Creation and evaluation of full-text literature-derived, feature-weighted disease models of genetically determined developmental disorders.

There are >2500 different genetically determined developmental disorders (DD), which, as a group, show very high levels of both locus and allelic heterogeneity. This has led to the wide-spread use of evidence-based filtering of genome-wide sequence data as a diagnostic tool in DD. Determining whether the association of a filtered variant at a specific locus is a plausible explanation of the phenotype in the proband is crucial and commonly requires extensive manual literature review by both clinical scientists and clinicians. Access to a database of weighted clinical features extracted from rigorously curated literature would increase the efficiency of this process and facilitate the development of robust phenotypic similarity metrics. However, given the large and rapidly increasing volume of published information, conventional biocuration approaches are becoming impractical. Here, we present a scalable, automated method for the extraction of categorical phenotypic descriptors from the full-text literature. Papers identified through literature review were downloaded and parsed using the Cadmus custom retrieval package. Human Phenotype Ontology terms were extracted using MetaMap, with 76-84% precision and 65-73% recall. Mean terms per paper increased from 9 in title + abstract, to 68 using full text. We demonstrate that these literature-derived disease models plausibly reflect true disease expressivity more accurately than widely used manually curated models, through comparison with prospectively gathered data from the Deciphering Developmental Disorders study. The area under the curve for receiver operating characteristic (ROC) curves increased by 5-10% through the use of literature-derived models. This work shows that scalable automated literature curation increases performance and adds weight to the need for this strategy to be integrated into informatic variant analysis pipelines. Database URL: https://doi.org/10.1093/database/baac038.

FISH mapping of de novo apparently balanced chromosome rearrangements identifies characteristics associated with phenotypic abnormality.

We report fluorescence in situ hybridization (FISH) mapping of 152, mostly de novo, apparently balanced chromosomal rearrangement (ABCR) breakpoints in 76 individuals, 30 of whom had no obvious phenotypic abnormality (control group) and 46 of whom had an associated disease (case group). The aim of this study was to identify breakpoint characteristics that could discriminate between these groups and which might be of predictive value in de novo ABCR (DN-ABCR) cases detected antenatally. We found no difference in the proportion of breakpoints that interrupted a gene, although in three cases, direct interruption or deletion of known autosomal-dominant or X-linked recessive Mendelian disease genes was diagnostic. The only significant predictor of phenotypic abnormality in the group as a whole was the localization of one or both breakpoints to an R-positive (G-negative) band with estimated predictive values of 0.69 (95% CL 0.54-0.81) and 0.90 (95% CL 0.60-0.98), respectively. R-positive bands are known to contain more genes and have a higher guanine-cytosine (GC) content than do G-positive (R-negative) bands; however, whether a gene was interrupted by the breakpoint or the GC content in the 200 kB around the breakpoint had no discriminant ability. Our results suggest that the large-scale genomic context of the breakpoint has prognostic utility and that the pathological mechanism of mapping to an R-band cannot be accounted for by direct gene inactivation.

Distinct methylation of the interferon gamma (IFN-gamma) and interleukin 3 (IL-3) genes in newly activated primary CD8+ T lymphocytes: regional IFN-gamma promoter demethylation and mRNA expression are heritable in CD44(high)CD8+ T cells.

