A premature termination codon within an alternative exon affecting only the metabolism of transcripts that retain this exon

Protein 4.1 pre‐mRNA splicing is regulated in tissue‐ and development‐specific manners. Exon 16, which encodes the N‐terminal region of the spectrin/actin‐binding domain, is one of the alternatively spliced sequence motifs. It is present in late differentiated erythroid cells but absent from early erythroblasts and from lymphoid cells. We describe a single nucleotide deletion of the erythroid protein 4.1 gene associated with hereditary elliptocytosis. The deletion located in exon 16 leads to a frameshift and a premature termination codon within the same exon. In an effort to examine the premature stop codon effect in relationship with exon 16 alternative splicing, we analyzed erythroid and lymphoid protein 4.1 mRNAs using the mutation and a linked downstream polymorphism as markers. We found that the premature stop codon does not affect the tissue‐specific alternative splicing among the two cell types analyzed and that the resulting alteration of mRNA metabolism correlates with the retention of exon 16 in reticulocytes. Conversely, skipping of exon 16 in lymphoid cells converts the mutant mRNA to a normal lymphoid‐specific mRNA isoform, hence bypassing the nonsense codon. Consistent with data obtained on constitutive nonsense exons, our observations argue in favor of a stop codon recognition mechanism that occurs after the regulated splicing status of the nonsense exon has been achieved. Hum Mutat 14:145–155, 1999. © 1999 Wiley‐Liss, Inc.     

Alteration of branch site consensus sequence and enhanced pre‐mRNA splicing of an NMDAR1 intron not associated with schizophrenia

Aberrant splicing of pre‐mRNA is recognized to account for a significant minority of disease‐causing mutations. The N‐methyl‐D ‐aspartate receptor (NMDA) subunit gene R1 (NMDAR1) is alternatively spliced to produce eight length variants. In an examination of the NMDAR1 as a candidate gene in schizophrenia, a presumed microdeletion/insertion (del/ins) was observed in intron 10 of an African‐American male near a weak putative branch‐site consensus sequence. Although exon 10 is not known to be alternatively spliced, the del/ins was posited to alter splicing efficiency. If splicing were abolished and intron retention occurred, an in‐frame translation product of more than 250 amino acids was predicted. To explore splicing efficiency, mini genes were examined through primer‐extension analyses in NIH293 embryonic kidney cell cultures. Rather than disruption of splicing, the del/ins allele exhibited a fivefold enhancement in splicing. In an association analysis with additional schizophrenic cases and unaffected controls, all of African‐American descent, the mutant allele was observed at equivalent frequencies. A family study also did not support cosegregation of the variant allele with psychiatric disease. © 2002 Wiley‐Liss, Inc.     

A rapid screening for steroid 21‐hydroxylase mutations in patients with congenital adrenal hyperplasia

Steroid 21‐hydroxylase deficiency is the major cause of congenital adrenal hyperplasia (CAH). CAH due to 21‐hydroxylase deficiency is divided into three classes: salt‐wasting (classical), non‐classical and simple virilizing, reflecting different degrees of clinical severity. Using polymerase chain reaction (PCR) and allele‐specific oligonucleotide hybridisation (ASO), we screened the DNA of 62 Caucasian CAH families (heterozygous parents and children) for 14 different and frequently‐found CYP21‐mutations (HGMD). Of the 62 patients (21 males, 41 females), 26 females and 11 males had the classical or salt‐wasting form, 3 females and 1 male had the non‐classical form and 14 females and 7 males had simple virilizing CAH. More than 60% of the patients were compound‐heterozygous. We found the mutations on 110 alleles (out of 124 alleles). There were 30 CYP21 gene deletions/conversions, 3 substitutions (P30L) in exon 1, 30 splice mutations (c.93‐13A/C>G) in intron 2, 26 point mutations (I172N) in exon 4, one cluster of mutations (I236N, V237E, M239K) in exon 6, 8 mutations (V281L and 1760‐1761insT) in exon 7, and 8 nonsense (Q318X) and 4 missense (R356W) mutations in exon 8. Our study supports the case for using this rapid technique for CAH‐family screening as long as alleles from both affected patients and parents are screened in parallel. Hum Mutat 13:505, 1999. © 1999 Wiley‐Liss, Inc.     

