Progressive multifocal leukoencephalopathy genetic risk variants for pharmacovigilance of immunosuppressant therapies.

Background: Progressive multifocal leukoencephalopathy (PML) is a rare and often lethal brain disorder caused by the common, typically benign polyomavirus 2, also known as JC virus (JCV). In a small percentage of immunosuppressed individuals, JCV is reactivated and infects the brain, causing devastating neurological defects. A wide range of immunosuppressed groups can develop PML, such as patients with: HIV/AIDS, hematological malignancies (e.g., leukemias, lymphomas, and multiple myeloma), autoimmune disorders (e.g., psoriasis, rheumatoid arthritis, and systemic lupus erythematosus), and organ transplants. In some patients, iatrogenic (i.e., drug-induced) PML occurs as a serious adverse event from exposure to immunosuppressant therapies used to treat their disease (e.g., hematological malignancies and multiple sclerosis). While JCV infection and immunosuppression are necessary, they are not sufficient to cause PML. Methods: We hypothesized that patients may also have a genetic susceptibility from the presence of rare deleterious genetic variants in immune-relevant genes (e.g., those that cause inborn errors of immunity). In our prior genetic study of 184 PML cases, we discovered 19 candidate PML risk variants. In the current study of another 152 cases, we validated 4 of 19 variants in both population controls (gnomAD 3.1) and matched controls (JCV+ multiple sclerosis patients on a PML-linked drug ≥ 2 years). Results: The four variants, found in immune system genes with strong biological links, are: C8B, 1-57409459-C-A, rs139498867; LY9 (alias SLAMF3), 1-160769595-AG-A, rs763811636; FCN2, 9-137779251-G-A, rs76267164; STXBP2, 19-7712287-G-C, rs35490401. Carriers of any one of these variants are shown to be at high risk of PML when drug-exposed PML cases are compared to drug-exposed matched controls: P value = 3.50E-06, OR = 8.7 [3.7-20.6]. Measures of clinical validity and utility compare favorably to other genetic risk tests, such as BRCA1 and BRCA2 screening for breast cancer risk and HLA-B*15:02 pharmacogenetic screening for pharmacovigilance of carbamazepine to prevent Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis. Conclusion: For the first time, a PML genetic risk test can be implemented for screening patients taking or considering treatment with a PML-linked drug in order to decrease the incidence of PML and enable safer use of highly effective therapies used to treat their underlying disease.

NUBPL mitochondrial disease: new patients and review of the genetic and clinical spectrum.

BACKGROUND: The nucleotide binding protein-like (NUBPL) gene was first reported as a cause of mitochondrial complex I deficiency (MIM 613621, 618242) in 2010. To date, only eight patients have been reported with this mitochondrial disorder. Five other patients were recently reported to have NUBPL disease but their clinical picture was different from the first eight patients. Here, we report clinical and genetic findings in five additional patients (four families). METHODS: Whole exome sequencing was used to identify patients with compound heterozygous NUBPL variants. Functional studies included RNA-Seq transcript analyses, missense variant biochemical analyses in a yeast model (Yarrowia lipolytica) and mitochondrial respiration experiments on patient fibroblasts. RESULTS: The previously reported c.815-27T>C branch-site mutation was found in all four families. In prior patients, c.166G>A [p.G56R] was always found in cis with c.815-27T>C, but only two of four families had both variants. The second variant found in trans with c.815-27T>C in each family was: c.311T>C [p.L104P] in three patients, c.693+1G>A in one patient and c.545T>C [p.V182A] in one patient. Complex I function in the yeast model was impacted by p.L104P but not p.V182A. Clinical features include onset of neurological symptoms at 3-18 months, global developmental delay, cerebellar dysfunction (including ataxia, dysarthria, nystagmus and tremor) and spasticity. Brain MRI showed cerebellar atrophy. Mitochondrial function studies on patient fibroblasts showed significantly reduced spare respiratory capacity. CONCLUSION: We report on five new patients with NUBPL disease, adding to the number and phenotypic variability of patients diagnosed worldwide, and review prior reported patients with pathogenic NUBPL variants.

Loss-of-Function NUBPL Mutation May Link Parkinson's Disease to Recessive Complex I Deficiency.