Differential genomic DNA methylation has the potential to influence the development of T cell cytokine production profiles. Therefore, we have conducted a clonal analysis of interferon (IFN)-gamma and interleukin (IL)-3 gene methylation and messenger (m)RNA expression in primary CD8+ T cells during the early stages of activation, growth, and cytokine expression. Despite similar distributions and densities of CpG methylation sites, the IFN-gamma and IL-3 promoters exhibited differential demethylation in the same T cell clone, and heterogeneity between clones. Methylation patterns and mRNA levels were correlated for both genes, but demethylation of the IFN-gamma promoter was widespread across >300 basepairs in clones expressing high levels of IFN-gamma mRNA, whereas demethylation of the IL-3 promoter was confined to specific CpG sites in the same clones. Conversely, the majority of clones expressing low or undetectable levels of IFN-gamma mRNA exhibited symmetrical methylation of four to six of the IFN-gamma promoter CpG sites. Genomic DNA methylation also has the potential to influence the maintenance or stability of T cell cytokine production profiles. Therefore, we also tested the heritability of IFN-gamma gene methylation and mRNA expression in families of clones derived from resting CD44(low)CD8+ T cells or from previously activated CD44(high)CD8+ T cells. The patterns of IFN-gamma gene demethylation and mRNA expression were faithfully inherited in all clones derived from CD44(high) cells, but variable in clones derived from CD44(low) cells. Overall, these findings suggest that differential genomic DNA methylation, including differences among cytokine genes, among individual T cells, and among T cells with different activation histories, is an important feature of cytokine gene expression in primary T cells.

Clinically-relevant postzygotic mosaicism in parents and children with developmental disorders in trio exome sequencing data.

Mosaic genetic variants can have major clinical impact. We systematically analyse trio exome sequence data from 4,293 probands from the DDD Study with severe developmental disorders for pathogenic postzygotic mosaicism (PZM) in the child or a clinically-unaffected parent, and use ultrahigh-depth sequencing to validate candidate mosaic variants. We observe that levels of mosaicism for small genetic variants are usually equivalent in both saliva and blood and ~3% of causative de novo mutations exhibit PZM; this is an important observation, as the sibling recurrence risk is extremely low. We identify parental PZM in 21 trios (0.5% of trios), resulting in a substantially increased sibling recurrence risk in future pregnancies. Together, these forms of mosaicism account for 40 (1%) diagnoses in our cohort. Likely child-PZM mutations occur equally on both parental haplotypes, and the penetrance of detectable mosaic pathogenic variants overall is likely to be less than half that of constitutive variants.

Antisense oligonucleotides specific for transforming growth factor beta2 inhibit the growth of malignant mesothelioma both in vitro and in vivo.

Transforming growth factor beta (TGF-beta) is a potent growth-regulatory and immunomodulatory cytokine that exerts a diverse range of effects on many types of cells. High levels of TGF-beta are produced by several human and mouse malignant mesothelioma (MM) cell lines, and it is known to act as a growth factor for these cells. Antisense oligonucleotides (ODNs), targeted against specific TGF-beta mRNA, were used to block TGF-beta production from MM cells in vitro and in vivo. TGF-beta antisense ODNs were encapsulated in liposomes and transfected into MM cells or delivered intratumorally. TGF-beta2 mRNA levels, assessed by semiquantitative PCR, and TGF-beta2 protein secretion were reduced after TGF-beta2 antisense ODN transfection. MM cell proliferation, assessed by tritiated thymidine uptake, was specifically inhibited by both TGF-beta1- and TGF-beta2-specific antisense ODNs. In vivo administration of TGF-beta2 antisense ODNs, delivered locally, reduced tumor growth. These data show that the blockade of TGF-beta2 within this tumor reduces tumor growth and raises the possibility that TGF-beta2 antisense ODNs may be useful as a therapy for this disease.

Transforming growth factor beta in infectious disease: always there for the host and the pathogen.

At the portals of pathogen entry, there are pools of the latent form of a potent cytokine, transforming growth factor beta (TGF-beta). Many infections activate these pools and stimulate further TGF-beta expression. As well as potent immunomodulation, activated TGF-beta might have important effects on pathogen entry, replication, persistence, latency and oncogenesis.

Nature versus nurture in T cell cytokine production.

Both extrinsic and intrinsic factors influence the development of cytokine expression patterns in T lymphocytes. The models proposed to accommodate these factors are often separated into two types: deterministic (or instructional) and probabilistic (or stochastic). In this review we compare these two types of models and examine how they account for different stages of T cell cytokine responses to antigen stimulation. We conclude by showing how a reconciliation of the two types of models may be possible, perhaps through the regulatory potential of heritable epigenetic mechanisms. This type of reconciliation may open up new avenues for manipulating T cell cytokine expression and redirecting immune responses.

