Splice Mutants Research Articles

A multiscale approach reveals the molecular architecture of the autoinhibited kinesin KIF5A

Kinesin-1 is a microtubule motor that transports cellular cargo along microtubules. KIF5A is one of three kinesin-1 isoforms in humans, all of which are autoinhibited by an interaction between the motor and an IAK motif in the proximal region of the C-terminal tail. The C-terminal tail of KIF5A is ∼80 residues longer than the other two kinesin-1 isoforms (KIF5B and KIF5C) and it is unclear if it contributes to autoinhibition. Mutations in KIF5A cause neuronal diseases and could affect autoinhibition, as reported for a mutation that skips exon 27, altering its C-terminal sequence. Here, we combined negative-stain electron microscopy, crosslinking mass spectrometry (XL-MS) and AlphaFold2 structure prediction to determine the molecular architecture of the full-length autoinhibited KIF5A homodimer, in the absence of light chains. We show that KIF5A forms a compact, bent conformation, through a bend between coiled coils 2 and 3, around P687. XL-MS of WT KIF5A revealed extensive interactions between residues in the motor, between coiled-coil 1 and the motor, between coiled-coils 1 and 2, with coiled coils 3 and 4, and the proximal region of the C-terminal tail and the motor in the autoinhibited state, but not between the distal C-terminal region and the rest of the molecule. While negative stain electron microscopy of exon-27 KIF5A splice mutant showed the presence of autoinhibited molecules, XL-MS analysis suggested that its autoinhibited state is more labile. Our model offers a conceptual framework for understanding how mutations within the motor and stalk domain may affect motor activity.

Identification and characterization of adipokinetic hormone and its receptor in bumble bee queens, Bombus lantschouensis

Adipokinetic hormone (AKH) and its receptor (AKHR) are crucial for energy homeostasis in insects, while their identification and functional characterization are controversial in bee species since the transition of energy storage from the solitary to the eusocial groups. As the primitively eusocial bee species, the bumble bee is an ideal organism for the evolution analysis of AKH and AKHR. In this current study, the identification and characterization of AKH of Bombus lantschouensis (Bomla_AKH) and its receptor (Bomla_AKHR) in queens exhibit that both Bomla_AKH and Bomla_AKHR are highly conserved with their counterparts in Bombus terrestris and Bombus impatiens. Notably, the alternative splicing that takes place with Bomla_akhr results in two putative proteins representing the amino and carboxyl fragments of Bomla_AKHR, respectively. Furthermore, the Bomla_akh expresses in the brain (with corpora cardiaca and corpora allata), while the Bomla_akhr expresses in the fat body and/or ovary depending on the specific development phase. Although the detailed mechanisms remain to be clarified, we can infer that the two putative proteins encoded by the splice mutant might participate in the functional regulation of Bomla_akhr in response to diverse demands in different tissues. The functional characterization and subcellular localization shows that the activation of Bomla_AKHR is AKH dose-dependent. Unfortunately, the response is too weak to do further signaling analysis, which might be due to the poor cytomembrane localization. Meanwhile, the activation mechanisms of Bomla_AKHR might be different from AKHRs of other insect species.

Mutational Landscape of DDR2 Gene in Lung Squamous Cell Carcinoma Using Next-generation Sequencing

BackgroundLung cancer represents the leading cause of cancer-related death worldwide. Despite great advances in lung cancer management with the recent emergence of molecular targeted therapies for non-squamous non–small-cell lung cancer, no dramatic improvements have been achieved in lung squamous cell carcinoma (SCC). Mutations in discoidin domain receptor 2 (DDR2) gene were recently identified as promising molecular targets in this histology. The aim of this study is to describe the DDR2 mutational landscape of lung SCC and investigate the associated clinical factors. MethodsNext-generation sequencing of the DDR2 gene was performed on 271 samples of lung SCC. Patients followed in our institution from January 2011 to August 2014 were retrospectively selected for data collection. Other driver gene alterations (EGFR, KRAS, BRAF, HER2, and PI3KCA) were analyzed using pyrosequencing. ResultsA total of 11 patients harboring a DDR2 mutation was detected among the 271 sequenced lung SCC samples (4%). We describe 10 unreported mutations, comprising a novel DDR2 exon 7 splice mutant. DDR2 mutations were not mutually exclusive with other driver gene alterations. One hundred thirty-six patients were included for clinical comparison and logistic regression analysis. No difference was detected between DDR2-mutant and DDR2 wild-type lung SCC regarding clinical characteristics or survival. ConclusionDDR2 mutations were observed in 4% of cases of lung SCC of European descent. DDR2-mutated tumors can exhibit other driver gene alterations. No clinical characteristics were significantly associated with DDR2 mutation.