In an unbiased genome-wide screen for copy number variants (CNVs) on a cohort of Parkinson's disease (PD) patients, we identified in one patient a complex chromosomal rearrangement involving the nucleotide binding protein-like (NUBPL) gene on chromosome 14q12. We noted that mutations in the NUBPL gene had been reported as causing autosomal recessive (AR) mitochondrial Complex I (CI) deficiency in children. The precise breakpoints of the rearrangement in our PD case were found to be identical to those described in a patient with AR CI deficiency who also harbored a second pathogenic mutation in NUBPL. Mitochondrial dysfunction has long been considered a strong contributor to PD, and there is substantial evidence that decreased CI activity plays a central role in PD pathogenesis. We hypothesize that pathogenic NUBPL variants may increase the risk for PD analogous to variants in the glucosylceramidase beta (GBA) gene that increase the risk of developing PD in heterozygous carriers.

Germline Genetic Risk Variants for Progressive Multifocal Leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a rare demyelinating disorder of the brain caused by reactivation of the JC virus (JCV), a polyomavirus that infects at least 60% of the population but is asymptomatic or results in benign symptoms in most people. PML occurs as a secondary disease in a variety of disorders or as a serious adverse event from immunosuppressant agents, but is mainly found in three groups: HIV-infected patients, patients with hematological malignancies, or multiple sclerosis (MS) patients on the immunosuppressant therapy natalizumab. It is severely debilitating and is deadly in ~50% HIV cases, ~90% of hematological malignancy cases, and ~24% of MS-natalizumab cases. A PML risk prediction test would have clinical utility in all at risk patient groups but would be particularly beneficial in patients considering therapy with immunosuppressant agents known to cause PML, such as natalizumab, rituximab, and others. While a JC antibody test is currently used in the clinical decision process for natalizumab, it is suboptimal because of its low specificity and requirement to periodically retest patients for seroconversion or to assess if a patient's JCV index has increased. Whereas a high specificity genetic risk prediction test comprising host genetic risk variants (i.e., germline variants occurring at higher frequency in PML patients compared to the general population) could be administered one time to provide clinicians with additional risk prediction information that is independent of JCV serostatus. Prior PML case reports support the hypothesis that PML risk is greater in patients with a genetically caused immunodeficiency disorder. To identify germline PML risk variants, we performed exome sequencing on 185 PML cases (70 in a discovery cohort and 115 in a replication cohort) and used the gnomAD variant database for interpretation. Our study yielded 19 rare variants (maximum allele frequency of 0.02 in gnomAD ethnically matched populations) that impact 17 immune function genes (10 are known to cause inborn errors of immunity). Modeling of these variants in a PML genetic risk test for MS patients considering natalizumab treatment indicates that at least a quarter of PML cases may be preventable.

Genetic fine-mapping of the Iowan SNCA gene triplication in a patient with Parkinson's disease.

The "Iowa kindred," a large Iowan family with autosomal-dominant Parkinson's disease, has been followed clinically since the 1920s at the Mayo Clinic. In 2003, the genetic cause was determined to be a 1.7 Mb triplication of the alpha-synuclein genomic locus. Affected individuals present with an early-onset, severe parkinsonism-dementia syndrome. Here, we present a descendant of the Iowa kindred with novel, disease-associated non-motor findings of reduced heart rate variability, complete anosmia, and a rare skin condition called colloid milium. At autopsy, key neuropathological findings were compatible with diffuse Lewy body disease. Using high-resolution comparative genomic hybridization (CGH) array analysis to fine-map the genomic breakpoints, we observed two independent recombination events of the SNCA locus that resulted in a genomic triplication of twelve genes, including SNCA, and the disruption of two genes, HERC6 and CCSER1, at the genomic breakpoints. In conclusion, we provide further evidence that the mere two-fold overexpression of alpha-synuclein leads to a fulminant alpha-synucleinopathy with rapid progression and severe clinical and neuropathological features.

Microduplications at the pseudoautosomal SHOX locus in autism spectrum disorders and related neurodevelopmental conditions.