MLYCD mutation analysis: evidence for protein mistargeting as a cause of MLYCD deficiency.

Malonyl-CoA decarboxylase (MLYCD) deficiency is an autosomal recessive disorder characterized by malonic aciduria, developmental delay, seizure disorder, hypoglycemia, and cardiomyopathy. Genomic sequencing of MLYCD in nine unrelated patients identified 16 of 18 pathogenic alleles, which are documented in the newly created Human MLYCD Allelic Variant Database (http://mlycd.hgu.mrc.ac.uk/). Fibroblast cell lines were available from eight of these patients and two previously reported patients with homozygous MLYCD mutations. Western blot analysis using antisera raised to a C-terminal peptide detected a 66-kDa band that was absent in six patients and substantially reduced in three patients. One patient showed an increase in protein levels with a prominent smeary 68-l83-kDa band. Immunocytochemical analysis of MLYCD-expressing patient cell lines showed apparent intracellular mislocalization. An extreme N-terminal mutation c.8G>A (p.G3D) mislocalized to the plasma membrane, suggesting that a novel targeting signal may reside in a four-amino acid conserved N-terminal motif. A 25-base deletion between the putative mitochondrial and peroxisomal initiating codons (M1 and M40) and a point mutation ablating the second of these (c.119T>C, p.M40T) both showed punctate perinuclear staining. As none of the three mislocalizing mutations are predicted to alter the catalytic function of the peptide, it seems likely that correct subcellular localization of MLYCD is critical for it to function normally.

Potential for interferon-alpha-based therapy in mesothelioma: assessment in a murine model.

Malignant mesothelioma is an aggressive tumor, usually induced by asbestos exposure, that has a poor prognosis and is unresponsive to conventional therapy. The present study was aimed at assessing the potential for interferon-alpha (IFN-alpha)-based therapies in a murine model for malignant mesothelioma. The effect of recombinant human IFN-alpha B/D on tumor growth, alone and in combination with either of two immunomodulatory and antiproliferative agents beta-carotene or alpha-difluoromethylornithine (DFMO), was assessed. The data suggest that IFN-alpha treatment is most efficacious when commenced early in tumor development. Combination of IFN-alpha with either DFMO or dietary beta-carotene supplementation improved the effect of an otherwise suboptimal IFN-alpha therapy regimen. Both IFN-alpha and beta-carotene had in vivo stimulatory effects on immune cells, perhaps indirectly by inhibiting TGF-beta generation. The immunomodulatory effects may contribute, at least in part, to the positive antitumor and clinical activities of the treatments in this model.

Dengue virus binding to human leukocyte cell lines: receptor usage differs between cell types and virus strains.

Monocyte macrophages (Mphi) are thought to be the principal target cells for the dengue viruses (DV), the cause of dengue fever and hemorrhagic fever. Cell attachment is mediated by the virus envelope (E) protein, but the host-cell receptors remain elusive. Currently, candidate receptor molecules include proteins, Fc receptors, glycosaminoglycans (GAGs) and lipopolysaccharide binding CD14-associated molecules. Here, we show that in addition to Mphi, cells of the T- and B-cell lineages, and including cells lacking GAGs, can bind and become infected with DV. The level of virus binding varied widely between cell lines and, notably, between virus strains within a DV serotype. The latter difference may be ascribable to one or more amino acid differences in domain II of the E protein. Heparin had no significant effect on DV binding, while heparinase treatment of cells in all cases increased DV binding, further supporting the contention that GAGs are not required for DV binding and infection of human cells. In contrast to a recent report, we found that lipopolysaccharide (LPS) had either no effect or enhanced DV binding to, and infection of, various human leukocyte cell lines, while in all virus-cell combinations, depletion of Ca(2+)/Mg(2+) enhanced DV binding. This argues against involvement of beta(2) integrins in virus-host cell interactions, a conclusion in accord with the demonstration of three virus binding membrane proteins of < 75 kDa. Collectively, the results of this study question the purported exclusive importance of the E protein domain III in DV binding to host cells and point to a far more complex interaction between various target cells and, notably, individual DV strains.

