A new nonsyndromic X-linked sensorineural hearing impairment linked to Xp21.2.

X-linked deafness is a rare cause of hereditary hearing impairment. We have identified a family with X-linked dominant sensorineural hearing impairment, characterized by incomplete penetrance and variable expressivity in carrier females, that is linked to the Xp21.2, which contains the Duchenne muscular dystrophy (DMD) locus. The auditory impairment in affected males was congenital, bilateral, profound, sensorineural, affecting all frequencies, and without evidence of radiographic abnormality of the temporal bone. Adult carrier females manifested bilateral, mild-to-moderate high-frequency sensorineural hearing impairment of delayed onset during adulthood. Eighteen commercially available, polymorphic markers from the X chromosome, generating a 10-15-cM map, were initially used for identification of a candidate region. DXS997, located within the DMD gene, generated a two-point LOD score of 2.91 at theta = 0, with every carrier mother heterozygous at this locus. Recombination events at DXS992 (located within the DMD locus, 3' to exon 50 of the dystrophin gene) and at DXS1068 (5' to the brain promoter of the dystrophin gene) were observed. No recombination events were noted with the following markers within the DMD locus: 5'DYS II, intron 44, DXS997, and intron 50. There was no clinical evidence of Duchenne or Becker muscular dystrophy in any family member. It is likely that this family represents a new locus on the X chromosome, which when mutated results in nonsyndromic sensorineural hearing loss and is distinct from the heterogeneous group of X-linked hearing losses that have been previously described.     

Partial deletion of a dystrophin gene leads to exon skipping and to loss of an intra-exon hairpin structure from the predicted mRNA precursor.

In dystrophin Kobe exon 19 of the dystrophin gene is skipped during the process of mRNA precursor splicing even though the splice sites are unchanged (Matsuo et al. J. Clin. Invest. 87:2127-2131,1991). In the predicted secondary structure of the mRNA precursor, exon 19 of dystrophin Kobe is paired with intron sequences, whereas a large part of exon sequence from wild type is paired with itself and folded into a large hairpin structure. As all of 22 additional dystrophin exons analyzed also form intra-exon hairpin structures, these structures may be considered essential components of exons. We suggest that the abolishment of a hairpin structure in the truncated exon of dystrophin Kobe might prevent the splicing machinery from recognizing the splice sites and induce exon skipping.     

Insertion of an LrDNA gene fragment and of filler DNA at a mitochondrial exon-intron junction in Podospora.

A rearrangement of the mitochondrial genome of a long lived mutant of Podospora anserina is presented. It consists in the insertion of 191 bp of the LrDNA gene (coding for the large ribosomal RNA) at the junction between exon1 and intron alpha of gene co1 (coding for subunit 1 of cytochrome oxidase). This insertion is accompanied by a 53 bp deletion of the junction and the presence of extra A and T nucleotides at both sides of the inserted sequence. We discuss possible mechanisms of production of this rearrangement. The presence of extra nucleotides at the recombination junctions suggests that it may pass through a stage of free DNA ends originating from a DNA break at the junction between exon1 and intron alpha of gene co1. The possibility that such a DNA break plays a major role in the instability of the mitochondrial genome is envisaged.     

Two alternative exons can result from activation of the cryptic splice acceptor site deep within intron 2 of the dystrophin gene in a patient with as yet asymptomatic dystrophinopathy

Intron 2 of the dystrophin gene is unusually large, extending 157 kb on the X-chromosome, and is known to contain one cryptic exon 2a. Here, we report that a single nucleotide change in the middle of this huge intron is a source of two novel extra exons. A novel point mutation changing T to A nucleotide was identified at 5591 bp downstream from the 3' end of exon 2 (T310+5591A) in genomic DNA of an asymptomatic dystrophinopathy case. The mutation identification was initiated by detection of two novel dystrophin mRNAs containing a 132-nucleotide or 46-nucleotide insertion between exons 2 and 3 in lymphocytes but one with a 132-nucleotide insertion in skeletal muscle. It was concluded that T310+5591A created a novel consensus sequence for a splice acceptor site leading to the formation of two novel exon structures by using two cryptic splice donor sites at 132 bp or 46 bp downstream. The former maintained the dystrophin reading frame and was expected to insert 44 amino acids in the N-terminal domain of dystrophin, whereas the latter created a premature stop codon. An immunohistochemical study of the skeletal muscle of the patient disclosed that the N-terminal domain of dystrophin was not stained, but the rod- and C-terminal domains were stained in a patchy and discontinuous manner, indicating that the in-frame mRNA was functional. Creation of a splice acceptor site by a single nucleotide change leading to extra exon structures is a novel molecular mechanism in human disease.     