BACKGROUND: The pseudoautosomal short stature homeobox-containing (SHOX) gene encodes a homeodomain transcription factor involved in cell-cycle and growth regulation. SHOX/SHOX enhancers deletions cause short stature and skeletal abnormalities in a female-dominant fashion; duplications appear to be rare. Neurodevelopmental disorders (NDDs), such as autism spectrum disorders (ASDs), are complex disorders with high heritability and skewed sex ratio; several rare (<1% frequency) CNVs have been implicated in risk. METHODS: We analysed data from a discovery series of 90 adult ASD cases, who underwent clinical genetic testing by array-comparative genomic hybridisation (CGH). Twenty-seven individuals harboured CNV abnormalities, including two unrelated females with microduplications affecting SHOX. To determine the prevalence of SHOX duplications and delineate their associated phenotypic spectrum, we subsequently examined array-CGH data from a follow-up sample of 26 574 patients, including 18 857 with NDD (3541 with ASD). RESULTS: We found a significant enrichment of SHOX microduplications in the NDD cases (p=0.00036; OR 2.21) and, particularly, in those with ASD (p=9.18×10(-7); OR 3.63) compared with 12 594 population-based controls. SHOX duplications affecting the upstream or downstream enhancers were enriched only in females with NDD (p=0.0043; OR 2.69/p=0.00020; OR 7.20), but not in males (p=0.404; OR 1.38/p=0.096; OR 2.21). CONCLUSIONS: Microduplications at the SHOX locus are a low penetrance risk factor for ASD/NDD, with increased risk in both sexes. However, a concomitant duplication of SHOX enhancers may be required to trigger a NDD in females. Since specific SHOX isoforms are exclusively expressed in the developing foetal brain, this may reflect the pathogenic effect of altered SHOX protein dosage on neurodevelopment.

The 22q11 PRODH/DGCR6 deletion is frequent in hyperprolinemic subjects but is not a strong risk factor for ASD.

The proline dehydrogenase (PRODH) gene maps to 22q11.2 in the region deleted in the velo-cardio-facial syndrome (VCFS). A moderate to severe reduction (>50%) in PRODH activity resulting from recessive deletions and/or missense mutations has been shown to cause type 1 hyperprolinemia (HPI). Autistic features have been reported as a common clinical manifestation of HPI. Here we studied the frequency of a recurrent small 22q11.2 deletion encompassing PRODH and the neighboring DGCR6 gene in three case-control studies, one comprising HPI patients (n = 83), and the other two comprising autism spectrum disorder (ASD) patients (total of n = 2800), analyzed with high-resolution microarrays. We found that the PRODH deletion is a strong risk factor for HPI (OR = 50.7; 95%CI = 7.5-2147) but not for ASD (P = 0.4, OR = 0.6-3.3). This result indicates either that the suggested association between ASD and HPI is spurious and results from a bias leading to the preferential inclusion of patients with autistic features in HPI series, or that HPI is present in only a very small subset of ASD patients. In this latter case, a very large sample size would be required to detect an association between the PRODH deletion and ASD in a case-control study.

Disruption of the ASTN2/TRIM32 locus at 9q33.1 is a risk factor in males for autism spectrum disorders, ADHD and other neurodevelopmental phenotypes.

Rare copy number variants (CNVs) disrupting ASTN2 or both ASTN2 and TRIM32 have been reported at 9q33.1 by genome-wide studies in a few individuals with neurodevelopmental disorders (NDDs). The vertebrate-specific astrotactins, ASTN2 and its paralog ASTN1, have key roles in glial-guided neuronal migration during brain development. To determine the prevalence of astrotactin mutations and delineate their associated phenotypic spectrum, we screened ASTN2/TRIM32 and ASTN1 (1q25.2) for exonic CNVs in clinical microarray data from 89 985 individuals across 10 sites, including 64 114 NDD subjects. In this clinical dataset, we identified 46 deletions and 12 duplications affecting ASTN2. Deletions of ASTN1 were much rarer. Deletions near the 3' terminus of ASTN2, which would disrupt all transcript isoforms (a subset of these deletions also included TRIM32), were significantly enriched in the NDD subjects (P = 0.002) compared with 44 085 population-based controls. Frequent phenotypes observed in individuals with such deletions include autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), speech delay, anxiety and obsessive compulsive disorder (OCD). The 3'-terminal ASTN2 deletions were significantly enriched compared with controls in males with NDDs, but not in females. Upon quantifying ASTN2 human brain RNA, we observed shorter isoforms expressed from an alternative transcription start site of recent evolutionary origin near the 3' end. Spatiotemporal expression profiling in the human brain revealed consistently high ASTN1 expression while ASTN2 expression peaked in the early embryonic neocortex and postnatal cerebellar cortex. Our findings shed new light on the role of the astrotactins in psychopathology and their interplay in human neurodevelopment.

A discovery resource of rare copy number variations in individuals with autism spectrum disorder.