Altered CD3 chain and cytokine gene expression in tumor infiltrating T lymphocytes during the development of mesothelioma.

The mechanisms whereby tumors escape immunosurveillance remain poorly understood. De-activation or deviation of T lymphocyte responses may occur following exposure to tumor-associated or -derived signals. In the present study it is demonstrated that during development of syngeneic malignant mesothelioma in mice, the relative CD3 delta, CD3 gamma and CD3 zeta mRNA levels expressed by tumor infiltrating lymphocytes (TIL) decrease, while CD3 epsilon mRNA levels remain relatively constant. Expression of IFN gamma mRNA by TIL decreased during tumor development, while IL-2 mRNA levels showed slight increases. IL-3 mRNA was not detected at any time during tumor development and IL-4 transcripts were detected in the later stages of tumor development. In the spleens of tumor-bearing mice, IL-2 transcripts were detected throughout the time course from days 1 to 22(24), while IFN gamma mRNA was only detected at early times from days 0-13. Previous work demonstrated a role for tumor cell-derived TGF beta in the immunobiology of mesothelioma. Here it is shown that the suppression of CD3-subunit expression by TIL was ameliorated in tumors where TGF beta -expression was reduced by inducible TGF beta-specific antisense-RNA, thus, suggesting that lymphocytes may become de-activated upon infiltration of the tumor micro-environment.

Carey-Fineman-Ziter (CFZ) syndrome: report on affected sibs.

We describe a sib pair with craniofacial anomalies, micrognathia, Mobius sequence, generalised myopathy, relative macrocephaly, and developmental delay. They appear to have the Carey-Fineman-Ziter syndrome (MIM 254940), which has been reported in only four children, a sib pair and two sporadic cases. This report on an additional affected brother and sister pair supports autosomal inheritance as the likely cause. These cases also confirm that scoliosis, talipes equinovarus, and a non-specific primary myopathy are important manifestations of Carey-Fineman-Ziter syndrome.

Clinical phenotype of desmosterolosis.

We describe a child with lethal multiple malformations and generalised accumulation of desmosterol. The infant had macrocephaly, a hypoplastic nasal bridge, thick alveolar ridges, gingival nodules, cleft palate, total anomalous pulmonary venous drainage, ambiguous genitalia, short limbs, and generalised osteosclerosis. Gas chromatography-mass spectrometry demonstrated an abnormal accumulation of desmosterol in kidney, liver. and brain. Higher than normal levels of the same sterol were detected in plasma samples obtained from both parents. The biochemical phenotype in this infant is highly suggestive of a novel inborn error of cholesterol biosynthesis caused by an autosomal recessive deficiency of 3betahydroxysterol-delta24-reductase. A phenotypic overlap of this case with Raine syndrome was noted; however, desmosterol accumulation was not found on postmortem tissue samples from a previously reported case of this disorder.

Presymptomatic testing for Huntington's disease by linkage and by direct mutation analysis: comparison of uptake of testing and characteristics of test applicants.

In Edinburgh, we have compared presymptomatic testing by linkage and by direct mutation analysis by investigating the demand for testing and characteristics of test applicants. Annual new requests for the direct test (DT) are now double the peak with the linkage test (LT) but only 6% individuals have requested re-testing. DT applicants were older with a smaller proportion having lived with an affected relative that LT applicants. This was because many were relatives of newly diagnosed first known cases in their family. This may also explain why DT applicants were less likely to expect a negative result and more likely to be uncertain about their risk. A greater proportion of DT applicants first heard about the test from relatives or their GP than LT applicants who were more likely to hear from Genetic Centre. The demand for follow-up by the Geneticist/Genetic Nurse was much less for DT than for LT applicants largely due to the support offered by the HD Advisors.

Immunologic relationships between equine herpesvirus type 1 (equine abortion virus) and type 4 (equine rhinopneumonitis virus).