A novel point mutation (G-1 to T) in a 5' splice donor site of intron 13 of the dystrophin gene results in exon skipping and is responsible for Becker muscular dystrophy.

The mutations in one-third of Duchenne and Becker muscular dystrophy patients remain unknown, as they do not involve gross rearrangements of the dystrophin gene. We now report a defect in the splicing of precursor mRNA (pre-mRNA), resulting from a maternally inherited mutation of the dystrophin gene in a patient with Becker muscular dystrophy. This defect results from a G-to-T transversion at the terminal nucleotide of exon 13, within the 5' splice site of intron 13, and causes complete skipping of exon 13 during processing of dystrophin pre-mRNA. The predicted polypeptide encoded by the aberrant mRNA is a truncated dystrophin lacking 40 amino acids from the amino-proximal end of the rod domain. This is the first report of an intraexon point mutation that completely inactivates a 5' splice donor site in dystrophin pre-mRNA. Analysis of the genomic context of the G-1-to-T mutation at the 5' splice site supports the exon-definition model of pre-mRNA splicing and contributes to the understanding of splice-site selection.     

The human thyroglobulin gene is over 300 kb long and contains introns of up to 64 kb.

Thyroglobulin (Tg), the precursor of thyroid hormones, is a 660.000 Da dimeric glycoprotein synthesized exclusively in the thyroid gland. We have cloned the human thyroglobulin gene from cosmid and phage libraries and constructed a complete restriction map. The gene encodes an 8.7 kb mRNA, covers at least 300 kb DNA and contains at least 37 exons separated by large introns of up to 64 kb. A striking difference in structure between the 5' and 3' part of the gene suggests that it is composed of two evolutionarily different regions. The first 30 kb DNA encode 3 kb of the mRNA, yielding an exon:intron ratio of 1:10, whereas the remaining 270 kb encodes 5.7 kb of the mRNA with an exon:intron ratio of 1:47. In thyroid cells, the Tg gene is not rearranged and nuclear RNA homologous with sequences internal to the 64 kb intron is present, suggesting that the Tg gene is transcribed as a 300 kb RNA.     

Two polymorphic dinucleotide repeats in intron 44 of the dystrophin gene

Duchenne muscular dystrophy (DMD) is one of the most common and severe X-linked disorders with an incidence of approximately 1 in 3500 newborn males. In more than 60% of DMD patients, deletions of part or all of the dystrophin gene have been shown. Despite this, carrier detection still poses a problem in some cases, because of the enormous size of the gene and the lack of sufficient numbers of informative markers. Here, we report the isolation and characterization of two additional microsatellite markers (IVS44SK12 and IVS44SK21) in intron 44 of the dystrophin gene. Both markers are useful for carrier detection either by indirect DNA analysis or by direct proof of loss of heterozygosity.     

Molecular basis of the Cd36 chromosomal deletion underlying SHR defects in insulin action and fatty acid metabolism

The human insulin resistance syndromes—type 2 diabetes, obesity, combined hyperlipidemia, and essential hypertension—are genetically complex disorders whose molecular basis is largely unknown. The spontaneously hypertensive rate (SHR) is a model of these human syndromes. In the SHR/NCrlBR strain, a chromosomal deletion event that occurred at the Cd36 locus during the evolution of this SHR strain has been proposed as a cause of defective insulin action and fatty acid metabolism. In this study, three copies of the Cd36 gene, one transcribed copy and two pseudogenes, were identified in normal rat strains, but only a single gene in SHR/NCrlBR. Analysis of SHR genomic sequence localized the chromosomal deletion event between intron 4 of the normally transcribed copy of the gene and intron 4 of the second pseudogene. The deletion led to the creation of a single chimeric Cd36 gene in SHR/NCrlBR. The boundaries of the recombination/deletion junction identified within intron 4 were surrounded by long interspersed nuclear elements (LINEs) and DNA topoisomerase I recognition sequences. An 8-bp deletion at the intron 14/exon 15 boundary of the second pseudogene abolishes the putative splice acceptor site and is the cause of an aberrant 3′ UTR previously observed in SHR/NCrlBR. We conclude that in SHR/NCrlBR, the complex trait of insulin resistance and defective fatty acid metabolism is caused by Cd36 deficiency, resulting from a chromosomal deletion caused by unequal recombination. This demonstrates that chromosomal deletions caused by unequal recombination can be a cause of quantitative or complex mammalian phenotypes. Received: 7 September 2001 / Accepted: 3 October 2001     

An error in dystrophin mRNA processing in golden retriever muscular dystrophy, an animal homologue of Duchenne muscular dystrophy.

