Second international guidelines for the diagnosis and management of hereditary hemorrhagic telangiectasia

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disease with an estimated prevalence of 1 in 5000 that is characterized by the presence of vascular malformations (VMs). These result in chronic bleeding, acute hemorrhage, and complications from shunting through VMs. The goal of the Second International HHT Guidelines process was to develop evidence-based consensus guidelines for the management and prevention of HHT-related symptoms and complications. The guidelines were developed using the AGREE II (Appraisal of Guidelines for Research and Evaluation II) framework and GRADE (Grading of Recommendations Assessment, Development and Evaluation) methodology. The guidelines expert panel included expert physicians (clinical and genetic) in HHT from 15 countries, guidelines methodologists, health care workers, health care administrators, patient advocacy representatives, and persons with HHT. During the preconference process, the expert panel generated clinically relevant questions in 6 priority topic areas. A systematic literature search was done in June 2019, and articles meeting a priori criteria were included to generate evidence tables, which were used as the basis for recommendation development. The expert panel subsequently convened during a guidelines conference to conduct a structured consensus process, during which recommendations reaching at least 80% consensus were discussed and approved. The expert panel generated and approved 6 new recommendations for each of the following 6 priority topic areas: epistaxis, gastrointestinal bleeding, anemia and iron deficiency, liver VMs, pediatric care, and pregnancy and delivery (36 total). The recommendations highlight new evidence in existing topics from the first International HHT Guidelines and provide guidance in 3 new areas: anemia, pediatrics, and pregnancy and delivery. These recommendations should facilitate implementation of key components of HHT care into clinical practice.

Basal Ganglia T1 Hyperintensity in Hereditary Hemorrhagic Telangiectasia.

BACKGROUND AND PURPOSE: The implications of basal ganglia T1 hyperintensity remain unclear in patients with hereditary hemorrhagic telangiectasia. This study was performed to assess the prevalence of this imaging finding in a large cohort of patients with hereditary hemorrhagic telangiectasia and to identify any association between this phenomenon and other disease manifestations. MATERIALS AND METHODS: In this retrospective study, we identified all patients at our institution diagnosed with definite hereditary hemorrhagic telangiectasia from 2001 to 2017. Patients who did not undergo brain MR imaging were excluded. Patient demographics, laboratory results, and hereditary hemorrhagic telangiectasia disease characteristics were noted. Basal ganglia hyperintensity was evaluated both qualitatively and quantitatively relative to the signal intensity in the ipsilateral thalami. Statistical analysis was performed with commercially available software. RESULTS: A total of 312 patients (41% men, 59% women; mean age, 51 ± 18 years) with definite hereditary hemorrhagic telangiectasia were identified. Basal ganglia T1 hyperintensity was present in 23.4% of patients and demonstrated a statistically significant association with older age (P < .001), increased hepatic AVMs (P < .001), high cardiac output state (P < .001), hepatic failure (P = .01), elevated peak serum alkaline phosphatase level (P = .03), and increased total bilirubin count (P = .03). There was no significant association with sex, hereditary hemorrhagic telangiectasia genetic mutation status, parkinsonism, or serum transaminase levels. CONCLUSIONS: Basal ganglia T1 hyperintensity occurs in >23% of patients with hereditary hemorrhagic telangiectasia and is associated with hepatic vascular malformations, hepatic dysfunction, and elevated cardiac output. The presence of this finding on screening MR imaging in patients with hereditary hemorrhagic telangiectasia should prompt further evaluation for visceral lesions causing arteriovenous shunting.

Neurovascular Manifestations of Hereditary Hemorrhagic Telangiectasia: A Consecutive Series of 376 Patients during 15 Years.