The identification of rare inherited and de novo copy number variations (CNVs) in human subjects has proven a productive approach to highlight risk genes for autism spectrum disorder (ASD). A variety of microarrays are available to detect CNVs, including single-nucleotide polymorphism (SNP) arrays and comparative genomic hybridization (CGH) arrays. Here, we examine a cohort of 696 unrelated ASD cases using a high-resolution one-million feature CGH microarray, the majority of which were previously genotyped with SNP arrays. Our objective was to discover new CNVs in ASD cases that were not detected by SNP microarray analysis and to delineate novel ASD risk loci via combined analysis of CGH and SNP array data sets on the ASD cohort and CGH data on an additional 1000 control samples. Of the 615 ASD cases analyzed on both SNP and CGH arrays, we found that 13,572 of 21,346 (64%) of the CNVs were exclusively detected by the CGH array. Several of the CGH-specific CNVs are rare in population frequency and impact previously reported ASD genes (e.g., NRXN1, GRM8, DPYD), as well as novel ASD candidate genes (e.g., CIB2, DAPP1, SAE1), and all were inherited except for a de novo CNV in the GPHN gene. A functional enrichment test of gene-sets in ASD cases over controls revealed nucleotide metabolism as a potential novel pathway involved in ASD, which includes several candidate genes for follow-up (e.g., DPYD, UPB1, UPP1, TYMP). Finally, this extensively phenotyped and genotyped ASD clinical cohort serves as an invaluable resource for the next step of genome sequencing for complete genetic variation detection.

Acquired copy number alterations in adult acute myeloid leukemia genomes.

Cytogenetic analysis of acute myeloid leukemia (AML) cells has accelerated the identification of genes important for AML pathogenesis. To complement cytogenetic studies and to identify genes altered in AML genomes, we performed genome-wide copy number analysis with paired normal and tumor DNA obtained from 86 adult patients with de novo AML using 1.85 million feature SNP arrays. Acquired copy number alterations (CNAs) were confirmed using an ultra-dense array comparative genomic hybridization platform. A total of 201 somatic CNAs were found in the 86 AML genomes (mean, 2.34 CNAs per genome), with French-American-British system M6 and M7 genomes containing the most changes (10-29 CNAs per genome). Twenty-four percent of AML patients with normal cytogenetics had CNA, whereas 40% of patients with an abnormal karyotype had additional CNA detected by SNP array, and several CNA regions were recurrent. The mRNA expression levels of 57 genes were significantly altered in 27 of 50 recurrent CNA regions <5 megabases in size. A total of 8 uniparental disomy (UPD) segments were identified in the 86 genomes; 6 of 8 UPD calls occurred in samples with a normal karyotype. Collectively, 34 of 86 AML genomes (40%) contained alterations not found with cytogenetics, and 98% of these regions contained genes. Of 86 genomes, 43 (50%) had no CNA or UPD at this level of resolution. In this study of 86 adult AML genomes, the use of an unbiased high-resolution genomic screen identified many genes not previously implicated in AML that may be relevant for pathogenesis, along with many known oncogenes and tumor suppressor genes.

Quantification of microRNAs, splicing isoforms, and homologous mRNAs with the invader assay.

The understanding of physiology and pathology requires accurate quantification of intracellular concentrations of important molecules such as unique RNA species. Accurate quantification of highly homologous messenger RNAs (mRNAs) (1-3), alternatively spliced mRNAs (4), and the short microRNAs (miRNAs) (5,6) has been successfully achieved using the Invader assay. This method directly detects specific RNA molecules in preparations of pure total cellular RNA (1- 100 ng) or in crude cell lysate (10(3)-10(4) cells) samples using an isothermal signal amplification process with a fluorescence resonance energy transfer (FRET)-based fluorescence readout. Features of the Invader assay include the ability to detect 1-10 RNA molecules per cell, to discriminate between RNAs that differ by a single base, and to precisely measure 1.2-fold changes in RNA expression. Further, an isothermal format and the ability to detect two different RNA molecules with a biplex format make the Invader assay suitable for high-throughput screening applications.

wuHMM: a robust algorithm to detect DNA copy number variation using long oligonucleotide microarray data.

Copy number variants (CNVs) are currently defined as genomic sequences that are polymorphic in copy number and range in length from 1000 to several million base pairs. Among current array-based CNV detection platforms, long-oligonucleotide arrays promise the highest resolution. However, the performance of currently available analytical tools suffers when applied to these data because of the lower signal:noise ratio inherent in oligonucleotide-based hybridization assays. We have developed wuHMM, an algorithm for mapping CNVs from array comparative genomic hybridization (aCGH) platforms comprised of 385 000 to more than 3 million probes. wuHMM is unique in that it can utilize sequence divergence information to reduce the false positive rate (FPR). We apply wuHMM to 385K-aCGH, 2.1M-aCGH and 3.1M-aCGH experiments comparing the 129X1/SvJ and C57BL/6J inbred mouse genomes. We assess wuHMM's performance on the 385K platform by comparison to the higher resolution platforms and we independently validate 10 CNVs. The method requires no training data and is robust with respect to changes in algorithm parameters. At a FPR of <10%, the algorithm can detect CNVs with five probes on the 385K platform and three on the 2.1M and 3.1M platforms, resulting in effective resolutions of 24 kb, 2-5 kb and 1 kb, respectively.

