthritis of the proximal interphalangeal joint of the right fourth digit, without signs of septic illness or meningitis, due to group B Streptococcus agalactiae.

Establishment of an oriP/EBNA1-based episomal vector transcribing human genomic beta-globin in cultured murine fibroblasts.

A novel oriP/EBNA1-based episomal vector has been constructed that persists episomally in cultured murine fibroblasts. The vector, pBH148, is equipped with the entire 185-kb human beta-globin gene locus. After amplification in bacteria, column-purified episomal pBH148 was transfected into both cultured EBNA1-expressing human D98/Raji positive control fusion cells (DRpBH148) and cultured EBNA1-negative murine fibroblast cells (A9pBH148). Cell cultures were maintained concurrently with and without hygromycin selection for a period of 3 months. We show long-term stable episome maintenance of the full-size 200-kb circular double-stranded pBH148 in both the DRpBH148 cultures and the A9pBH148 cultures, regardless of selective pressure by agarose gel electrophoresis and Southern blot. EBNA1 transgene was detected by PCR in all transfected cultures. In addition, we were able to detect correctly spliced human beta-globin mRNA by RT-PCR in all transfected late-passage DRpBH148 and A9pBH148 cell cultures. These findings illustrate that this oriP/EBNA1-based episomal vector is stable in a previously nonpermissive murine cell line and is a potential vector for human gene therapy.

Development of a competitive PCR method for physical titration of recombinant EBV vector in a helper-dependent packaging system.

Epstein-Barr virus (EBV) is a gamma-herpesvirus with B lymphotropism and a double-stranded DNA genome of 172 kb that is episomally maintained in permissive cells during latency. EBV-based vectors containing minimal cis elements for replication, amplification, and helper-dependent packaging in a producer cell line HH514 have been developed to deliver therapeutic/suicide transgenes as infectious viral particles (miniEBV) to EBV-transformed B lymphoblastoid cells or B lymphoma cells. A quantitative, competitive PCR-based assay was developed to determine the relative packaging efficiencies of miniEBV and helper P3HR1 coproduced in HH514 cells. This provides a rapid and accurate quantitation of the physical titer of the virus preparation, which helps preserve the biological titer of the virus preparation and increase the efficiency of transgene delivery by miniEBV infection. In addition, it provides a sensitive and accurate way to evaluate future development of a helper-free packaging system by detecting any possible helper virus contamination.

Rapid and Systematic Transfer and Recovery of Large BACs/PACs into Mammalian Cells by HAEC Retrofitting.

Studies of the human genome have prompted the development of several cloning systems that can manipulate large fragments of human DNA as functional units. The discoveries in yeast of the sequences for replication (autonomously replicating sequence [ARS]), centromeres, and telomeres have allowed the creation of large linear molecules (yeast artificial chromosome [YAC]), which can replicate and segregate as chromosomes (1). YACs have been used to generate genomic libraries from different organisms with megabase insert size and have become an essential tool for physical and genetic mapping of various mammalian genomes (2). Nevertheless, the instabilities associated with YAC libraries (chimerism and deletions) have led to the development of alternative cloning systems (3).

[Sensitive and specific screening for detection of spina bifida by echography in the second trimester; systematic review and meta-analysis]. / Sensitieve én specifieke screening ter detectie van spina bifida door echografie in het tweede trimester; systematische review en meta-analyse.

OBJECTIVE: Evaluation of the diagnostic accuracy of ultrasound in the second trimester for the detection of spina bifida. DESIGN: Systematic review and meta-analysis. METHOD: Medline and Embase were searched to identify prospective studies in a general pregnant population. Also, in the Cochrane Library, references of identified reports and recent reviews were checked for relevant studies. Retrieved abstracts were selected independently by 2 authors using predefined criteria. Assessment of quality and generalizability of the included studies and data extraction were performed by 3 authors independently. The data were tested for heterogeneity and pooled estimates of sensitivity and specificity were calculated for all studies. RESULTS: 13 studies were included. Specificity was approximately 100% in all cases. Sensitivity varied from 40% to 100%. The summary point estimate for sensitivity was 71% with a 95% confidence interval of 59%-81%. CONCLUSION: Ultrasound in the second trimester is a specific investigation to detect spina bifida, which may detect seven out of ten defects. Used as a screening tool, it could contribute to a significant reduction in the number of children born with spina bifida.

Therapeutic mammalian artificial episomal chromosomes.

Two general strategies are being developed to engineer mammalian artificial chromosomes (MACs) as therapeutic vectors: (i) in vitro MAC cloning by enzymatic ligation of the individual MAC components followed by propagation in single cell organisms such as bacteria or yeast; and (ii) in situ MAC assembly by co-introduction of the various MAC elements into an 'incubator' mammalian tissue culture cell and use of it as a 'foster parental' donor cell. Because of their organizational compactness, in vitro built MACs are stuitable for somatic-based human gene therapy. In contrast, the long-term persistence of in situ built MACs can be capitalized on to generate husbandry transgenic animals expressing therapeutic genes. While current MAC systems generally rely on cis-elements exclusively from viral or genomic origin, the next generation of MACs may combine both into chimeric systems. As illustration of the genetic flexibility and technological potential of chimeric MACs, the herpes viral oriP/EBNA1 system, paradigm of a self-replicating and self-segregating episome in human cells is discussed in terms of future therapeutic applications.