Protein-protein interaction between human UGT1A1 and its splice mutant

Protein-protein interaction between human UGT1A1 and its splice mutant

Dimerization of human uridine diphosphate glucuronosyltransferase allozymes 1A1 and 1A9 alters their quercetin glucuronidation activities.

Uridine diphosphate glucuronosyltransferase 1A (UGT1A) is a major phase II drug-metabolism enzyme superfamily involved in the glucuronidation of endobiotics and xenobiotics in humans. Many polymorphisms in UGT1A genes are reported to inhibit or decrease UGT1A activity. In this study, two UGT1A1 allozymes, UGT1A1 wild-type and a splice mutant, as well as UGT1A9 wild-type and its three UGT1A9 allozymes, UGT1A9*2(C3Y), UGT1A9*3(M33T), and UGT1A9*5(D256N) were single- or double-expressed in a Bac-to-Bac expression system. Dimerization of UGT1A1 or UGT1A9 allozymes was observed via fluorescence resonance energy transfer (FRET) and co-immunoprecipitation analysis. SNPs of UGT1A altered the ability of protein-protein interaction, resulting in differential FRET efficiencies and donor-acceptor r distances. Dimerization changed the chemical regioselectivity, substrate-binding affinity, and enzymatic activity of UGT1A1 and UGT1A9 in glucuronidation of quercetin. These findings provide molecular insights into the consequences of homozygous and heterozygous UGT1A1 and UGT1A9 allozymes expression on quercetin glucuronidation.

Allelic variation of the COMT gene in a despotic primate society: A haplotype is related to cortisol excretion in Macaca fuscata

Sequence variations in genes of the monoamine neurotransmitter system and their common function in human and non-human primate species are an ongoing issue of investigation. However, the COMT gene, coding for the catechol-O-methyltransferase, has not yet attracted much scientific attention regarding its functional role in non-human primates. Considering that a polymorphism of the human COMT gene affects the enzyme activity and cortisol level in response to a social stressor, this study investigated the impact of COMT on endocrine stress and behavioural parameters in Japanese macaques (Macaca fuscata). The species exemplifies a despotic hierarchy in which males' social rank positions require an adaptation of behaviour strategies. During the mating period steroid secretion and the frequency of aggressive encounters between males increase. We addressed i) whether this species exhibits potential functional COMT variants, ii) whether these variants are associated with faecal cortisol excretion of males, iii) how they are distributed among different social rank positions and iv) whether they are associated with behavioural strategies during times of mate competition. By genotyping 26 males we identified three COMT haplotypes (HT), including a putative splice mutant (HT3). This variant was associated with increased cortisol excretion. Given the observed inverse correlation between cortisol and physical aggression, we assume that different COMT haplotypes may predispose individuals to pursue more or less aggressive strategies. How these gene-stress effects might favour a specific social role is discussed. Our study of non-invasive genotyping in combination with behavioural and endocrine parameters represents an important step towards the understanding of gene-stress effects in a hierarchically organised primate society.

Androgen Receptor Activation in Castration-Recurrent Prostate Cancer: The Role of Src-Family and Ack1 Tyrosine Kinases

There is growing appreciation that castration-recurrent prostate cancer (CR-CaP) is driven by the continued expression of androgen receptor (AR). AR activation in CR-CaP through various mechanisms, including AR overexpression, expression of AR splice variants or mutants, increased expression of co-regulator proteins, and by post-translational modification, allows for the induction of AR-regulated genes in response to very low levels of tissue-expressed, so-called intracrine androgens, resulting in pathways that mediate CaP proliferation, anti-apoptosis and oncogenic aggressiveness. The current review focuses on the role played by Src-family (SFK) and Ack1 non-receptor tyrosine kinases in activating AR through direct phosphorylation, respectively, on tyrosines 534 or 267, and how these modifications facilitate progression to CR-CaP. The fact that SFK and Ack1 are central mediators for multiple growth factor receptor signaling pathways that become activated in CR-CaP, especially in the context of metastatic growth in the bone, has contributed to recent therapeutic trials using SFK/Ack1 inhibitors in monotherapy or in combination with antagonists of the AR activation axis.