Discovery of a previously unrecognized microdeletion syndrome of 16p11.2-p12.2.

We have identified a recurrent de novo pericentromeric deletion in 16p11.2-p12.2 in four individuals with developmental disabilities by microarray-based comparative genomic hybridization analysis. The identification of common clinical features in these four individuals along with the characterization of complex segmental duplications flanking the deletion regions suggests that nonallelic homologous recombination mediated these rearrangements and that deletions in 16p11.2-p12.2 constitute a previously undescribed syndrome.

Characterization of a recurrent 15q24 microdeletion syndrome.

We describe multiple individuals with mental retardation and overlapping de novo submicroscopic deletions of 15q24 (1.7-3.9 Mb in size). High-resolution analysis showed that in three patients both proximal and distal breakpoints co-localized to highly identical segmental duplications (>51 kb in length, > 94% identity), suggesting non-allelic homologous recombination as the likely mechanism of origin. Sequencing studies in a fourth individual provided base pair resolution and showed that both breakpoints in this case were located in unique sequence. Despite the differences in the size and location of the deletions, all four individuals share several major features (growth retardation, microcephaly, digital abnormalities, hypospadias and loose connective tissue) and resemble one another facially (high anterior hair line, broad medial eyebrows, hypertelorism, downslanted palpebral fissures, broad nasal base, long smooth philtrum and full lower lip), indicating that this represents a novel syndrome caused by haploinsufficiency of one or more dosage-sensitive genes in the minimal deletion region. Our results define microdeletion of 15q24 as a novel recurrent genomic disorder.

A high-resolution map of segmental DNA copy number variation in the mouse genome.

Submicroscopic (less than 2 Mb) segmental DNA copy number changes are a recently recognized source of genetic variability between individuals. The biological consequences of copy number variants (CNVs) are largely undefined. In some cases, CNVs that cause gene dosage effects have been implicated in phenotypic variation. CNVs have been detected in diverse species, including mice and humans. Published studies in mice have been limited by resolution and strain selection. We chose to study 21 well-characterized inbred mouse strains that are the focus of an international effort to measure, catalog, and disseminate phenotype data. We performed comparative genomic hybridization using long oligomer arrays to characterize CNVs in these strains. This technique increased the resolution of CNV detection by more than an order of magnitude over previous methodologies. The CNVs range in size from 21 to 2,002 kb. Clustering strains by CNV profile recapitulates aspects of the known ancestry of these strains. Most of the CNVs (77.5%) contain annotated genes, and many (47.5%) colocalize with previously mapped segmental duplications in the mouse genome. We demonstrate that this technique can identify copy number differences associated with known polymorphic traits. The phenotype of previously uncharacterized strains can be predicted based on their copy number at these loci. Annotation of CNVs in the mouse genome combined with sequence-based analysis provides an important resource that will help define the genetic basis of complex traits.

Discovery of previously unidentified genomic disorders from the duplication architecture of the human genome.

Genomic disorders are characterized by the presence of flanking segmental duplications that predispose these regions to recurrent rearrangement. Based on the duplication architecture of the genome, we investigated 130 regions that we hypothesized as candidates for previously undescribed genomic disorders. We tested 290 individuals with mental retardation by BAC array comparative genomic hybridization and identified 16 pathogenic rearrangements, including de novo microdeletions of 17q21.31 found in four individuals. Using oligonucleotide arrays, we refined the breakpoints of this microdeletion, defining a 478-kb critical region containing six genes that were deleted in all four individuals. We mapped the breakpoints of this deletion and of four other pathogenic rearrangements in 1q21.1, 15q13, 15q24 and 17q12 to flanking segmental duplications, suggesting that these are also sites of recurrent rearrangement. In common with the 17q21.31 deletion, these breakpoint regions are sites of copy number polymorphism in controls, indicating that these may be inherently unstable genomic regions.