Control of the heat stress‐induced alternative splicing of a subset of genes by hnRNP K

Pre‐mRNA splicing is widely repressed upon heat shock in eukaryotic cells. However, it has been shown that HSP105 pre‐mRNA is alternatively spliced in response to heat stress. Using RNAi screening in HeLa cells, we found that RNA‐binding proteins hnRNP K and PSF/SFPQ are necessary for the exon 12 exclusion of HSP105 during heat stress. Moreover, exon array analyses showed that a group of genes is alternatively spliced during heat stress in an hnRNP K‐dependent manner, whereas hnRNP K is not necessary for the stress‐induced alternative splicing of the remaining genes. Among the latter group, we found that SRp38/SRSF10 and SC35/SRSF2 are necessary for the inclusion of exon 13 of TNRC6A during heat stress. Thus, our study clearly showed that several RNA‐binding proteins are involved in the splicing regulation in response to heat stress in mammalian cells.     

A "silent" nucleotide substitution in exon 4 is responsible for the "alternative expression" of HLA-A*01:01:38L through aberrant splicing

A Welsh Bone Marrow Donor Registry donor was serologically typed, using both alloantisera and monoclonal antibodies, as human leukocyte antigen (HLA)-A2, A-, but typed by polymerase chain reaction sequence-specific priming as HLA-A*01, A*02. Full gene sequencing of the A*01 separated allele indicated an apparently normal A*01:01:01:01 apart from a silent change at nucleotide 705 in exon 4, codon 211 (alanine: normally GCG but GCA in this donor). Sequence analysis of the amplified A*01 allele in cDNA synthesized from RNA indicated that exons 1, 2, 3, and 5 had typical A*01:01 sequences. However, exon 4 was truncated in this allele (87 nucleotides shorter), beginning just after the single nucleotide polymorphism (SNP) identified in genomic DNA sequencing. The nucleotide sequence up to, and 1 nucleotide after, the SNP is homologous with the 3' end of human leukocyte antigen (HLA)-A intron 3 and thus resembles a splice site. However, a small amount of "normal" HLA-A1 was detected on the surface of cells from an Epstein-Barr virus transformed B-cell line (BCL), but not on peripheral blood mononuclear cells, by flow cytometry. Additionally, a trace amount of "normal sized" A*01 was amplified from cDNA. We suggest that in this A*01 variant allele (A*01:01:38L) intron 3 is largely spliced out with a part of exon 4; exon 4 is still in-frame but the protein is smaller than the wild type. This is likely to affect folding and assembly of the "wild type" mature protein on the cell surface, thus explaining the apparent null phenotype when assayed by conventional serology. However, a small amount of A1 protein is made from correctly spliced A*01 mRNA and is detectable on BCLs using flow cytometry.     

Steroid 21‐hydroxylase deficiency: Mutational spectrum in Denmark,three novel mutations,and in vitro expression analysis

We have investigated 68 unrelated 21‐hydroxylase deficient Danish patients, representing 136 alleles, and determined the mutational spectrum of the CYP21 gene. The most frequent mutations detected were deletion of CYP21 and the splice mutation in intron 2 (I2‐splice). Segregation analysis showed evidence of a de novo mutation in each of two patients. Three novel mutations were detected: G64E in exon 1, Q262X in exon 7, and A362V in exon 8. G64E and A362V were introduced in the CYP21 cDNA by in vitro site‐directed mutagenesis, and the two corresponding proteins were transiently expressed in COS‐7 cells. The activity of 21‐hydroxylase was determined using the two hormone substrates 17‐hydroxyprogesterone and progesterone. The analysis showed no enzyme activity for any of the substrates, a result that correlates well with the severity of the patients' disease. Hum Mutat 13:482–486, 1999. © 1999 Wiley‐Liss, Inc.     