BACKGROUND AND PURPOSE: Hereditary hemorrhagic telangiectasia is associated with a wide range of neurovascular abnormalities. The aim of this study was to characterize the spectrum of cerebrovascular lesions, including brain arteriovenous malformations, in patients with hereditary hemorrhagic telangiectasia and to study associations between brain arteriovenous malformations and demographic variables, genetic mutations, and the presence of AVMs in other organs. MATERIALS AND METHODS: Consecutive patients with definite hereditary hemorrhagic telangiectasia who underwent brain MR imaging/MRA, CTA, or DSA at our institution from 2001 to 2015 were included. All studies were re-evaluated by 2 senior neuroradiologists for the presence, characteristics, location, and number of brain arteriovenous malformations, intracranial aneurysms, and nonshunting lesions. Brain arteriovenous malformations were categorized as high-flow pial fistulas, nidus-type brain AVMs, and capillary vascular malformations and were assigned a Spetzler-Martin score. We examined the association between baseline clinical and genetic mutational status and the presence/multiplicity of brain arteriovenous malformations. RESULTS: Three hundred seventy-six patients with definite hereditary hemorrhagic telangiectasia were included. One hundred ten brain arteriovenous malformations were noted in 48 patients (12.8%), with multiple brain arteriovenous malformations in 26 patients. These included 51 nidal brain arteriovenous malformations (46.4%), 58 capillary vascular malformations (52.7%), and 1 pial arteriovenous fistula (0.9%). Five patients (10.4%) with single nidal brain arteriovenous malformation presented with hemorrhage. Of brain arteriovenous malformations, 88.9% (88/99) had a Spetzler-Martin score of ≤2. Patients with brain arteriovenous malformations were more likely to be female (75.0% versus 57.6%, P = .01) and have a family history of hereditary hemorrhagic telangiectasia (95.8% versus 84.8%, P = .04). The prevalence of brain arteriovenous malformation was 19.7% in endoglin (ENG) mutations and 12.5% in activin receptor-like kinase (1ACVRL1) mutations. CONCLUSIONS: Our study of 376 patients with hereditary hemorrhagic telangiectasia demonstrated a high prevalence of brain arteriovenous malformations. Nidal brain arteriovenous malformations and capillary vascular malformations occurred in roughly equal numbers.

Analysis of pCU1 replication origins: dependence of oriS on the plasmid-encoded replication initiation protein RepA.

The broad-host-range replicon of the plasmid pCU1 has three origins of vegetative replication called oriB, oriS, and oriV. In the multi-origin replicon, individual origins can distinguish among replication factors provided by the host. It has been found that during replication in Escherichia coli polA(-) host, oriS was the only active origin of a mutant pCU1 derivative bearing a mutation in the gene encoding replication initiation protein RepA. To further investigate the capacity of oriS to function in an E. coli polA(-) host we constructed a number of clones of the basic replicon of pCU1 containing oriS as the only replication origin. An oriS construct created with pUC18 could transform the polA(-) strain when RepA was supplied in trans. When the oriS region (between nucleotides 290 and 832) was ligated to an antibiotic resistance Omega fragment, the construct could be recovered as a plasmid from polA(+) strain if functional RepA was provided in trans. Our results therefore indicate that the basic replicon of pCU1, containing oriS as the sole origin, does require RepA to initiate plasmid replication in E. coli

A tobacco cryptic constitutive promoter, tCUP, revealed by T-DNA tagging.

We have isolated a constitutive promoter sequence, tCUP, from tobacco by T-DNA tagging using a promoterless GUS-nos3' reporter gene construct. The T-DNA integration event produced a translational fusion with the GUS gene that is expressed widely in organs, at both the mRNA and enzyme activity levels. In tobacco transformed with a tCUP-GUS-nos3' gene, GUS specific activity in leaves was within a range of values similar to those of plants transformed with the widely used constitutive promoter gene fusion, CaMV 35S promoter-GUS-nos3'. Characteristics of the tCUP promoter sequence differ from those of other plant constitutive promoters; for instance, the tCUP sequence lacks a TATA box. Transcription initiates at a single site within the tCUP sequence which is similar to a transcriptional start site consensus sequence determined for plant genes. The tCUP promoter is cryptic as RNA accumulation at the transcriptional start site is not detected in untransformed tobacco. Thus, tCUP is the first example of a cryptic, constitutive promoter isolated from plants. The tCUP-GUS-nos3' gene fusion produced GUS activity in tissues of all species tested suggesting that tCUP may utilize fundamental transcription mechanisms found in plants.

Conditionally lethal genes in the N pilus region of plasmid pCU1.

Plasmid genes or regions that are conditionally lethal to Escherichia coli have been called kil and those lethal to Klebsiella but not to E. coli have been called kik. Both classes of genes are found in or close to the N pilus region of the plasmid pCU1 and the closely related plasmid pKM101. Here we describe two new and overlapping lethal genes that are located between kikA and traA of the plasmid pCU1 and display host specificity. KilC is lethal in E. coli and Klebsiella while kikC is lethal only in Klebsiella. The previously identified korA gene is sufficient to override the lethality of kilC in trans or in cis but is insufficient to override kikC. kilC expression in E. coli leads to cell death accompanied by an increase in average cell length without affecting septum formation.

