LNA modification of single-stranded DNA oligonucleotides allows subtle gene modification in mismatch-repair-proficient cells

Synthetic single-stranded DNA oligonucleotides (ssODNs) can be used to generate subtle genetic modifications in eukaryotic and prokaryotic cells without the requirement for prior generation of DNA double-stranded breaks. However, DNA mismatch repair (MMR) suppresses the efficiency of gene modification by >100-fold. Here we present a commercially available ssODN design that evades MMR and enables subtle gene modification in MMR-proficient cells. The presence of locked nucleic acids (LNAs) in the ssODNs at mismatching bases, or also at directly adjacent bases, allowed 1-, 2-, or 3-bp substitutions in MMR-proficient mouse embryonic stem cells as effectively as in MMR-deficient cells. Additionally, in MMR-proficient Escherichia coli, LNA modification of the ssODNs enabled effective single-base-pair substitution. In vitro, LNA modification of mismatches precluded binding of purified E. coli MMR protein MutS. These findings make ssODN-directed gene modification particularly well suited for applications that require the evaluation of a large number of sequence variants with an easy selectable phenotype.

Polymerase ε1 mutation in a human syndrome with facial dysmorphism, immunodeficiency, livedo, and short stature (“FILS syndrome”)

DNA polymerase ε (Polε) is a large, four-subunit polymerase that is conserved throughout the eukaryotes. Its primary function is to synthesize DNA at the leading strand during replication. It is also involved in a wide variety of fundamental cellular processes, including cell cycle progression and DNA repair/recombination. Here, we report that a homozygous single base pair substitution in POLE1 (polymerase ε 1), encoding the catalytic subunit of Polε, caused facial dysmorphism, immunodeficiency, livedo, and short stature (“FILS syndrome”) in a large, consanguineous family. The mutation resulted in alternative splicing in the conserved region of intron 34, which strongly decreased protein expression of Polε1 and also to a lesser extent the Polε2 subunit. We observed impairment in proliferation and G1- to S-phase progression in patients’ T lymphocytes. Polε1 depletion also impaired G1- to S-phase progression in B lymphocytes, chondrocytes, and osteoblasts. Our results evidence the developmental impact of a Polε catalytic subunit deficiency in humans and its causal relationship with a newly recognized, inherited disorder.

Differences in biomarker expression in HNSCC according to p53 status.

5523 Background: Patients with p53 wildtype head and neck squamous cell carcinoma (HNSCC) tend to be HPV-positive, which associates with better prognosis. The purpose of this study was to explore biomarker expression profiles for insight into molecular differences in HNSCC patients based on p53 status. Methods: TP53 gene sequencing using the AmpliChip p53 microarray (Roche Molecular Systems, Inc.) was attempted on 61 HNSCC patients previously tested with Caris Target Now tumor profiling service. DNA was extracted from a FFPE sample, amplified and processed on the AmpliChip p53 microarray to detect single base pair substitution and deletion mutations in exons 2 - 11 and their flanking splice sites in the TP53 gene (GenBank X54156). EGFR FISH , HER2 IHC and 22 other predictive biomarkers, e.g. TS, TOPO2A, MGMT, etc., were assayed and retrospectively analyzed. All tests were performed in a CLIA-certified lab and interpreted by board-certified pathologists or cytogeneticists. Statistical analysis was performed using SPSS (PASW statistics17) for parametric and non-parametric tests of independence. Results: 52 cases provided sufficient quality DNA for p53 analysis and results revealed a mutation rate of 25% in HNSCC patients. Interestingly, only EGFR FISH and HER2 IHC (p=.002 and p=.004, respectively) were differentially expressed in wildtype vs. mutated p53. Matched-pair analysis in the p53 mutated subgroup (n=13) showed no significant trend regarding EGFR status (p=.763) but a slight trend towards HER-2 negativity (p=.020). In the p53 wildtype subgroup (n=39), a strong association with EGFR FISH non-amplification (n=28, 71.8%, p<.001) as well as HER-2 negativity (n=38, 97.4%, p<.001) was shown. Conclusions: To our knowledge, this is the first analysis of differential biomarker expression profiles in HNSCC based on p53 status. We hypothesize that the absence of EGFR amplification in the p53 wildtype cancers may be a contributing factor to the improved prognosis observed in HPV-positive HNSCC. Additionally, the strong association between p53 wildtype HNSCC patients and EGFR non-amplification suggests EGFR-targeted therapies like cetuximab would likely fail in p53 wildtype patients.

