Ina Vandenbroucke

Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders and is caused by mutations in the NF1 gene. Mutation detection is complex due to the large size of the NF1 gene, the presence of pseudogenes and the great variety of possible lesions. Although there is no evidence for locus heterogeneity in NF1, mutation detection rates rarely exceed 50%. We studied 67 unrelated NF1 patients fulfilling the NIH diagnostic criteria, 29 familial and 38 sporadic cases, using a cascade of complementary techniques. We performed a protein truncation test starting from puromycin‐treated EBV cell lines and, if no mutation was found, continued with heteroduplex, FISH, Southern blot and cytogenetic analysis. We identified the germline mutation in 64 of 67 patients and 32 of the mutations are novel. This is the highest mutation detection rate reported in a study of typical NF1 patients. All mutations were studied at the genomic and RNA level. The mutational spectrum consisted of 25 nonsense, 12 frameshift, 19 splice mutations, six missense and/or small in‐frame deletions, one deletion of the entire NF1 gene, and a translocation t(14;17)(q32;q11.2). Our data suggest that exons 10a‐10c and 37 are mutation‐rich regions and that together with some recurrent mutations they may account for almost 30% of the mutations in classical NF1 patients. We found a high frequency of unusual splice mutations outside of the AG/GT 5¢ and 3¢ splice sites. As some of these mutations form stable transcripts, it remains possible that a truncated neurofibromin is formed. Hum Mutat 15:541–555, 2000.

Quantification of splice variants using real-time PCR

A reliable and robust method for measuring the expression of alternatively spliced transcripts is an important step in investigating the significance of each variant. So far, accurate quantification of splice variants has been laborious and difficult due to the intrinsic limitations of conventional methods. The many advantages of real-time PCR have made this technique attractive to study its application in quantification of splice isoforms. We use skipping of exon 37 in the NF1 gene as a model to compare and evaluate the different strategies for quantitating splice variants using real-time PCR. An overview of three different possibilities for detecting alternative transcripts is given. We propose the use of a boundary-spanning primer to quantify isoforms that differ greatly in abundance. We describe here a novel method for creating a reliable standard curve using one plasmid containing both alternative transcripts. In addition, we validate the use of an absolute standard curve based on a dilution series of fluorometrically quantified PCR products.

Tracking the Evolution of Multiple In Vitro Hepatitis C Virus Replicon Variants under Protease Inhibitor Selection Pressure by 454 Deep Sequencing

ABSTRACT Resistance to hepatitis C virus (HCV) inhibitors targeting viral enzymes has been observed in in vitro replicon studies and during clinical trials. The factors determining the emergence of resistance and the changes in the viral quasispecies population under selective pressure are not fully understood. To assess the dynamics of variants emerging in vitro under various selective pressures with TMC380765, a potent macrocyclic HCV NS3/4A protease inhibitor, HCV genotype 1b replicon-containing cells were cultured in the presence of a low, high, or stepwise-increasing TMC380765 concentration(s). HCV replicon RNA from representative samples thus obtained was analyzed using (i) population, (ii) clonal, and (iii) 454 deep sequencing technologies. Depending on the concentration of TMC380765, distinct mutational patterns emerged. In particular, culturing with low concentrations resulted in the selection of low-level resistance mutations (F43S and A156G), whereas high concentrations resulted in the selection of high-level resistance mutations (A156V, D168V, and D168A). Clonal and 454 deep sequencing analysis of the replicon RNA allowed the identification of low-frequency preexisting mutations possibly contributing to the mutational pattern that emerged. Stepwise-increasing TMC380765 concentrations resulted in the emergence and disappearance of multiple replicon variants in response to the changing selection pressure. Moreover, two different codons for the wild-type amino acids were observed at certain NS3 positions within one population of replicons, which may contribute to the emerging mutational patterns. Deep sequencing technologies enabled the study of minority variants present in the HCV quasispecies population present at baseline and during antiviral drug pressure, giving new insights into the dynamics of resistance acquisition by HCV.