The specificity of selected immune responses to equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4) was examined in 3 colostrum-deprived specific-pathogen-free foals. Single foals were vaccinated with inactivated EHV-1, inactivated EHV-4, or control cell lysate plus adjuvant followed by successive intranasal challenge exposures with EHV-1 and EHV-4 or with EHV-4 and EHV-1. Vaccination with inactivated virus preparations elicited cellular immune responses and antibody which were augmented by subsequent challenge exposures. Cellular immune responses, as measured by in vitro lymphocyte blastogenesis, were cross-reactive after foals were given either EHV-1 or EHV-4. Serum virus-neutralizing antibody responses were type-specific for foals given EHV-1, but were cross-reactive after EHV-4 administrations. It was concluded that diseases caused by EHV-1 and EHV-4 may be more effectively controlled with a bivalent vaccine containing both EHV-1 and EHV-4 than with the presently used monovalent vaccines based on EHV-1 alone.

Symmetrical upper limb peromelia and lower limb phocomelia associated with a de novo apparently balanced reciprocal translocation: 46,XX,t(2; 12)(p25.1;q24.1).

We report a female fetus of 20 weeks gestation with severe symmetrical deformity affecting all four limbs. These deformities were unusual in that there was upper limb peromelia and lower limb phocomelia. No additional major malformations were identified on postmortem examination. In particular there was no evidence of splenogonadal fusion or micrognathia and hypoglossia. The limb malformations in this case are associated with a de novo apparently balanced reciprocal translocation 46,XX,t(2;12)(p25.1;q24.1). The cytogenetic features of Roberts-SC phocomelia syndrome were not detected. Unfortunately, the fibroblast line died and no FISH or DNA analysis could be carried out. In spite of this, the case is presented as it may be useful to other researchers in the selection of candidate genes for mendelian forms of peromelia and phocomelia.

Genomic instability associated with limb defects: case report and review of the literature.

We report a child in whom we observed markedly increased genome-wide spontaneous chromosomal breakage in both leucocytes and fibroblasts associated with severe growth retardation, radial aplasia, leucopenia, mild hydrocephalus and an unusual trichodystrophy. Exposure to DNA cross-linking agents diepoxybutane, mitomycin-C and mustine hydrochloride in this case did not result in the increased chromosome breakage seen in Fanconi anaemia. It may be that this child has a defect in postreplicative DNA repair interacting with the protein components deficient in FA.

Molecular epidemiology and pathogenesis of some equine herpesvirus type 1 (equine abortion virus) and type 4 (equine rhinopneumonitis virus) isolates.

Representative strains of EHV isolated from an aborted foetus and from a horse with rhinopneumonitis in New Zealand had restriction endonuclease DNA fingerprints typical of those usually associated with these syndromes elsewhere and now designated EHV1 and 4 respectively. EHV1 was isolated from the brain and spinal cord of a 4-year-old gelding that died of myeloencephalitis. A mare on the same farm, at about the same time as the gelding developed myeloencephalitis, aborted and EHV1 was isolated from the tissues of the aborted foetus. Restriction endonuclease DNA fingerprints of the viruses isolated from myeloencephalitis and abortion were indistinguishable by Bam HI but were distinguishable using Bgl I, Pvu II, Xho I and Hind III. The restriction endonuclease DNA fingerprints of 3 EHV1 strains known to cause myeloencephalitis were compared with each other and with EHV1 strains not known to be associated with myeloencephalitis. The Bgl I Pvu II and Hind III DNA fingerprints of the 3 myeloencephalogenic strains appear distinguishable from non-myeloencephalogenic strains. Abortion was induced in a mare by intrauterine inoculation of EHV4. The Bam HI, Bgl I, Pvu II, Xho I and Hind III restriction endonuclease DNA fingerprints of the inoculum virus were indistinguishable from virus recovered from the foetus. It was concluded that passage of the virus through the foetus did not detectably alter the restriction endonuclease DNA fingerprint.