Population Shift Underlies Ca2+-induced Regulatory Transitions in the Sodium-Calcium Exchanger (NCX)

In eukaryotic Na(+)/Ca(2+) exchangers (NCX) the Ca(2+) binding CBD1 and CBD2 domains form a two-domain regulatory tandem (CBD12). An allosteric Ca(2+) sensor (Ca3-Ca4 sites) is located on CBD1, whereas CBD2 contains a splice-variant segment. Recently, a Ca(2+)-driven interdomain switch has been described, albeit how it couples Ca(2+) binding with signal propagation remains unclear. To resolve the dynamic features of Ca(2+)-induced conformational transitions we analyze here distinct splice variants and mutants of isolated CBD12 at varying temperatures by using small angle x-ray scattering (SAXS) and equilibrium (45)Ca(2+) binding assays. The ensemble optimization method SAXS analysis demonstrates that the apo and Mg(2+)-bound forms of CBD12 are highly flexible, whereas Ca(2+) binding to the Ca3-Ca4 sites results in a population shift of conformational landscape to more rigidified states. Population shift occurs even under conditions in which no effect of Ca(2+) is observed on the globally derived Dmax (maximal interatomic distance), although under comparable conditions a normal [Ca(2+)]-dependent allosteric regulation occurs. Low affinity sites (Ca1-Ca2) of CBD1 do not contribute to Ca(2+)-induced population shift, but the occupancy of these sites by 1 mM Mg(2+) shifts the Ca(2+) affinity (Kd) at the neighboring Ca3-Ca4 sites from ∼ 50 nM to ∼ 200 nM and thus, keeps the primary Ca(2+) sensor (Ca3-Ca4 sites) within a physiological range. Thus, Ca(2+) binding to the Ca3-Ca4 sites results in a population shift, where more constraint conformational states become highly populated at dynamic equilibrium in the absence of global conformational transitions in CBD alignment.

Activation of Src and transformation by an RPTPα splice mutant found in human tumours

Receptor protein tyrosine phosphatase α (RPTPα)-mediated Src activation is required for survival of tested human colon and oestrogen receptor-negative breast cancer cell lines. To explore whether mutated RPTPα participates in human carcinogenesis, we sequenced RPTPα cDNAs from five types of human tumours and found splice mutants in ∼30% of colon, breast, and liver tumours. RPTPα245, a mutant expressed in all three tumour types, was studied further. Although it lacks any catalytic domain, RPTPα245 expression in the tumours correlated with Src tyrosine dephosphorylation, and its expression in rodent fibroblasts activated Src by a novel mechanism. This involved RPTPα245 binding to endogenous RPTPα (eRPTPα), which decreased eRPTPα-Grb2 binding and increased eRPTPα dephosphorylation of Src without increasing non-specific eRPTPα activity. RPTPα245-eRPTPα binding was blocked by Pro210 → Leu/Pro211 → Leu mutation, consistent with the involvement of the structural 'wedge' that contributes to eRPTPα homodimerization. RPTPα245-induced fibroblast transformation was blocked by either Src or eRPTPα RNAi, indicating that this required the dephosphorylation of Src by eRPTPα. The transformed cells were tumourigenic in nude mice, suggesting that RPTPα245-induced activation of Src in the human tumours may have contributed to carcinogenesis.