Minor variant detection in amplicons using 454 massive parallel pyrosequencing: experiences and considerations for successful applications

Ultra-deep sequencing (UDS) of amplicons is a major application for next-generation sequencing technologies, even more so for the 454 Genome Sequencer FLX. Especially for this application, errors that might be introduced during any of the sample processing or data analysis steps should be avoided or at least recognized, as they might lead to aberrant sequence variant calling. Since 454 pyrosequencing relies on PCR-driven target amplification, it is key to differentiate errors introduced during the amplification step from genuine minority variants. Thereto, optimal primer design is imperative because primer selection, primer dimer formation, and nonspecific binding may all affect the quality and outcome of amplicon-based deep sequencing. Also, other intrinsic PCR characteristics including amplification drift and the formation of secondary structures may influence sequencing data quality. We illustrate these phenomena using real life case studies and propose experimental and analytical evidence-based solutions for effective practice. Furthermore, because accuracy of the DNA polymerase is vital for reliable UDS results, a comparative analysis of error profiles from seven different DNA polymerases was performed and experimentally assessed in parallel by 454 sequencing. Finally, intra and interrun variability evaluation of the 454 sequencing protocol revealed highly reproducible results in amplicon-based UDS.

CXCR4-using HIV type 1 variants are more commonly found in peripheral blood mononuclear cell DNA than in plasma RNA.

Objective:To compare the distribution of R5-like and X4-like HIV-1envelope sequences in plasma and peripheral blood mononuclear cell (PBMC). Methods:Clonal sequencing of the HIV-1 glycoprotein 120 region was performed on PBMC DNA and plasma RNA of 11 HIV-1 subtype B-infected patients with high probability of carrying X4 virus. Coreceptor use was predicted using the position-specific scoring matrix (PSSM). Results:A total of 330 and 427 clonal envelope sequences were obtained from PBMC and plasma, respectively. PSSM interpretation revealed the presence of a mixture of predicted X4 and R5 sequences in 10 patients and pure R5 sequences in 1. The X4 sequences were significantly more represented in PBMC (with an average of 52.2% of the clonal proviral sequences scored X4) compared with plasma (19.7% X4 sequences) (P < 0.0001). At the single patient level, the higher representation of X4 sequences in PBMC reached statistical significance (P < 0.002) in 6 individuals. Conclusions:Mixtures of X4 and R5 sequences with highly divergent PSSM scores are present in both plasma and PBMC, but a shift toward a more abundant representation of X4-like PSSM scores in PBMC-derived DNA was apparent. Additional studies are needed to evaluate the clinical importance of these findings with regard to tropism prediction and the use of CCR5 anatagonists.

Study of Genotypic and Phenotypic HIV-1 Dynamics of Integrase Mutations During Raltegravir Treatment: A Refined Analysis by Ultra-Deep 454 Pyrosequencing

BACKGROUND The dynamics of raltegravir-resistant variants and their impact on virologic response in 23 HIV-1-infected patients, who started a salvage raltegravir-containing regimen, were investigated. METHODS Integrase population sequencing and Ultra-Deep-454 Pyrosequencing (UDPS) were performed on plasma samples at baseline and at raltegravir failure. All integrase mutations detected at a frequency ≥1% were considered to be reliable for the UDPS analyses. Phylogenetic and phenotypic resistance analyses were also performed. RESULTS At baseline, primary resistance mutations were not detected by both population and UDPS genotypic assays; few secondary mutations (T97A-V151I-G163R) were rarely detected and did not show any statistically association either with virologic response at 24-weeks or with the development of resistant variants at failure. At UDPS, not all resistant variants appearing early during treatment evolved as major populations during failure; only specific resistance pathways (Y143R-Q148H/R-N155H) associated with an increased rate of fitness and phenotypic resistance were selected. CONCLUSIONS Resistance to raltegravir in integrase strand transfer inhibitor-naive patients remains today a rare event, which might be changed by future extensive use of such drugs. In our study, pathways of resistance at failure were not predicted by baseline mutations, suggesting that evolution plus stochastic selection plays a major role in the appearance of integrase-resistance mutations, whereas fitness and resistance are dominant factors acting for the late selection of resistant quasispecies.