Genetic analysis of the mobilization and leading regions of the IncN plasmids pKM101 and pCU1.

The conjugative IncN plasmids pKM101 and pCU1 have previously been shown to contain identical oriT sequences as well as conserved restriction endonuclease cleavage patterns within their tra regions. Complementation analysis and sequence data presented here indicate that these two plasmids encode essentially identical conjugal DNA-processing proteins. This region contains three genes, traI, traJ, and traK, transcribed in the same orientation from a promoter that probably lies within or near the conjugal transfer origin (oriT). Three corresponding proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and complementation analysis confirmed that this region contains three tra complementation groups. All three proteins resemble proteins of the IncW plasmid R388 and other plasmids thought to have roles in processing of plasmid DNA during conjugation. The hydropathy profile of TraJ suggests a transmembrane topology similar to that of several homologous proteins. Both traK and traI were required for efficient interplasmid site-specific recombination at oriT, while traJ was not required. The leading region of pKM101 contains three genes (stbA, stbB, and stbC), null mutations in which cause elevated levels of plasmid instability. Plasmid instability was observed only in hosts that are proficient in interplasmid recombination, suggesting that this recombination can potentially lead to plasmid loss and that Stb proteins somehow overcome this, possibly via site-specific multimer resolution.

Localization of the nic site of IncN conjugative plasmid pCU1 through formation of a hybrid oriT.

The N-type oriT of plasmid pMUR274 was cloned on a 474-bp RsaI-SspI fragment, and the nucleotide sequence was determined. A comparison of the pMUR274 oriT sequence and the sequence of the oriTs of IncN plasmid pCU1 and IncW plasmid R388 demonstrated 57 and 28% identity, respectively. Intramolecular, site-specific recombination between the pCU1 oriT and the oriT of pMUR274 resulted in the formation of a hybrid oriT containing one half of each parental sequence. The junction point of the hybrid occurred within a 10-bp sequence, GCTATACACC, present in both parental sequences and represents the nic site of each oriT. Mutation of the first A or second T residue within the 10-bp junction sequence reduced transfer less than 20-fold, while mutation of either the second or third A residue reduced transfer over 1,000-fold. Site-specific recombination between a wild-type pCU1 oriT and these four mutant pCU1 oriTs demonstrated that nic lies between the second T and second A residues of the 10-bp junction sequence. Site-specific recombination between wild-type and mutant pCU1 oriTs also demonstrated that point mutations to the right of nic reduced both initiation and termination of transfer while point mutations to the left of nic reduced termination but had little or no effect on initiation. A 28-bp deletion within the AT-rich region 39 bases to the right of nic reduced both initiation and termination, while deletion of a 6-bp inverted repeat sequence at the right-most boundary of the minimal oriT region reduced initiation but not termination.

Structure and mode of action of kikA, a genetic region lethal to Klebsiella oxytoca and associated with conjugative antibiotic-resistance plasmids of the IncN group.

Transmission of conjugative plasmids of the IncN group into Klebsiella oxytoca, but not into Escherichia coli, results in the marked reduction of viability of the recipients. In the plasmid pCU1 a 500-bp locus called kikA has the major role in determining this phenotype. Expression of two open reading frames (orf104 and orf70) is required for the Kik+ phenotype and they can function in cis or in trans. orf104 encodes a 8.9-kDa soluble protein which is translocated into the periplasm. orf70 encodes a 7.6-kDa soluble protein which is found in the cytoplasm. The expression of the kikA region from its natural promoter(s) is positively regulated at the level of transcription by an additional plasmid locus which is located within the tra region. Further experiments show that the action of kikA causes reversible growth inhibition but does not affect cellular respiration and does not induce any morphological changes of the host cell.

A novel plasmid gene involved in bacteriophage PRD1 infection and conjugative host-range.

PRD1 infects bacteria carrying IncN plasmids by binding to their conjugative pili. Mutations in a plasmid locus kikA close to the pilus region result in PRD1 resistance and reduced conjugation proficiency to Klebsiella but not to Escherichia coli. One of the two genes of kikA is sufficient to restore both normal phenotypes. PRD1 binds to cells carrying the mutant plasmid but fails to inject its genome.