A system for shuttling 200-kb BAC/PAC clones into human cells: stable extrachromosomal persistence and long-term ectopic gene activation.

A novel shuttle vector, pBH140, has been constructed that allows stable maintenance of large genomic inserts as human artificial episomal chromosomes (HAECs) in mammalian cells. The vector, essentially a hybrid BAC-HAEC, contains an F-based replication system as in a bacterial artificial chromosome (BAC) and the Epstein-Barr virus (EBV) latent origin of replication system, oriP, for replication in human cells. A 185-kb DNA insert containing the entire human beta-globin locus, including its locus control region (LCR), was retrofitted into this vector. The resulting beta-globin BAC-HAEC clone, p148BH, was transfected into human cells and analyzed for episomal maintenance and expression of the beta-globin gene. FISH revealed an association of the vector with different human chromosomes but no integration. The beta-globin BAC-HAECs were present at an average copy number of 11-15 per nucleus in the stably transformed human cells. After 1 year of continuous in vitro cultivation, the HAECs persisted as structurally intact 200-kb episomes. While no beta-globin transcription could be detected in the parental D98/Raji cells, correctly spliced RT-PCR products were produced at significant levels in long-term cultures of the BAC-HAEC-transduced cells. The wide availability of BAC and PAC libraries, the ease in manipulating cloned DNA in bacteria, and the episomal stability of the pBH140 vector make this system ideal for studies on gene expression and other genomic functions in human cells. The potential significance of large, functionally active episomes for gene therapy is discussed.

Epstein-Barr-based episomal chromosomes shuttle 100 kb of self-replicating circular human DNA in mouse cells.

We describe the microcell fusion transfer of 100-200 kb self-replicating circular human minichromosomes from human into mouse cells. This experimental approach is illustrated through the shutting of the latent 170 kb double-stranded DNA genome from the human herpesvirus, Epstein-Barr virus, into nonpermissive rodent cells. Using this interspecies transfer strategy, circular episomes carrying 95-105 kb of human DNA were successfully established at low copy number in mouse A9 cells. Selected episomes were stably maintained for 6 months, and unselected episomes were characterized by a 95% episomal retention per cell division. The establishment of a mouse artificial episomal chromosome system should facilitate evolutionary and therapeutic studies of large human DNA in rodent genetic backgrounds.

Mammalian artificial chromosomes as tools for gene therapy.

Mammalian artificial chromosomes (MACs) represent powerful tools for human gene therapy and animal transgenesis. First-generation linear genomic human artificial chromosomes (HACs) and circular chimeric genomic/viral mouse artificial episomal chromosomes (MAECs) have been developed. HACs have been shuttled from human into mouse embryonal stem cells and human trans-chromosomic mice have been generated. The potential of new genetic cis-elements and epigenetic phenomena for de novo segregation and replication activities on MACs are points for discussion. Once the size and delivery constraints of HACs are circumvented, therapeutic applications will be numerous, particularly for recessive syndromes involving large genes and multigenic diseases.

Defective bypass replication of a leading strand cyclobutane thymine dimer in xeroderma pigmentosum variant cell extracts.

Xeroderma pigmentosum variant (XP-V) is an inherited disorder resulting in hypersensitivity to the cytotoxic, mutagenic, and carcinogenic effects of UV light. There is evidence suggesting that XP-V cells carry a defect in the replication of UV-induced DNA damage, leading to mutations in genes, e.g., proto-oncogenes and tumor suppressor genes, of exposed skin cells. Using an in vitro assay to quantitatively evaluate replication of the most prevalent UV-derived DNA lesion, the cis,syn-thymine dimer (T x T), we have recently found that a T x T located on the leading strand can be bypassed by a bona fide human replication fork but can also induce fork uncoupling with selective synthesis of the undamaged lagging strand (D. Svoboda and J-M. Vos, Proc. Natl. Acad. Sci. USA, 92: 11975-11979, 1995). We now report the application and further refinement of this sensitive assay to the replication of a T x T-containing template by XP-V cell-free extracts. In comparison to normal controls, a 10-26-fold deficiency in the bypass replication of T x T was observed in XP-V cell extracts. In contrast, the disease extracts were as competent as controls for replication of the undamaged TT plasmid and for leading T x T-induced fork uncoupling. Besides mismatch repair and nucleotide excision repair, the bypass replication defect of XP-V may represent a novel category of hereditary mutator phenotypes affecting DNA damage processing.