Three novel alternative splicing mutations in BCR‐ABL1 detected in CML patients with resistance to kinase inhibitors

Multiple types of mutations in the BCR-ABL1 kinase domain have been reported. We previously reported a common alternatively spliced BCR-ABL mRNA with a 35-nucleotide insertion (35INS). We report three novel alternative splicing mutants expressed as the dominant transcripts in patient with chronic myelogenous leukemia and resistance to kinase inhibitors. We screened RNA from more than 200 patients with resistance to more than one of the three kinase inhibitors for ABL1 kinase domain mutations by direct sequencing. We found three not previously described splice mutants. All three showed >90% mutant transcript. The first resulted from the insertion of 79 nucleotides into the ABL1 exon 8-9 junction. The inserted sequence contained a sequence from regions of intron 8, located 120 bp apart: the 35-nucleotide sequence previously described, and an additional 44-nucleotide segment downstream from 35INS. The combined 79-nucleotide insertion splice mutant showed the same protein change as 35INS (p C475YfsX11). The second splice mutation comprised an 84-nucleotide sequence from intron 7 inserted into the ABL1 exon7-8 junction, also causing a frameshift and protein truncation (p A424EfsX18). The third splice derived from a 231-nucleotide sequence from intron 4 retained in the ABL1 exon 4-5 junction adding 40 intron-encoded amino acids and leading to a frameshift and early termination (p E275LfsX41). These findings, when combined with the data on 35INS, support the concept that loss of the C-terminus of BCR-ABL1 is associated with significant resistance to kinase inhibitors; this mechanism appears to be a major source of resistance to kinase inhibitors.

Multiple splicing defects caused by hERG splice site mutation 2592+1G>A associated with long QT syndrome

Long QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). Cryptic splice site activation in hERG has recently been identified as a novel pathogenic mechanism of LQT2. In this report, we characterize a hERG splice site mutation, 2592+1G>A, which occurs at the 5' splice site of intron 10. Reverse transcription-PCR analyses using hERG minigenes transfected into human embryonic kidney-293 cells and HL-1 cardiomyocytes revealed that the 2592+1G>A mutation disrupted normal splicing and caused multiple splicing defects: the activation of cryptic splice sites within exon 10 and intron 10 and complete intron 10 retention. We performed functional and biochemical analyses of the major splice product, hERGΔ24, in which 24 amino acids within the cyclic nucleotide binding domain of the hERG channel COOH-terminus is deleted. Patch-clamp experiments revealed that the splice mutant did not generate hERG current. Western blot and immunostaining studies showed that mutant channels did not traffic to the cell surface. Coexpression of wild-type hERG and hERGΔ24 resulted in significant dominant-negative suppression of hERG current via the intracellular retention of the wild-type channels. Our results demonstrate that 2592+1G>A causes multiple splicing defects, consistent with the pathogenic mechanisms of long QT syndrome.

Mutations at the Intron 9 Donor Splice Site in hERG Lead to Cryptic Splicing in LQT2

Long QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). More than 30% of LQT2 mutations are nonsense, frameshift, or splice site mutations that may affect mRNA stability and splicing. To date, relatively few studies have focused on the pathogenesis of hERG splice site mutations. We characterized three LQT2 mutations in the 5′ donor splice site of intron 9: 2398G>T, 2398+3A>T, and 2398+5G>T. G2398 is the last nucleotide of exon 9 and 2398G>T has been previously classified as a missense mutation (G800W). The functional consequences of these mutations were studied by RT-PCR analysis of RNA collected from HEK293 cells transfected with minigenes containing the wild-type or mutant genomic sequence spanning exon 8 to exon 11 of hERG. All three splice site mutants disrupt normal splicing and produce an aberrantly spliced transcript. Sequence analysis showed that this transcript results from the use of a cryptic 5′ donor splice site in intron 9 located 54 nt downstream of the normal site. Translation of this transcript would result in an in-frame insertion of 18 amino acids in the cyclic nucleotide binding domain. A full length hERG cDNA construct including the 2398G>T mutation and the additional 54 nt from intron 9 was expressed in HEK293 cells. Patch clamp studies revealed that the splice mutant channels did not produce hERG current. Western blot analysis showed that the mutant expressed the immature form of the hERG protein indicating defective channel trafficking. These studies underscore the importance of RNA analysis in describing the pathogenesis of LQT2. The intron 9 donor splice site appears to be a localized hot-spot for LQT2 mutations.