Use of a DNA polymerase III bypass mutant of Escherichia coli, pcbA1, to isolate potentially useful mutations of a complex plasmid replicon.

The essential replicon region of plasmid pCU1 has, within 1.2 kb, two origins of replication that can function in the absence of Escherichia coli DNA polymerase I and one that requires this polymerase. To isolate mutants in the replicon pathway that uses the PolI-dependent origin in the presence of the two other origins, we examined the feasibility of exploiting E. coli strains carrying a polymerase c bypass mutation (pcbA) and which can survive and form colonies with the polymerase activity of polC inactivated at 42 degrees C. The selection scheme that is described was successful and resulted in the isolation of a mutant replicon that is not maintained at 42 degrees C in a PcbA-PolC+tsPolA+ strain. Nucleotide sequencing indentified the mutated region to be within the origin (OriV) that was known to be polA-dependent. Electron microscopy of mutant plasmid molecules replicating in a Pcb+ strain confirmed that OriV is inactivated.

Lethality and survival of Klebsiella oxytoca evoked by conjugative IncN group plasmids.

The transmission of plasmid pCU1 (or other IncN group plasmid) into a population of Klebsiella oxytoca cells reduces the viability of the population. A 2,400-bp region adjacent to traA is responsible for this phenotype and includes two regions, called kikA and kikC. Klebsiella cells which received this region and survived were found to acquire a chromosomal mutation which renders them immune to killing even after the plasmid is cured from the cells. To obtain insight into the mode of this apparent lethality, an appropriate pCU1lacZ derivative was constructed. It could be introduced with high efficiency into Klebsiella cells. Analyses of the resultant colonies indicate that the loss of viability is not a consequence of the death of plasmid-free segregants. On the contrary and unlike postsegregational killing by plasmids, cells survived by losing the plasmid or by acquiring, secondarily, a chromosomal mutation which confers immunity to killing.

Determination of the binding sites of RepA, a replication initiator protein of the basic replicon of the IncN group plasmid pCU1.

A 2kb DNA region of the broad-host-range plasmid pCU1 carries all of the information essential for the stable maintenance of the plasmid and to express the same host-range specificity. It was predicted that the protein required to initiate replication from at least one of the three origins of the plasmid is encoded by the longest open-reading frame (ORF239) of the three overlapping in-frame ORFs located within the 2 kb region. The product of ORF239 has been named RepA. The initiator protein was overexpressed, purified and used for in vitro binding studies. Gel mobility shift experiments were performed to localize RepA binding sites. The DNA sequence protected by the bound RepA molecule(s) was determined by DNase I footprinting and 19 of a 20 bp long sequence that is part of the protected sequence were located in two clusters flanking the repA gene. A plasmid created by linking a 310 bp fragment (nucleotides 238 to 547) of the 2 kb region to the antibiotic resistance genes carried by the omega fragment, can be maintained stably if the RepA protein is supplied in trans. We conclude that this 310 bp DNA fragment, which consists of a short G+C and a long A+T rich region and the cluster of five RepA binding sites, carries a functional origin of the plasmid-protein dependent replication. The position of this origin indicates that it is oriB, one of the three origins previously identified by electron microscopy. The second cluster of RepA binding sites is downstream of the repA gene and consists of 14 sites that are in inverted orientation compared with the binding sites located in the oriB region. They are part of the region that was shown formerly to be involved in controlling the copy number of the plasmid. In contrast to oriB, binding of RepA to neither the oriS nor oriV region was detected.

The incN plasmid replicon: two pathways of DNA polymerase I-independent replication.

The 2,053-bp broad-host-range incompatibility group N replicon of plasmid pCU1 has two components: a region of 1,200 bp that is sufficient for its replication in Escherichia coli PolA+ and PolA- hosts and a regulatory region called the group I iteron region that contains 13 39-bp iterons. Within the 1,200-bp region, there are three replication origins, two of which, called oriB and oriS, function in PolA+ and PolA- hosts and a third, called oriV, which functions only in PolA+ hosts. The region also specifies a protein called RepA. We now show that both oriB and oriS can function in a delta polA strain but that in such a strain, only oriB has an absolute requirement for RepA. oriS can function without RepA and polymerase I provided that the iteron region is deleted and that in this circumstance, it is the only origin, the usage of which is detected. The requirements for oriB usage can thus be distinguished from those for oriS usage. The oriB region can be recovered as a plasmid only if RepA is provided in trans. These complex features of this replicon are also shown to be shared by the IncN replicons of other antibiotic resistance plasmids. Functionally distinguishable origins in a small replicon may be a way of endowing such a replicon with a broad host range.

Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis.

Brassica napus cv. Topas microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. We show that this switch in developmental pathways is accompanied by the induction of high levels of napin seed storage protein gene expression. Changes in the plant growth or microspore culture conditions were not by themselves sufficient to induce napin gene expression. Specific members of the napin multigene family were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), constitute the highly conserved BnmNAP subfamily of napin genes. Both RNA gel blot analysis, using a subfamily-specific probe, and histochemical analysis of transgenic plants expressing a BngNAP1 promoter-beta-glucuronidase gene fusion demonstrated that the BnmNAP subfamily is expressed in embryogenic microspores as well as during subsequent stages of microsporic embryo development.

T-DNA tagging of a seed coat-specific cryptic promoter in tobacco.

T-DNA tagging with a promoterless beta-glucuronidase (GUS) gene generated a transgenic Nicotiana tabacum plant that expressed GUS activity only in developing seed coats. Cloning and deletion analysis of the GUS fusion revealed that the promoter responsible for seed coat specificity was located in the plant DNA proximal to the GUS gene. A 3.3 kb fragment corresponding to the insertion site was isolated from untransformed plants. No long open reading frames were detected in this region. Northern blots and RNase protection assays failed to detect transcripts from this region in untransformed plants. Furthermore, the insertion site was situated within the N. tomentosiformis genome of the allotetraploid species N. tabacum, in a region which is not conserved within the genus Nicotiana. It is concluded that seed coat-specific GUS expression in this transgenic plant resulted from T-DNA insertion next to a cryptic promoter. These results suggest that at least some of the fusions generated to marker genes in promoter trapping studies are not associated with conventional gene promoters. The possibility that similar insertion events play a role in gene evolution is discussed.

Three clustered origins of replication in a promiscuous-plasmid replicon and their differential use in a PolA+ strain and a delta PolA strain of Escherichia coli K-12.

A 1,197-bp region of the broad-host-range plasmid pCU1 is adequate for its replication. Analysis of replicating molecules containing this region reveals three clustered origins of vegetative replication and replication proceeds bidirectionally from each in a theta mode. In an Escherichia coli polymerase I deletion mutant, utilization of one of these three origins was not detected. The potentiality for origin utilization may therefore be a determinant of replicon host range.

DNA cassettes containing the origin of transfer (oriT) of two broad-host-range transfer systems.

Plasmid constructs are described that carry retrievable DNA cassettes containing the origin of transfer region (oriT) from two broad-host-range plasmids. Restriction of these high copy number plasmids with any one of a variety of enzymes yields a linear DNA fragment of convenient size containing the oriT region of either pCUI or RK2. This DNA can be ligated into any vector or recombinant plasmid containing a compatible enzyme site and can be easily identified by size on an agarose gel. Any plasmid can therefore be mobilized using a number of helper strains or conjugative plasmids derived from the parental plasmids. In addition, the cassettes can be used for a variety of genetic manipulations including "selectable" linker mutagenesis.

Isolation and characterization of kikA, a region on IncN group plasmids that determines killing of Klebsiella oxytoca.

Transfer of the IncN group plasmid pCU1 from Escherichia coli to Klebsiella oxytoca by conjugation kills a large proportion (90 to 95%) of the recipients of plasmid DNA, whereas transfer to E. coli or even to the closely related Enterobacter aerogenes does not. Two regions, kikA and kikB, have been identified on pCU1 that contribute to the Kik (killing in klebsiellas) phenotype. We have localized the kikA region to 500 bp by deletion analysis and show by DNA-DNA hybridization that kikA is highly conserved among the plasmids of incompatibility group N. The expression in K. oxytoca of kikA under the control of the strong inducible E. coli tac promoter results in loss of cell viability. The nucleotide sequence showed two overlapping open reading frames (ORFs) within the kikA region. The first ORF codes for a putative polypeptide of 104 amino acids (ORF104). The second ORF codes for a 70-amino-acid polypeptide (ORF70). The properties of the putative protein encoded by ORF104 and gene fusions of kikA to alkaline phosphatase by using TnphoA suggest that killing may involve an association with the bacterial membrane; however, we could not rule out the possibility that ORF70 plays a role in the Kik phenotype.