Genetic diagnosis of 21‐hydroxylase deficiency: DGGE‐based mutation scanning of CYP21

Congenital adrenal hyperplasia (CAH) due to 21‐hydroxylase deficiency is caused by mutations in the gene CYP21 encoding the enzyme steroid 21‐hydroxylase. In addition to deletions, approximately 20 different point mutations have been reported, and still novel mutations are detected. This makes genetic diagnosis as well as carrier detection of 21‐hydroxylase deficiency a complicated matter. We developed a simple nonradioactive assay based on the polymerase chain reaction (PCR) in combination with denaturing gradient gel electrophoresis (DGGE) to screen for mutations in the CYP21 gene. DGGE allows a fast scanning of PCR‐amplified segments of genes for the presence or absence of any single base pair alterations. We have performed this technique on the coding sequence and intron‐exon junctions of CYP21. Our results emphasize that this procedure constitutes a fast and reliable approach when performing diagnosis of 21‐hydroxylase deficiency. Hum Mutat 13:385–389, 1999. © 1999 Wiley‐Liss, Inc.     

Lysosomal acid lipase mutations that determine phenotype in Wolman and cholesterol ester storage disease

Mechanisms producing the divergent phenotypes, Wolman disease (WD) and cholesterol ester storage disease (CESD), associated with the genetic deficiency of human lysosomal acid lipase/cholesterol ester hydrolase (hLAL) function were investigated with the determination of HLAL activity levels, mRNA and protein expression, and defects in structural gene sequences in cells from three WD and five CESD patients. Measured with natural substrates, HLAL activities were all below 2% of normal, regardless of phenotype. Immunoblotting showed a lack of detectable hLAL protein in all mutant fibroblasts. Four CESD, but no WD genomes contained at least one allele with a specific exon 8 splice junction mutation, c.894 G>A, that encodes a shortened form of hLAL mRNA. Other CESD mutations were identical in type to the WD defects: nucleotide deletions (positions 397, 684, 980), insertions (594), or substitutions (193, 347) that result in premature terminations precluding any function. The only exception was a substitution at nucleotide 866 in the CESD case without an exon 8 splicing mutation; expression of the predicted S289C change in a transfection assay produced a low, but clearly measurable, level of acid esterase activity. Although it is not easily demonstrated in conventional assays, CESD is distinct from WD in that at least one mutant allele has the potential to produce enough residual enzymatic function to ameliorate the phenotype; in the majority of CESD cases this may come from a single, easily detected, splicing mutation in one allele.     

A novel intronic splice site deletion of the IL‐2 receptor common gamma chain results in expression of a dysfunctional protein and T‐cell‐positive X‐linked Severe combined immunodeficiency

X‐linked severe combined immunodeficiency is caused by mutations in the IL‐2 receptor common gamma chain and classically presents in the first 6 months of life with predisposition to bacterial, viral and fungal infections. In most instances, affected individuals are lymphopenic with near complete absence of T cells and NK cells. We report a boy who presented at 12 months of age with Pneumocystis jiroveci pneumonia and a family history consistent with X‐linked recessive inheritance. He had a normal lymphocyte count including the presence of T cells and a broad T‐cell‐receptor diversity, as well as normal surface expression of the common gamma chain (CD132) protein. He however had profound hypogammaglobulinaemia, and IL‐2‐induced STAT5 phosphorylation was absent. Sequencing of IL‐2RG demonstrated a 12‐base pair intronic deletion close to the canonical splice site of exon 5, which resulted in a variety of truncated IL2RG mRNA species. A review of the literature identified 4 other patients with T‐cell‐positive X‐SCID, with the current patient being the first associated with an mRNA splicing defect. This case raises the question of how a dysfunctional protein incapable of mediating STAT5 phosphorylation might nonetheless support T‐cell development. Possible explanations are that STAT5‐mediated signal transduction may be less relevant to IL7‐receptor‐mediated T‐cell development than are other IL7R‐induced intracellular transduction pathways or that a low level of STAT5 phosphorylation, undetectable in the laboratory, may be sufficient to support some T‐cell development.     