Pathophysiology and therapy for haemoglobinopathies; Part I: sickle cell disease

In sickle cell disease, a single base pair substitution in the gene encoding the β-globin chain of the haemoglobin molecule gives rise to a surprisingly broad spectrum of pathophysiological and clinical manifestations. Inflammation, endothelial activation, red blood cell membrane abnormalities and altered availability of vasoactive factors characterise this disorder. Clinically, patients suffer from a host of seemingly unrelated maladies, from pain episodes to strokes, life-threatening infections and pulmonary hypertension. Deepened understanding of this complex disease now allows us to begin to turn away from simple supportive treatments, and move towards therapies aimed at specific pathophysiological targets. This article, the first of two reviews on the pathophysiology of haemoglobinopathies, discusses the molecular basis of sickle cell disease, and elaborates on the many factors that exacerbate or ameliorate the disease process. It then focuses on the promising targeted therapies currently in use or under investigation. An accompanying article on haemoglobinopathies (Part II) focuses on thalassaemias.

Neurofilament M gene in a French-Canadian Population with Parkinson’s Disease

Background:Recently, a single base pair substitution (G1747A) mutation of the neurofilament M (NF-M) gene was reported in a French-Canadian patient with early onset Parkinson’s disease (PD). Three unaffected siblings were found to be heterozygotes for the NF-M Gly336Ser mutation but, to date, no other affected PD individuals have been found with a similar mutation. No other individuals with Parkinson’s disease and of similar ethnic background have been screened for this mutation.Methods:We screened 102 French-Canadian patients with definite PD and 45 French-Canadian controls for this substitution in the NF-M gene using a PCR-restriction enzyme digestion method.Results:None of the patients or controls carried this mutation.Conclusion:Our results would indicate that this mutation is not common even in a PD population of similar ethnic background and suggest this change represents a rare variant. However, these results do not exclude the possibility that other mutations in this gene could be present.

Two Different Molecular Defects in the Tva Receptor Gene Explain the Resistance of Two tvar Lines of Chickens to Infection by Subgroup A Avian Sarcoma and Leukosis Viruses

ABSTRACT The subgroup A to E avian sarcoma and leukosis viruses (ASLVs) are highly related and are thought to have evolved from a common ancestor. These viruses use distinct cell surface proteins as receptors to gain entry into avian cells. Chickens have evolved resistance to infection by the ASLVs. We have identified the mutations responsible for the block to virus entry in chicken lines resistant to infection by subgroup A ASLVs [ASLV(A)]. The tva genetic locus determines the susceptibility of chicken cells to ASLV(A) viruses. In quail, the ASLV(A) susceptibility allele tvas encodes two forms of the Tva receptor; these proteins are translated from alternatively spliced mRNAs. The normal cellular function of the Tva receptor is unknown; however, the extracellular domain contains a 40-amino-acid, cysteine-rich region that is homologous to the ligand binding region of the low-density lipoprotein receptor (LDLR) proteins. The chicken tvas cDNAs had not yet been fully characterized; we cloned the chicken tva cDNAs from two lines of subgroup A-susceptible chickens, line H6 and line 0. Two types of chicken tvas cDNAs were obtained. These cDNAs encode a longer and shorter form of the Tva receptor homologous to the Tva forms in quail. Two different defects were identified in cDNAs cloned from two different ASLV(A)-resistant inbred chickens, line C and line 72. Line C tvar contains a single base pair substitution, resulting in a cysteine-to-tryptophan change in the LDLR-like region of Tva. This mutation drastically reduces the binding affinity of TvaR for the ASLV(A) envelope glycoproteins. Line 72 tvar2 contains a 4-bp insertion in exon 1 that causes a change in the reading frame, which blocks expression of the Tva receptor.