FcRn binding properties of an abnormal truncated analbuminemic albumin variant

Background: The major histocompatibility class I-related neonatal Fc receptor, FcRn, salvages both IgG and albumin from degradation and thus contributes to maintain high serum levels of these proteins. Analbuminemia is a rare autosomal recessive disorder characterized by clinically observed allelic albumin variants that are absent or found in very low concentrations in the blood circulation. Such variants may have altered FcRn binding properties that affect their half-life, biodistribution and thereby transport ability. Methods: We established an easy cloning, expression and purification strategy to obtain recombinant GST-tagged human serum albumin (HSA) variants for evaluation of pH dependent FcRn binding properties using an enzyme-linked immunosorbent assay (ELISA) and a real time surface plasmon resonance (SPR) biosensor system. Results: The strategy yielded purified GST-tagged albumin variants. A recombinant truncated HSA variant similar to a clinically observed splice mutant denoted Bartin, here abrogated HSA Bartin, showed no detectable pH dependent FcRn binding compared to a fully functional albumin wild type variant, HSA Wt, and a truncated HSA variant consisting of only the carboxy terminal domain III (HSA DIII). Conclusions: The approach described can be used to rapidly screen clinically observed truncated or otherwise mutant or modified HSA variants regarding their pH dependent FcRn binding properties. Here, we demonstrate that a recombinant truncated HSA variant, HSA Bartin, does not interact with FcRn, which gives a molecular explanation for the low serum levels. In addition, DIII of HSA alone was shown to retain its FcRn binding property.

Three Novel Alternative Splicing Mutations in BCR-ABL1 detected in CML Patients with Resistance to Kinase Inhibitors.

Abstract 1107Poster Board I-129The BCR-ABL1 tyrosine kinase is the target for kinase inhibitors in patients with chronic myeloid leukemia (CML). Multiple types of mutations in the BCR-ABL1 kinase domain have been reported. We previously reported a common alternatively spliced BCR-ABL mRNA with a 35-nucleotide insertion (35INS) between ABL1 kinase domain exons 8 and 9 that is expressed at various levels in CML patients with resistance to kinase inhibitors. Here we report 3 novel alternative splicing mutants expressed as the dominant transcripts in patient with CML and resistance to kinase inhibitors. We initially screened for ABL1 kinase domain mutations in approximately 200 patients with resistance to more than one of the three kinase inhibitors (imatinib, nilotinib, or dasatinib). Screening was conducted by first amplifying the fusion BCR-ABL1 transcript, to ensure that normal ABL1 was not amplified, then sequencing the ABL1 kinase domain coding region. We discovered 3 not previously described splice mutants, present in one patient each. All 3 showed >90% mutant transcript. The first resulted from the insertion of 79 nucleotides into the ABL1 exon 8-9 junction. The inserted sequence in fact contained a sequence from regions of intron 8, located 120 bp apart: the 35-nucleotide sequence previously described, and an additional 44-nucleotide segment downstream from 35INS. The combined 79-nucleotide insertion splice mutant showed the same protein change as 35INS (p C475YfsX11). The second splice mutation comprised an 84-nucleotide sequence from intron 7 inserted into the ABL1 exon7-8 junction, also causing a frameshift and protein truncation (p A424EfsX18). The third splice derived from a 231–nucleotide sequence from intron 4 retained in the ABL1 exon 4-5 junction. This insertion added 40 intron-encoded amino acids after K274, leading to a frameshift and early termination of BCR-ABL1 in the p-loop (p E275LfsX41). Exposure to kinase inhibitors may lead to the expression of alternatively spliced mRNA that is significantly elongated but causes a truncated fusion BCR-ABL1 that confers resistance to kinase inhibitors. These findings, when combined with the data on 35INS, support the concept that loss of the C-terminus of BCR-ABL1 is associated with significant resistance to kinase inhibitors; this mechanism appears to be a major source of resistance to kinase inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Splice Variant JAK2 Transcript Deleting Exon 14 in Patients with Chronic Myeloproliferative Neoplasms.