HLA‐A locus allelic dropout in Sanger sequence‐based typing due to the single nucleotide polymorphism of exon 1

The DNA‐based method is used widely for HLA genotyping in routine work, but some allele may be dropout in the genotyping procedure. Here, we reported a case with HLA‐A allele dropout in the Sanger PCR‐SBT test. The initial PCR‐SBT method with a commercial agent kit was not characterized, and the result of Luminex technology indicated the dropout as a HLA‐A*02 allele. Subsequently, the sequences of exons 2–4 were fully matched with the A*02:07 and A*11:01:01 by allele group‐specific primer amplification PCR‐SBT. On further analysis, a novel allele A*02:07:07 was identified, which has one nucleotide difference from A*02:07:01 at position 6 C>G of exon 1. According to the sequencing for 5′‐UTR to 3′‐UTR, the novel single nucleotide polymorphism of exon 1 was contributed to HLA‐A locus allele dropout in the sample. Our results indicated multiplatform analysis is necessary when a conclusive HLA type cannot be determined by a single methodology.     

SF2 and SRp55 regulation of CD45 exon 4 skipping during T cell activation

CD45 is an alternatively spliced membrane phosphatase required for T cell activation. Exons 4, 5 and 6 can be included or skipped from spliced mRNA resulting in several protein isoforms that include or exclude epitopes referred to as RA, RB or RC, respectively. T cells reciprocally express CD45RA or CD45RO (lacking all three exons), corresponding to naive versus memory T cells. Overexpression of the alternative splicing regulators, SF2 or SWAP, induces skipping of CD45 exon 4 in transfected COS cells. We show here that the arginine/serine-rich domain of SWAP and the RNA recognition motifs of SF2 are required for skipping of CD45 exon 4. Unlike SWAP, SF2 specifically regulated alternative splicing of CD45 exon 4, having no effect on a non-regulated exon of CD45 (exon 9). Like SF2 and SWAP, the SR proteins SC35, SRp40 and SRp75, but not SRp55 also induced CD45 exon 4 skipping. In contrast, antisense inhibition of SRp55 induced exon 4 skipping. SF2 and SRp55 proteins were not detectable or expressed at a very low level in freshly isolated CD45RA+ and CD45RO+ T cells. Activation of CD45RA+ T cells shifted CD45 expression from CD45RA to CD45RO, and induced a large increase in expression of both SF2 and SRp55. Thus, SF2 at least in part determines splicing of CD45 exon 4 during T cell activation. SRp55, SR protein phosphorylation, or other splicing factors likely regulate CD45 splicing in CD45RO+ memory T cells. The different SR proteins expressed by memory and activated T cells emphasize the different phenotypes of these cell types that both express CD45RO.     

A G-->A transition creates a branch point sequence and activation of a cryptic exon, resulting in the hereditary disorder neurofibromatosis 2

We describe a G-->A transition within intron 5 of the NF2 gene. This mutation creates a consensus splice branch point sequence. To our knowledge this is the first report of a mutation that creates a functional branch point sequence in a human hereditary disorder. The new branch point sequence is located 18 bp upstream of a consensus splice acceptor site. A consensus splice donor site is found 106 bp 3' of the acceptor site. Asa consequence the G-->A transition results in an alternatively spliced mRNA containing an additional exon 5a of 106 bp derived from intron sequences. We cloned the mutant cDNA and show that due to an in-frame stop codon the cDNA codes for a truncated NF2 protein. The mutation was observed in three affected members of an NF2 family. In a tumour of one of the family members both alternatively spliced and wild-type mRNA were found, although the wild-type allele of the gene is absent due to an interstitial deletion on chromosome 22. We also show that immunoprecipitations reveal the presence of full-length wild-type NF2 protein in the tumour lysate. These data support the hypothesis that some degree of normal splicing of the mutant precursor RNA is taking place. It is therefore likely that this residual activity of the mutant allele explains the relatively mild phenotype in the family. These data also indicate that complete inactivation of the gene is not required for tumour formation.