Homozygous Cys542-->Arg substitution in GPIIIa in a Swiss patient with type I Glanzmann's thrombasthenia

Glanzmann's thrombasthenia (GT) arises from a qualitative or quantitative defect in the GPIIb-IIIa complex (integrin alphaIIbbeta3), the mediator of platelet aggregation. We describe a patient in whom clinical and laboratory findings typical of type I GT were found together with a second pathology involving neurological and other complications symptomatic of tuberous sclerosis. Analysis of platelet proteins by Western blotting revealed trace amounts of normally migrating GPIIb and equally small amounts of GPIIIa of slightly slower than normal migration. Flow cytometry confirmed a much decreased binding to platelets of monoclonal antibodies to GPIIb, GPIIIa or GPIIb-IIIa, and an antibody to the alphav subunit also showed decreased binding. Nonradioactive PCR single-strand conformation polymorphism analysis followed by direct sequencing of PCR-amplified DNA fragments showed a homozygous point mutation (T to C) at nucleotide 1722 of GPIIIa cDNA and which led to a Cys542-->Arg substitution in the GPIIIa protein. The mutation gave rise to a HinP1 I restriction site in exon 11 of the GPIIIa gene and allele-specific restriction enzyme analysis of family members confirmed that a single mutated allele was inherited from each parent. This amino acid substitution presumably changes the capacity for disulphide bond formation within the cysteine-rich core region of GPIIIa and its study will provide new information on GPIIb-IIIa and alphavbeta3 structure and biosynthesis.

Large deletions in the CFTR gene: clinics and genetics in Swiss patients with CF

Cystic fibrosis (CF) is the most common life-shortening autosomal recessive disorder in Caucasians, and is associated with at least one mutation on each CF transmembrane conductance regulator (CFTR) allele. Some patients, however, with only one identifiable point mutation carry on the other allele, a large deletion that is not detected by conventional screening methods. The overall frequency of large deletions in patients with CF is estimated to be 1-3%. Using the CFTR Multiplex Ligation dependent Probe Amplification Kit (MRC-Holland, Amsterdam, Netherlands) that allows the exact detection of copy numbers from all 27 exons in the CFTR gene, we screened 50 patients with only one identified mutation for large deletions in the CFTR gene. Each detected deletion was confirmed using our real-time polymerase chain reaction (PCR) assay and deletion-specific PCR reactions using junction fragment primers. We detected large deletions in eight patients (16%). These eight CF alleles belong to four different deletion types (CFTRindel2, CFTRdele14b-17b, CFTRdele17a-17b and CFTRdele 2-9) whereof the last is novel. Comparing detailed clinical data of all these patients with CF and the molecular genetic findings, we were able to elaborate criteria for deletion screenings and possible genotype-phenotype associations. In conclusion, we agree with other authors that deletion screenings should be implemented in routine genetic diagnostics of CF.

Genotypic and phenotypic characterization of Trypanosoma brucei gambiense isolates from Ibba, South Sudan, an area of high melarsoprol treatment failure rate

Resistance of trypanosomes to melarsoprol is ascribed to reduced uptake of the drug via the P2 nucleoside transporter. The aim of this study was to look for evidence of drug resistance in Trypanosoma brucei gambiense isolates from sleeping sickness patients in Ibba, South Sudan, an area of high melarsoprol failure rate. Eighteen T. b. gambiense stocks were phenotypically and only 10 strains genotypically characterized. In vitro, all isolates were sensitive to melarsoprol, melarsen oxide, and diminazene. Infected mice were cured with a 4 day treatment of 2.5mg/kg bwt melarsoprol, confirming that the isolates were sensitive. The gene that codes for the P2 transporter, TbATI, was amplified by PCR and sequenced. The sequences were almost identical to the TbAT1(sensitive) reference, except for one point mutation, C1384T resulting in the amino acid change proline-462 to serine. None of the described TbAT1(resistant)-type mutations were detected. In a T. b. gambiense sleeping sickness focus where melarsoprol had to be abandoned due to the high incidence of treatment failures, no evidence for drug resistant trypanosomes or for TbAT1(resistant)-type alleles of the P2 transporter could be found. These findings indicate that factors other than drug resistance contribute to melarsoprol treatment failures.