Abstract 2161Poster Board II-138The JAK2 V617F mutation in exon 14 is the most common mutation in chronic myeloproliferative neoplasms (MPNs). While other point mutations and small deletions and insertions in exons 12, 13, and 14 have been reported in the JAK2 JH2 domain, deletion of the entire exon 14 is rarely detected in patients with MPNs. In a series of >10,000 samples from patients with suspected MPNs tested for JAK2 mutations by direct sequencing of mRNA, we detected a complete exon 14 (88 bp) deletion mutation in <1% of those with JAK2 mutation. This appears to be an alternative splicing mutation, not detectable with DNA-based testing, leading to a frameshift deletion in the JAK2 JH2 domain and the expression of a truncated protein (S593I fsX8). Although this mutation was present at detectable levels (>15% of total JAK2 transcript) in a small proportion of MPN cases, we decided to investigate the possibility it may be expressed at low levels (<15% of JAK2 transcript) in patients with MPNs. Using a sensitive reverse transcription-PCR—based fluorescent fragment analysis method to quantify the percentage of truncated (minus exon 14) JAK2 mRNA as compared to wild-type, we tested 61 patients with confirmed MPN; 183 patients with suspected MPNs (93 V617F positive, 90 V617F negative); and 46 healthy normal control subjects. The wild-type PCR product was 273 bp, while the exon 14 splice mutant displayed a 185-bp peak. All samples with a splicing mutant expression to wild-type ratio >15% were confirmed by direct sequencing. The JAK2 exon 14 splice mutation was detected at low levels in 9 of the 61 MPN patients (15%), accounting for 3.96% to 33.85% (mean=12.04%) of JAK2 transcript in these individuals. Among the 183 suspected MPN patients, the exon 14 splice variant was detected in 20 of the 93 (21.5%) with V617F (mean expression level relative to wild type=5.41%, range=2.13%-26.22%) and 31 of the 90 (34.4%) without V617F (mean expression=3.88%; range=2.08%-12.22%). All 46 normal individuals were considered negative for the alternatively spliced transcript. In conclusion, the expression of an alternatively spliced JAK2 mRNA with deletion of exon 14, leading to a truncated JAK2 protein, is a common abnormality in patients with MPNs. This alternatively spliced transcript is more common in MPN patients without V617F mutation and might contribute to the leukemogenesis in these patients. Low levels of JAK2 exon 14 splice mutation may also contribute to the effects of the V617F mutation in patients with MPNs. However, this mutation is missed if DNA and not the RNA is used for testing for JAK2 mutations. Disclosures:No relevant conflicts of interest to declare.

A splice site mutation in hERG leads to cryptic splicing in human long QT syndrome

Mutations in the human ether-a-go-go-related gene (hERG) cause type 2 long QT syndrome. In this study, we investigated the pathogenic mechanism of the hERG splice site mutation 2398+1G>C and the genotype–phenotype relationship of mutation carriers in three unrelated kindreds with long QT syndrome. The effect of 2398+1G>C on mRNA splicing was studied by analysis of RNA isolated from lymphocytes of index patients and using minigenes expressed in HEK293 cells and neonatal rat ventricular myocytes. RT-PCR analysis revealed that the 2398+1G>C mutation disrupted the normal splicing and activated a cryptic splice donor site in intron 9, leading to the inclusion of 54 nt of the intron 9 sequence in hERG mRNA. The cryptic splicing resulted in an in-frame insertion of 18 amino acids in the middle of the cyclic nucleotide binding domain. In patch clamp experiments the splice mutant did not generate hERG current. Western blot and immunostaining studies showed that the mutant expressed an immature form of hERG protein that failed to reach the plasma membrane. Coexpression of the mutant and wild-type channels led to a dominant negative suppression of wild-type channel function by intracellular retention of heteromeric channels. Our results demonstrate that 2398+1G>C activates a cryptic site and generates a full-length hERG protein with an insertion of 18 amino acids, which leads to a trafficking defect of the mutant channel.

Genotype–Phenotype Correlation of MousePde6bMutations

To identify the underlying molecular defects causing retinal degeneration in seven N-ethyl-N-nitrosourea (ENU) induced mutant alleles of the Pde6b gene and to analyze the timescale of retinal degeneration in these new models of retinitis pigmentosa. Conformation sensitive capillary electrophoresis and DNA sequencing were used to identify the mutations in the Pde6b gene. Visual acuity testing was performed with a visual-tracking drum at ages ranging from postnatal day 25 to week 10. Retinal examination was performed with an indirect ophthalmoscope. Animals were killed and eyes were prepared for histologic analysis. Point mutations in the seven new alleles of Pde6b were identified: Three generated premature stop codons, two were missense mutations, and two were splice mutations. The three stop codon mutants and one of the splice mutants had phenotypes indistinguishable from the Pde6b(rd1) mouse in rapidity of onset of retinal degeneration, suggesting that they are null alleles. However, the remaining alleles showed slower onset of retinal degeneration, as determined by visual acuity testing, fundus examination, and histology, indicating that they are hypomorphic alleles. These data demonstrate a correlation between genotype and phenotype. Four of the mutants with severe genetic lesions have rapid onset of retinal degeneration, as determined by fundus examination. These mice were indistinguishable from Pde6b(rd1) mice, which are effectively blind by 3 weeks of age. In contrast, the milder genetic lesions show a slower progression of the disease and provide the community with models that more closely mimic human retinitis pigmentosa.

