Intronic Mutation

Abstract: Studies of genetically engineered flies and mice have revealed the role that orthologs of the human LIM homeobox LHX4 have in the control of motor-neuron–identity assignment and in pituitary development. Remarkably, these mouse strains, which bear a targeted modification of Lhx4 in the heterozygous state, are asymptomatic, whereas homozygous animals die shortly after birth. Nevertheless, we have isolated the human LHX4 gene, as well as the corresponding cDNA sequence, to test whether it could be involved in developmental defects of the human pituitary region. LHX4, which encodes a protein 99% identical to its murine counterpart, consists of six coding exons and spans >45 kb of the q25 region of chromosome 1. We report a family with an LHX4 germline splice-site mutation that results in a disease phenotype characterized by short stature and by pituitary and hindbrain (i.e., cerebellar) defects in combination with abnormalities of the sella turcica of the central skull base. This intronic mutation, which segregates in a dominant and fully penetrant manner over three generations, abolishes normal LHX4 splicing and activates two exonic cryptic splice sites, thereby predicting two different proteins deleted in their homeodomain sequence. These findings, which elucidate the molecular basis of a complex Mendelian disorder, reveal the fundamental pleiotropic role played by a single factor that tightly coordinates brain development and skull shaping during head morphogenesis.

Abstract: Background— Fabry disease is a treatable lysosomal storage disorder, which is often misdiagnosed or belatedly diagnosed. Methods and Results— To determine the disease incidence in the Taiwan Chinese population, a Fabry disease newborn screening study was initiated. A total of 110 027 newborns were screened by assaying the α-galactosidase A (α-Gal A) activity using dry blood spots. Low plasma α-Gal A activity and presence of a Fabry mutation was demonstrated in 45 neonates (3 females). Eight different mutations were identified, including 3 known missense mutations (R112H, A143T, and R356W), 4 novel missense mutations (G104V, M296L, G360C, and K391T), and one known intronic mutation (IVS4+919G→A). The IVS4+919G→A mutation was most common (82% of patients). A total of 20 maternal grandparents of infants harboring this intronic mutation were evaluated by echocardiography, mutation analysis and α-Gal A activity assay. The intronic mutation was found in 9 grandfathers and 11 grandmothers. Of these grandparents, 3 grandfathers (33%) but none of the grandmothers had hypertrophic cardiomyopathy. Additionally, 16 males who had been diagnosed with idiopathic hypertrophic cardiomyopathy were screened by mutation analysis and α-Gal A activity; 4 (25%) showed deficient plasma α-Gal A activity in combination with the intronic mutation. Conclusion— We found an unexpected high prevalence of the cardiac variant Fabry mutation IVS4+919G→A among both newborns (≈1 in 1600 males) and patients with idiopathic hypertrophic cardiomyopathy in the Taiwan Chinese population. The early identification of undiagnosed patients allows timely therapeutic intervention providing a better clinical outcome. Received March 5, 2009; accepted July 21, 2009. # CLINICAL PERSPECTIVE {#article-title-2}

Abstract: Fabry disease is an inborn error of glycosphingolipid catabolism, resulting from deficient activity of lysosomal α-galactosidase A (α-Gal A). A rare alternative splicing that introduces a 57-nucleotide (nt) intronic sequence to the α-Gal A transcript from intron 4 of the gene has been identified. In addition, a novel midintronic base substitution that results in substantially increased alternative splicing has been identified in a patient with Fabry disease who has the cardiac variant phenotype. The sequence of the patient's intron 4 contains a single G→A transversion at genomic nt 9331 (IVS4+919G→A), located at the −4 position of the 3′ end of the intronic insertion (nts 9278–9334 in the genomic sequence). Minigene constructs containing the entire intron 4 sequence with G, A, C, or T at nt 9331 within an α-Gal A complementary DNA expression vector were prepared and expressed in COS-1 cells. Whereas transfection of the G or T minigenes transcribed predominantly normal-sized transcripts, the transfection of the A or C minigenes produced a large amount of the alternatively spliced transcript. These results suggest that the G→A mutation, within an A/C-rich domain, results in increased recognition of the alternative splicing by an A/C-rich enhancer–type exonic splicing enhancer. The intronic mutation was not observed in 100 unrelated unaffected men but was present in 6 unrelated patients with cardiac Fabry disease. Reverse-transcriptase polymerase chain reaction of total RNA of various normal human tissues revealed that the alternatively spliced transcript was present in all of the samples, and especially at a higher ratio in the lung and muscle. The normal transcript was present in the patients' lymphoblasts and resulted in ∼10% residual enzyme activity, leading to a cardiac phenotype of Fabry disease.

Abstract: Leber congenital amaurosis (LCA) is the most severe form of inherited retinal degeneration, with an onset in the first year of life. The most frequent mutation that causes LCA, present in at least 10% of individuals with LCA from North-American and Northern-European descent, is an intronic mutation in CEP290 that results in the inclusion of an aberrant exon in the CEP290 mRNA. Here, we describe a genetic therapy approach that is based on antisense oligonucleotides (AONs), small RNA molecules that are able to redirect normal splicing of aberrantly processed pre-mRNA. Immortalized lymphoblastoid cells of individuals with LCA homozygously carrying the intronic CEP290 mutation were transfected with several AONs that target the aberrant exon that is incorporated in the mutant CEP290 mRNA. Subsequent RNA isolation and reverse transcription-PCR analysis revealed that a number of AONs were capable of almost fully redirecting normal CEP290 splicing, in a dose-dependent manner. Other AONs however, displayed no effect on CEP290 splicing at all, indicating that the rescue of aberrant CEP290 splicing shows a high degree of sequence specificity. Together, our data show that AON-based therapy is a promising therapeutic approach for CEP290-associated LCA that warrants future research in animal models to develop a cure for this blinding disease.