Inhibition of FcepsilonRI-dependent mediator release and calcium flux from human mast cells by sialic acid-binding immunoglobulin-like lectin 8 engagement

BACKGROUND: Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of glycan-binding inhibitory receptors, and among them, Siglec-8 is selectively expressed on human eosinophils, basophils, and mast cells. On eosinophils, Siglec-8 engagement induces apoptosis, but its function on mast cells is unknown. OBJECTIVE: We sought to study the effect of Siglec-8 engagement on human mast cell survival and mediator release responses. METHODS: Human mast cells were generated from CD34+ precursors. Apoptosis was studied by using flow cytometry. Mast cell mediator release or human lung airway smooth muscle contraction was initiated by FcepsilonRI cross-linking with or without preincubation with Siglec-8 or control antibodies, and release of mediators was analyzed along with Ca++ flux. RBL-2H3 cells transfected with normal and mutated forms of Siglec-8 were used to study how Siglec-8 engagement alters mediator release. RESULTS: Siglec-8 engagement failed to induce human mast cell apoptosis. However, preincubation with Siglec-8 mAbs significantly (P < .05) inhibited FcepsilonRI-dependent histamine and prostaglandin D(2) release, Ca++ flux, and anti-IgE-evoked contractions of human bronchial rings. In contrast, release of IL-8 was not inhibited. Siglec-8 ligation was also shown to inhibit beta-hexosaminidase release and Ca++ flux triggered through FcepsilonRI in RBL-2H3 cells transfected with full-length human Siglec-8 but not in cells transfected with Siglec-8 containing a tyrosine to phenylalanine point mutation in the membrane-proximal immunoreceptor tyrosine-based inhibitory motif domain. CONCLUSION: These data represent the first reported inhibitory effects of Siglec engagement on human mast cells.

Exon 17 skipping in CLCN1 leads to recessive myotonia congenita

Mutations in CLCN1, the gene encoding the ClC-1 chloride channel in skeletal muscle, lead to myotonia congenita. The effects on the intramembranous channel forming domains have been investigated more than that at the intracellular C-terminus. We have performed a mutation screen involving the whole CLCN1 gene of patients with myotonia congenita by polymerase chain reaction (PCR), single-strand conformation polymorphism studies, and sequencing. Two unrelated patients harbored the same homozygous G-to-T mutation on the donor splice site of intron 17. This led to the skipping of exon 17, as evidenced by the reverse transcriptase PCR. When the exon 17-deleted CLCN1 was expressed in Xenopus oocytes, no chloride current was measurable. This function could be restored by coexpression with the wild-type channel. Our data suggest an important role of this C-terminal region and that exon 17 skipping resulting from a homozygous point mutation in CLCN1 can lead to recessive myotonia congenita.

Integrative analysis of RUNX1 downstream pathways and target genes

BACKGROUND: The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia. RESULTS: Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFbeta, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFbeta. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes. CONCLUSION: This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications.

Looking the cow in the eye: deletion in the NID1 gene is associated with recessive inherited cataract in Romagnola cattle

Cataract is a known condition leading to opacification of the eye lens causing partial or total blindness. Mutations are known to cause autosomal dominant or recessive inherited forms of cataracts in humans, mice, rats, guinea pigs and dogs. The use of large-sized animal models instead of those using mice for the study of this condition has been discussed due to the small size of rodent lenses. Four juvenile-onset cases of bilateral incomplete immature nuclear cataract were recently observed in Romagnola cattle. Pedigree analysis suggested a monogenic autosomal recessive inheritance. In addition to the cataract, one of the cases displayed abnormal head movements. Genome-wide association and homozygosity mapping and subsequent whole genome sequencing of a single case identified two perfectly associated sequence variants in a critical interval of 7.2 Mb on cattle chromosome 28: a missense point mutation located in an uncharacterized locus and an 855 bp deletion across the exon 19/intron 19 border of the bovine nidogen 1 (NID1) gene (c.3579_3604+829del). RT-PCR showed that NID1 is expressed in bovine lenses while the transcript of the second locus was absent. The NID1 deletion leads to the skipping of exon 19 during transcription and is therefore predicted to cause a frameshift and premature stop codon (p.1164fs27X). The truncated protein lacks a C-terminal domain essential for binding with matrix assembly complexes. Nidogen 1 deficient mice show neurological abnormalities and highly irregular crystal lens alterations. This study adds NID1 to the list of candidate genes for inherited cataract in humans and is the first report of a naturally occurring mutation leading to non-syndromic catarct in cattle provides a potential large animal model for human cataract.

