Effects of splicing mutations on NF2-transcripts: transcript analysis and information theoretic predictions.

This study examined the effects of 22 putative splicing mutations in the NF2 gene by means of transcript analysis and information theory based prediction. Fourteen mutations were within the dinucleotide acceptor and donor regions, often referred to as (AG/GT) sequences. Six were outside these dinucleotide regions but within the more broadly defined splicing regions used in the information theory based model. Two others were in introns and outside the broadly defined regions. Transcript analysis revealed exon skipping or activation of one or more cryptic splicing sites for 17 mutations. No alterations were found for the two intronic mutations and for three mutations in the broadly defined splicing regions. Concordance and partial concordance between the calculated predictions and the results of transcript analysis were found for 14 and 6 mutations, respectively. For two mutations, the predicted alteration was not found in the transcripts. Our results demonstrate that the effects of splicing mutations in NF2 are often complex and that information theory based analysis is helpful in elucidating the consequences of these mutations.

Transfer of a mitochondrial DNA fragment to MCOLN1 causes an inherited case of mucolipidosis IV.

A patient with mucolipidosis-IV heterozygous for two mutations in MCOLN1 expressed only her father's cDNA mutation c.1207C>T predicting an R403C change in mucolipin. She inherited a 93bp segment from mitochondrial NADH dehydrogenase 5 (MTND5) from her mother that was inserted in-frame prior to the last nucleotide of exon 2 of MCOLN1 (c.236_237ins93). This alteration abolished proper splicing of MCOLN1. The splice site at the end of the exon was not used due to an inhibitory effect of the inserted segment, resulting in two aberrant splice products containing stop codons in the downstream intron. These products were eliminated via nonsense-mediated decay. This is the first report of an inherited transfer of mitochondrial nuclear DNA causing a genetic disease. The elimination of the splice site by the mitochondrial DNA requires a change in splicing prediction models.

Targeting of pancreatic glia in type 1 diabetes.

OBJECTIVE: Type 1 diabetes reflects autoimmune destruction of beta-cells and peri-islet Schwann cells (pSCs), but the mechanisms of pSC death and the T-cell epitopes involved remain unclear. RESEARCH DESIGN AND METHODS: Primary pSC cultures were generated and used as targets in cytotoxic T-lymphocyte (CTL) assays in NOD mice. Cognate interaction between pSC and CD8(+) T-cells was assessed by transgenic restoration of beta2-microglobulin (beta2m) to pSC in NOD.beta2m(-/-) congenics. I-A(g7) and K(d) epitopes in the pSC antigen glial fibrillary acidic protein (GFAP) were identified by peptide mapping or algorithms, respectively, and the latter tested by immunotherapy. RESULTS: pSC cultures did not express major histocompatibility complex (MHC) class II and were lysed by ex vivo CTLs from diabetic NOD mice. In vivo, restoration of MHC class I in GFAP-beta2m transgenics significantly accelerated adoptively transferred diabetes. Target epitopes in the pSC autoantigen GFAP were mapped to residues 79-87 and 253-261 for K(d) and 96-110, 116-130, and 216-230 for I-A(g7). These peptides were recognized spontaneously in NOD spleens as early as 2.5 weeks of age, with proliferative responses peaking around weaning and detectable lifelong. Several were also recognized by T-cells from new-onset type 1 diabetic patients. NOD mouse immunotherapy at 8 weeks with the CD8(+) T-cell epitope, GFAP 79-87 but not 253-261, significantly inhibited type 1 diabetes and was associated with reduced gamma-interferon production to whole protein GFAP. CONCLUSIONS: Collectively, these findings elucidate a role for pSC-specific CD8(+) T-cells in islet inflammation and type 1 diabetes pathogenesis, further supporting neuronal involvement in beta-cell demise.

Glycerol metabolism and the determination of triglycerides--clinical, biochemical and molecular findings in six subjects.