Mouse splice mutant generation from ENU-treated ES cells—A gene-driven approach

Mutant mice are important for elucidating mammalian gene functions and for modeling human disease phenotypes. In recent years, chemical mutagenesis has become an increasingly popular method to disrupt gene functions due to its high efficiency of inducing mutations throughout the genome. Mutagenesis of embryonic stem (ES) cells offers the possibility of gene-driven approaches, which, however, require efficient mutation detection procedures to screen archives of mutated samples for lesions in particular genes. We have developed an approach that focuses on the detection of splice mutations in highly pooled cDNA samples using exon-skipping PCR primers. As a proof of concept, splice mutants for the Kit gene were isolated from a library comprising approximately 40,000 ES cell clones treated with N-ethyl- N-nitrosourea followed by transmission through the mouse germ-line. The approach will be useful for the production of mouse models for human disease-related splice mutations and as a general gene disruption strategy.

Cellular activity of dopamine DA2 receptor splice variants and mutants

Two splice variants of the dopamine D2 (DA2) receptors—a long (DA2l) and short (DA2s) form—and two corresponding mutants (serine at position 311 replaced by a cysteine) have been described. Using CHO-cells transfected with the genes for the splice variants and their respective mutants and a bioassay based on the online registration of the extracellular acidification rate (ECAR) of intact cells, we investigated the cellular activity upon stimulation of the receptor. We first confirmed that the acute response upon short agonist stimulation was significantly higher for DA2s than for DA2l. However, in contrast to the ongoing opinion, we found that the desensitisation pattern upon long-term agonist treatment was indistinguishable for both wild-type receptors. As far as the corresponding ser311cys mutated receptors are concerned, the concentration–response curves and the desensitisation pattern were superimposable to the corresponding wild-type receptors. Inhibition of protein kinase C (PKC) had very little effect both on the concentration–response curves and on the desensitisation pattern. We conclude that signal transduction following stimulation of DA2 receptors in the CHO expression system is more effective for DA2s than for DA2l. The point mutation in position 311 has little impact on the downstream signalling of DA2 receptors.

Characterization of 17 novel endoglin mutations associated with hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia type 1 (HHT1) is a vascular dysplasia caused by mutations in the endoglin (ENG) gene and associated with epistaxis, telangiectases, and a high incidence of pulmonary arteriovenous malformations. To efficiently detect deletions and insertions, we optimized a quantitative multiplex polymerase chain reaction (QMPCR) analysis. We report 17 novel mutations, of which six were detected by QMPCR. Three deletions occurring in intronic sequences were associated with a single copy of exons 9a-14, exon 5, and exons 7-8, respectively. A transient 70kDa monomeric mutant protein resulted from the in-frame deletion of exons 7 and 8 but no mutant protein was present in the other cases. Deletion (in exon 10) or insertion (in exon 7) of two nucleotides, as well as a 1-bp deletion in the small exon 9a were found by QMPCR. Sequencing was required to detect single nucleotide deletions/insertions in exons 2, 5, 6, and 8. No mutant proteins were associated with these frame shift mutations. Two novel splice site mutations resulted in skipping of exons 2 and 4, respectively, while a previously reported intron 3 splice mutant was observed as a de novo mutation. We also report five novel nonsense and missense mutations, including one de novo. Review of the 80 HHT1 families reported to date indicates that 10% would not be resolved by sequencing and that an additional 25% could be revealed by QMPCR performed prior to sequencing. Thus the use of QMPCR accelerates genetic screening for HHT1 and resolves mutations affecting whole exons.