Drosophila valois encodes a divergent WD protein that is required for Vasa localization and Oskar protein accumulation

valois (vls) was identified as a posterior group gene in the initial screens for Drosophila maternal-effect lethal mutations. Despite its early genetic identification, it has not been characterized at the molecular level until now. We show that vls encodes a divergent WD domain protein and that the three available EMS-induced point mutations cause premature stop codons in the vls ORF. We have generated a null allele that has a stronger phenotype than the EMS mutants. The vlsnull mutant shows that vls+ is required for high levels of Oskar protein to accumulate during oogenesis, for normal posterior localization of Oskar in later stages of oogenesis and for posterior localization of the Vasa protein during the entire process of pole plasm assembly. There is no evidence for vls being dependent on an upstream factor of the posterior pathway, suggesting that Valois protein (Vls) instead acts as a co-factor in the process. Based on the structure of Vls, the function of similar proteins in different systems and our phenotypic analysis, it seems likely that vls may promote posterior patterning by facilitating interactions between different molecules.

A naturally occurring prfA truncation in a Listeria monocytogenes field strain contributes to reduced replication and cell-to-cell spread

Listeria (L.) monocytogenes is an environmental bacterium that may become an intracellular pathogen upon ingestion to cause gastroenteritis, septicaemia, abortions, and/or fatal infections of the central nervous system. We here describe a L. monocytogenes field strain (JF5171) isolated from a bovine placenta in the context of abortion, which exhibited attenuation in bovine brain-slice cultures. The whole genome of strain JF5171 was sequenced, and the invasion, replication, and intercellular spread of JF5171 were further analyzed by quantification of colony forming units and immunofluorescence studies. Phospholipase and hemolysis activity of JF5171 were also quantified along with transcription levels of actA, hly and prfA. The data obtained were compared to those of the widely used L. monocytogenes reference strain, EGD-e. JF5171 exhibited reduced replication and lower levels of phospholipase and hemolysis activity. Invasion and cell-to-cell spread was strongly decreased compared to EGD-e, and actin polymerization was absent. A frame shift deletion was identified in the JF5171 coding region of the major regulator for virulence, prfA. This resulted in a truncated C-terminus sequence (WEN* vs. WGKLN*). In addition, a point mutation resulted in a lysine to arginine substitution at amino acid position 197. Complementation with prfA from EGD-e and with (EGD-e) prfA-K197N increased the replication and spread efficiency of JF5171. In contrast, complementation with the truncated version of prfA had no effect. Taken together, these results suggest that the truncated C-terminus of prfA considerably contributes to the strongly attenuated phenotype observed in vitro.

Hairless Streaks in Cattle Implicate TSR2 in Early Hair Follicle Formation

Four related cows showed hairless streaks on various parts of the body with no correlation to the pigmentation pattern. The stripes occurred in a consistent pattern resembling the lines of Blaschko. The non-syndromic hairlessness phenotype observed occurred across three generations of a single family and was compatible with an X-linked mode of inheritance. Linkage analysis and subsequent whole genome sequencing of one affected female identified two perfectly associated non-synonymous sequence variants in the critical interval on bovine chromosome X. Both variants occurred in complete linkage disequilibrium and were absent in more than 3900 controls. An ERCC6L missense mutation was predicted to cause an amino acid substitution of a non-conserved residue. Analysis in mice showed no specific Ercc6l expression pattern related to hair follicle development and therefore ERCC6L was not considered as causative gene. A point mutation at the 5'-splice junction of exon 5 of the TSR2, 20S rRNA accumulation, homolog (S. cerevisiae), gene led to the production of two mutant transcripts, both of which contain a frameshift and generate a premature stop codon predicted to truncate approximately 25% of the protein. Interestingly, in addition to the presence of both physiological TSR2 transcripts, the two mutant transcripts were predominantly detected in the hairless skin of the affected cows. Immunohistochemistry, using an antibody against the N-terminal part of the bovine protein demonstrated the specific expression of the TSR2 protein in the skin and the hair of the affected and the control cows as well as in bovine fetal skin and hair. The RNA hybridization in situ showed that Tsr2 was expressed in pre- and post-natal phases of hair follicle development in mice. Mammalian TSR2 proteins are highly conserved and are known to be broadly expressed, but their precise in vivo functions are poorly understood. Thus, by dissecting a naturally occurring mutation in a domestic animal species, we identified TSR2 as a regulator of hair follicle development.