Ultra-deep sequencing of HIV-1 reverse transcriptase before start of an NNRTI-based regimen in treatment-naive patients

There are conflicting data on the impact of low frequency HIV-1 drug-resistant mutants on the response of first-line highly active antiretroviral therapy (HAART), more specifically containing a NNRTI. As population sequencing does not detect resistant viruses representing less than 15-25% of the viral population, more sensitive techniques have been developed but still need clinical validation. We evaluated ultra-deep sequencing (UDPS), recently more available and affordable, as a tool for the detection of HIV-1 minority species carrying drug resistant mutation (DRM) in a clinical setting. A retrospective analysis of the reverse transcriptase (RT) gene of plasma HIV-1 from 70 patients starting a NNRTI based regimen was performed. Minority populations were defined as representing > 1% and < 20% of the total viral population. Using UDPS, we could not confirm an association between the presence of low minority variants harbouring RT mutations at the start of therapy and primary or secondary therapeutic failure.

Quantification of NF1 transcripts reveals novel highly expressed splice variants

Previously, we have shown that the NF1 gene gives rise to multiple novel splice variants. In the present study, nine NF1 variants were quantified by real‐time PCR in various human tissues. Some of these variants were expressed at low to moderate low levels and possible implications of these findings are discussed. Interestingly, two variants (NF1‐ΔE4b and NF1‐ΔE43) were shown to be highly expressed in specific tissues. NF1‐ΔE43 lacks a nuclear targeting sequence and might be functionally different from full‐length NF1. These novel NF1 splice variants might expand our understanding of the role of neurofibromin.

Neurofibromin is actively transported to the nucleus

Mutations in the neurofibromatosis type 1 (NF1) tumor suppressor gene predispose individuals to a variety of benign and malignant tumors. Many tumor suppressors ‘shuttle’ between the nucleus and the cytoplasm, thus regulating their function. By expressing different NF1 constructs in COS‐7 cells (encompassing exons 28–49 and fused to the green fluorescent protein), we identified a functional nuclear localization signal (NLS) in exon 43. Mutation of the NLS completely abolishes the nuclear entry of the NF1‐derivative fusion protein. A highly expressed splice variant that lacks this NLS controls the localization and hence the function of neurofibromin. The localization of neurofibromin in the nucleus may provide novel clues to unknown functions for NF1.

Independent NF1 mutations in two large families with spinal neurofibromatosis

The neurofibromatoses are a group of neurocutaneous disorders that show extreme clinical heterogeneity and are characterised by growth abnormalities in tissues derived from the embryonic neural crest.1,2 Two main clinical forms exist, type 1 (NF1) and type 2 (NF2), as well as several alternate and related forms.2,3 NF1 and NF2 are the only clinically well defined disorders and both genes have been identified.4–8 The NIH diagnostic criteria for NF1, as defined by the conference statement,9 are met if two or more of the following are found: six or more CAL spots; two or more neurofibromas of any type or one plexiform neurofibroma; axillary or inguinal freckling; optic glioma; two or more Lisch nodules; a distinct osseous lesion; a first degree relative (parent, sib, or offspring) with NF1 according to the above criteria. Spinal nerve sheath tumours are described as symptomatic findings in only 5% of NF1 patients,10 although they can be observed by MRI in up to 36% of patients.11–13 The presence of a wide, symmetrical distribution of spinal neurofibromas, occurring in all adult affected members of the same family and segregating in an autosomal dominant fashion, is however extremely rare. This form, familial spinal NF (FSNF), has been considered an alternate form of NF since patients generally lack dermal neurofibromas and Lisch nodules, both typical hallmarks of NF1, and since symptomatic and generalised spinal neurofibromas are uncommon in classical NF1. FSNF has been reported in only four families.12,14,15 Three multigenerational families with spinal neurofibromas and CAL spots were shown to be linked to markers surrounding the NF1 locus.12,14,15 In the fourth family, presenting with spinal neurofibromas without CAL spots, linkage to the NF1 locus was excluded.14 Only in one FSNF family has …