Analysis of chromosome breakpoints in neuroblastoma at sub-kilobase resolution using fine-tiling oligonucleotide array CGH.

Understanding the genes and genetic pathways targeted by recurrent chromosomal imbalances in malignancy, along with the molecular mechanisms that generate the imbalances, are important problems in cancer biology. In this report, we demonstrate that oligonucleotide array CGH (oaCGH) analysis can routinely map chromosomal imbalance breakpoints at exon-level resolution, including imbalances that are single copy number genomic alterations. Different tiling-path array designs were used in this study: a whole-genome array with a 6-kb median probe spacing and fine-tiling arrays for selected genomic regions with either 50- or 140-bp median probe spacing. In both array formats, oligonucleotide probes were of isothermal design and were tiled through genic and inter-genic regions. Whole-genome oaCGH analysis of two neuroblastoma cell lines and three primary tumors led to the identification of 58 chromosomal breakpoints that generated 45 large-scale partial chromosomal imbalances (> 2 Mb). An unexpectedly high proportion (34%) of these breakpoint intervals mapped to regions containing segmental duplications. In addition, 88 smaller-sized regions (< 2 Mb) of imbalance were detected, the majority of which mapped to segmentally duplicated regions and may reflect constitutional copy number polymorphisms. The chromosomal breakpoints for 12 recurrent abnormalities exhibited in neuroblastoma tumors and cell lines, including MYCN amplicon boundaries, loss of 3p, loss of 11q, and gain of 17q, could be mapped to intervals ranging from 50 bp to 10 kb in size using high-density fine-tiling oligonucleotide microarrays. Fine-tiling oaCGH analysis provides an unprecedented level of resolution, allowing detailed mapping of recurrent unbalanced chromosomal abnormalities. Supplementary material for this article can be found on the Genes, Chromosomes, and Cancer website at http://www.interscience.wiley.com/jpages/1045-2257/suppmat/index.html.

Accumulation of miR-155 and BIC RNA in human B cell lymphomas.

We show that the microRNA miR-155 can be processed from sequences present in BIC RNA, a spliced and polyadenylated but non-protein-coding RNA that accumulates in lymphoma cells. The precursor of miR-155 is likely a transient spliced or unspliced nuclear BIC transcript rather than accumulated BIC RNA, which is primarily cytoplasmic. By using a sensitive and quantitative assay, we find that clinical isolates of several types of B cell lymphomas, including diffuse large B cell lymphoma (DLBCL), have 10- to 30-fold higher copy numbers of miR-155 than do normal circulating B cells. Similarly, the quantities of BIC RNA are elevated in lymphoma cells, but ratios of the amounts of the two RNAs are not constant, suggesting that the level of miR-155 is controlled by transcription and processing. Significantly higher levels of miR-155 are present in DLBCLs with an activated B cell phenotype than with the germinal center phenotype. Because patients with activated B cell-type DLBCL have a poorer clinical prognosis, quantification of this microRNA may be diagnostically useful.

Quantification of alternatively spliced FGFR2 RNAs using the RNA invasive cleavage assay.

The regulated splicing of fibroblast growth factor receptor-2 (FGFR2) transcripts leads to tissue-specific expression of distinct receptor isoforms. These isoforms contain two different versions of the ligand binding Ig-like domain III, which are encoded by exon IIIb or exon IIIc. The mutually exclusive use of exon IIIb and exon IIIc can be recapitulated in tissue culture using DT3 and AT3 rat prostate carcinoma cells. We used this well-characterized system to evaluate the precision and accuracy of the RNA invasive cleavage assay to specifically measure FGFR2 alternative splicing outcomes. Experiments presented here demonstrated that the RNA invasive cleavage assay could specifically detect isoforms with discrimination levels that ranged from 1 in 5 x 10(3) to 1 in 10(5). Moreover the assay could detect close to 0.01 amole of FGFR2 RNAs. The assay detected the expected levels of transcripts containing either exon IIIb or IIIc, but, surprisingly, it detected high levels of IIIb-IIIc double inclusion transcripts. This finding, which has important implications for the role of exon silencing and of mRNA surveillance mechanisms, had been missed by RT-PCR. Additionally, we used the RNA invasive cleavage assay to demonstrate a novel function for the regulatory element IAS2 in repressing exon IIIc inclusion. We also show here that purification of RNA is not necessary for the invasive cleavage assay, because crude cell lysates could be used to accurately measure alternative transcripts. The data presented here indicate that the RNA invasive cleavage assay is an important addition to the repertoire of techniques available for the study of alternative splicing.