Recent recommendations in the National Cholesterol Education Program Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (ATPIII) are expected to increase the number of triglyceride (TG) determinations and consequently the risk of misinterpretation of "non-blanked" results with co-determination of free glycerol. Glycerol-kinase deficiency (GKD) is one cause of falsely elevated TG results. The natural history of isolated GKD with symptom-free cases and cases with e.g. severe episodes of hypoglycemia and/or ketoacidosis challenges the laboratories to identify cases of GKD and family members at risk. "Blanked" methods reporting both glycerol and TG concentration are therefore desirable. Molecular studies of the glycerol kinase (GK) and DAX1 genes were performed on four cases of "persistent hypertriglyceridemia" found in an Italian population and on two pediatric cases with high serum glycerol concentration. Two new missense mutations were found (C358Y, T961). Molecular modeling on GK from E. coli, indicate that these mutations are located in parts of the enzyme important for enzyme formation or activity. One splice-site mutation, (IVS9A-1G>A), was found in two brothers. Splice-junction analysis indicates that it destroys the splice site and results in a mixture of mRNA. Deletion of the GK and DAX1 genes was found in one child with symptoms of adrenal failure. A female with glycerolemia and glyceroluria had normal GK activity but possibly slightly decreased ability to oxidize glycerol.

SLC7A9 mutations in all three cystinuria subtypes.

BACKGROUND: Cystinuria is an inherited disorder of cystine and dibasic amino acid transport in kidney. Subtypes are defined by the urinary cystine excretion patterns of the obligate heterozygous parents: Type I/N (fully recessive or silent); Type II/N (high excretor); Type III/N (moderate excretor). The first gene implicated in cystinuria (SLC3A1) is associated with the Type I urinary phenotype. A second cystinuria gene (SLC7A9) was recently isolated, and mutations of this gene were associated with dominant (non-Type I) cystinuria alleles. Here we report genotype-phenotype studies of SLC7A9 mutations in a cohort of well-characterized cystinuria probands and their family members. METHODS: Individual exons of the SLC7A9 gene were screened by single strand conformation polymorphism (SSCP) analysis and sequencing of abnormally migrating fragments. RESULTS: Seven mutations were identified. A single bp insertion (799insA) was present in four patients: on Type III alleles in two patients and on Type II alleles in two patients. These results suggest that Type II and Type III may be caused by the same mutation and, therefore, other factors must influence urinary cystine excretion. A 4bp deletion in intron 12 (IVS12+4delAGTA) and a missense mutation (1245G-->A, A354T) were identified on Type III alleles. A nonsense codon (1491G-->T, E436X) and a possible splicing mutation (IVS9-17G-->A) were seen in a Type I/III patient, but the mutations could not be assigned to particular alleles. Of additional interest were two missense mutations (316T-->C, I44T and 967C-->T, P261L) linked to Type I alleles. CONCLUSION: Our results provide evidence that some SLC7A9 mutations may be associated with fully recessive (Type I) forms of cystinuria. We also demonstrate SLC7A9 mutations in dominant Types II and III cystinuria. The finding of SLC7A9 mutations in all three subtypes underscores the complex interactions between specific cystinuria genes and other factors influencing cystine excretion. A simpler phenotypic classification scheme (recessive and dominant) for cystinuria is warranted.

Clinical heterogeneity and molecular findings in five Polish patients with glycerol kinase deficiency: investigation of two splice site mutations with computerized splice junction analysis and Xp21 gene-specific mRNA analysis.

Five cases of glycerol kinase deficiency are presented with clinical, biochemical, and genetic results. Two had the glycerol kinase deficiency as part of an Xp21 contiguous gene deletion syndrome-complex form-and three had an isolated form of the enzyme deficiency. In these we found two splice site mutations (IVS1+4A>G, IVS9-1G>T) and one insertion (1393_1394insG). In patients with the complex form, a deletion of the DAX1, GK genes and the distal part of the DMD gene was found. A computerized study was performed to predict the effects of the splice site mutations. It showed that the IVS9-1G>T mutation substantially altered and removed the wild-type site and enhanced a cryptic site seven nucleotides downstream, and that the IVS1+4A>G diminished the strength of the wild-type donor site from strong to leaky. To verify these predictions, we developed an RT-PCR system with gene-specific primers that exclusively amplifies the Xp21 glycerol kinase gene transcript. Identification of individuals at risk is motivated by a need to avoid delay in a correct diagnosis. For reliable identification of heterozygotes for isolated glycerol kinase deficiency, knowledge of the specific mutation in the proband is required. This is easily obtained with the RT-PCR analyses developed in this study.