Lethal chondrodysplasia in a family of Holstein cattle is associated with a de novo splice site variant of COL2A1

Background Lethal chondrodysplasia (bulldog syndrome) is a well-known congenital syndrome in cattle and occurs sporadically in many breeds. In 2015, it was noticed that about 12 % of the offspring of the phenotypically normal Danish Holstein sire VH Cadiz Captivo showed chondrodysplasia resembling previously reported bulldog calves. Pedigree analysis of affected calves did not display obvious inbreeding to a common ancestor, suggesting the causative allele was not a rare recessive. The normal phenotype of the sire suggested a dominant inheritance with incomplete penetrance or a mosaic mutation. Results Three malformed calves were examined by necropsy, histopathology, radiology, and computed tomography scanning. These calves were morphologically similar and displayed severe disproportionate dwarfism and reduced body weight. The syndrome was characterized by shortening and compression of the body due to reduced length of the spine and the long bones of the limbs. The vicerocranium had severe dysplasia and palatoschisis. The bones had small irregular diaphyses and enlarged epiphyses consisting only of chondroid tissue. The sire and a total of four affected half-sib offspring and their dams were genotyped with the BovineHD SNP array to map the defect in the genome. Significant genetic linkage was obtained for several regions of the bovine genome including chromosome 5 where whole genome sequencing of an affected calf revealed a COL2A1 point mutation (g.32473300 G > A). This private sequence variant was predicted to affect splicing as it altered the conserved splice donor sequence GT at the 5’-end of COL2A1 intron 36, which was changed to AT. All five available cases carried the mutant allele in heterozygous state and all five dams were homozygous wild type. The sire VH Cadiz Captivo was shown to be a gonadal and somatic mosaic as assessed by the presence of the mutant allele at levels of about 5 % in peripheral blood and 15 % in semen. Conclusions The phenotypic and genetic findings are comparable to a previously reported COL2A1 missense mutation underlying lethal chondrodysplasia in the offspring of a mosaic French Holstein sire (Igale Masc). The identified independent spontaneous splice site variant in COL2A1 most likely caused chondrodysplasia and must have occurred during the early foetal development of the sire. This study provides a first example of a dominant COL2A1 splice site variant as candidate causal mutation of a severe lethal chondrodysplasia phenotype. Germline mosaicism is a relatively frequent mechanism in the origin of genetic disorders and explains the prevalence of a certain fraction of affected offspring. Paternal dominant de novo mutations are a risk in cattle breeding, especially because the ratio of defective offspring may be very high and be associated with significant animal welfare problems.

A novel SCN5A mutation, F1344S, identified in a patient with Brugada syndrome and fever-induced ventricular fibrillation

OBJECTIVE: Brugada syndrome (BS) is an inherited electrical cardiac disorder characterized by right bundle branch block pattern and ST segment elevation in leads V1 to V3 on surface electrocardiogram that can potentially lead to malignant ventricular tachycardia and sudden cardiac death. About 20% of patients have mutations in the only so far identified gene, SCN5A, which encodes the alpha-subunit of the human cardiac voltage-dependent sodium channel (hNa(v)1.5). Fever has been shown to unmask or trigger the BS phenotype, but the associated molecular and the biophysical mechanisms are still poorly understood. We report on the identification and biophysical characterization of a novel heterozygous missense mutation in SCN5A, F1344S, in a 42-year-old male patient showing the BS phenotype leading to ventricular fibrillation during fever. METHODS: The mutation was reproduced in vitro using site-directed mutagenesis and characterized using the patch clamp technique in the whole-cell configuration. RESULTS: The biophysical characterization of the channels carrying the F1344S mutation revealed a 10 mV mid-point shift of the G/V curve toward more positive voltages during activation. Raising the temperature to 40.5 degrees C further shifted the mid-point activation by 18 mV and significantly changed the slope factor in Na(v)1.5/F1344S mutant channels from -6.49 to -10.27 mV. CONCLUSIONS: Our findings indicate for the first time that the shift in activation and change in the slope factor at a higher temperature mimicking fever could reduce sodium currents' amplitude and trigger the manifestation of the BS phenotype.

ENU-induced phenovariance in mice: inferences from 587 mutations

Background We present a compendium of N-ethyl-N-nitrosourea (ENU)-induced mouse mutations, identified in our laboratory over a period of 10 years either on the basis of phenotype or whole genome and/or whole exome sequencing, and archived in the Mutagenetix database. Our purpose is threefold: 1) to formally describe many point mutations, including those that were not previously disclosed in peer-reviewed publications; 2) to assess the characteristics of these mutations; and 3) to estimate the likelihood that a missense mutation induced by ENU will create a detectable phenotype. Findings In the context of an ENU mutagenesis program for C57BL/6J mice, a total of 185 phenotypes were tracked to mutations in 129 genes. In addition, 402 incidental mutations were identified and predicted to affect 390 genes. As previously reported, ENU shows strand asymmetry in its induction of mutations, particularly favoring T to A rather than A to T in the sense strand of coding regions and splice junctions. Some amino acid substitutions are far more likely to be damaging than others, and some are far more likely to be observed. Indeed, from among a total of 494 non-synonymous coding mutations, ENU was observed to create only 114 of the 182 possible amino acid substitutions that single base changes can achieve. Based on differences in overt null allele frequencies observed in phenotypic vs. non-phenotypic mutation sets, we infer that ENU-induced missense mutations create detectable phenotype only about 1 in 4.7 times. While the remaining mutations may not be functionally neutral, they are, on average, beneath the limits of detection of the phenotypic assays we applied. Conclusions Collectively, these mutations add to our understanding of the chemical specificity of ENU, the types of amino acid substitutions it creates, and its efficiency in causing phenovariance. Our data support the validity of computational algorithms for the prediction of damage caused by amino acid substitutions, and may lead to refined predictions as to whether specific amino acid changes are responsible for observed phenotypes. These data form the basis for closer in silico estimations of the number of genes mutated to a state of phenovariance by ENU within a population of G3 mice.

Bevacizumab, Pemetrexed, and Cisplatin, or Bevacizumab and Erlotinib for Patients With Advanced Non-Small-Cell Lung Cancer Stratified by Epidermal Growth Factor Receptor Mutation: Phase II Trial SAKK19/09

Treatment allocation by epidermal growth factor receptor mutation status is a new standard in patients with metastatic nonesmall-cell lung cancer. Yet, relatively few modern chemotherapy trials were conducted in patients characterized by epidermal growth factor receptor wild type. We describe the results of a multicenter phase II trial, testing in parallel 2 novel combination therapies, predefined molecular markers, and tumor rebiopsy at progression. Objective: The goal was to demonstrate that tailored therapy, according to tumor histology and epidermal growth factor receptor (EGFR) mutation status, and the introduction of novel drug combinations in the treatment of advanced nonesmall-cell lung cancer are promising for further investigation. Methods: We conducted a multicenter phase II trial with mandatory EGFR testing and 2 strata. Patients with EGFR wild type received 4 cycles of bevacizumab, pemetrexed, and cisplatin, followed by maintenance with bevacizumab and pemetrexed until progression. Patients with EGFR mutations received bevacizumab and erlotinib until progression. Patients had computed tomography scans every 6 weeks and repeat biopsy at progression. The primary end point was progression-free survival (PFS) ≥ 35% at 6 months in stratum EGFR wild type; 77 patients were required to reach a power of 90% with an alpha of 5%. Secondary end points were median PFS, overall survival, best overall response rate (ORR), and tolerability. Further biomarkers and biopsy at progression were also evaluated. Results: A total of 77 evaluable patients with EGFR wild type received an average of 9 cycles (range, 1-25). PFS at 6 months was 45.5%, median PFS was 6.9 months, overall survival was 12.1 months, and ORR was 62%. Kirsten rat sarcoma oncogene mutations and circulating vascular endothelial growth factor negatively correlated with survival, but thymidylate synthase expression did not. A total of 20 patients with EGFR mutations received an average of 16.