Golden retriever muscular dystrophy (GRMD) is a spontaneous, X-linked, progressively fatal disease of dogs and is also a homologue of Duchenne muscular dystrophy (DMD). Two-thirds of DMD patients carry detectable deletions in their dystrophin gene. The defect underlying the remaining one-third of DMD patients is undetermined. Analysis of the canine dystrophin gene in normal and GRMD dogs has failed to demonstrate any detectable loss of exons. Here, we have demonstrated a RNA processing error in GRMD that results from a single base change in the 3' consensus splice site of intron 6. The seventh exon is then skipped, which predicts a termination of the dystrophin reading frame within its N-terminal domain in exon 8. This is the first example of dystrophin deficiency caused by a splice-site mutation.     

Investigating the mechanism of chromosomal deletion: characterization of 39 deletion breakpoints in introns 47 and 48 of the human dystrophin gene

The region of the dystrophin gene containing introns 45-50 is characterized by a high rate of recombination events that give rise to large deletions causing dystrophinopathy. The nucleotide sequence of this intronic region has recently been released in GenBank. With the aim of further understanding the mechanism favoring the occurrence of these deletions, we have characterized the distribution of introns 47 and 48 deletion endpoints in 39 dystrophinopathy patients. In 14 of these patients we were able to sequence the break junction. On these sequences we were able to identify several intronic motifs that could predispose to DNA double-strand breaks. Our results, combined with other literature data, show that unequal homologous recombination is a very poorly represented event in the dystrophin gene, whereas junction features are suggestive of a model of recombination in which DNA double-strand breaks are incorrectly repaired by a nonhomologous end-joining mechanism. The correlation among recombination rate, deletion frequency, and percentage of repetitive elements is discussed.     

Extensive and prolonged restoration of dystrophin expression with vivo-morpholino-mediated multiple exon skipping in dystrophic dogs

Duchenne muscular dystrophy (DMD) is a severe and the most prevalent form of muscular dystrophy, characterized by rapid progression of muscle degeneration. Antisense-mediated exon skipping is currently one of the most promising therapeutic options for DMD. However, unmodified antisense oligos such as morpholinos require frequent (weekly or bi-weekly) injections. Recently, new generation morpholinos such as vivo-morpholinos are reported to lead to extensive and prolonged dystrophin expression in the dystrophic mdx mouse, an animal model of DMD. The vivo-morpholino contains a cell-penetrating moiety, octa-guanidine dendrimer. Here, we sought to test the efficacy of multiple exon skipping of exons 6-8 with vivo-morpholinos in the canine X-linked muscular dystrophy, which harbors a splice site mutation at the boundary of intron 6 and exon 7. We designed and optimized novel antisense cocktail sequences and combinations for exon 8 skipping and demonstrated effective exon skipping in dystrophic dogs in vivo. Intramuscular injections with newly designed cocktail oligos led to high levels of dystrophin expression, with some samples similar to wild-type levels. This is the first report of successful rescue of dystrophin expression with morpholino conjugates in dystrophic dogs. Our results show the potential of phosphorodiamidate morpholino oligomer conjugates as therapeutic agents for DMD.     

Polymorphism across an exon-intron boundary in an avian Mhc class II B gene.

Twenty-three sequence haplotypes spanning the boundary of the second exon and intron of a red-winged blackbird Mhc class II B gene, Agph-DAB1, are presented. The polymorphism of the exon segment is distributed in two divergent allelic lineages which appear to be maintained by balancing selection. The silent nucleotide diversity of the exon (pi = 0.101) is more than five times that of the intron (pi = 0.018) and decays rapidly across the exon-intron boundary. Additionally, genealogical reconstruction indicates that divergence from a common ancestor in the exon sample is over four times that of the intron. The intron sequences reveal a pattern of polymorphism which is characteristic of directional selection, rather than a pattern expected from linkage to a balanced polymorphism. These results suggest that the evolutionary histories of these two adjacent regions have been disassociated by recombination or gene conversion. The estimated population recombination parameter between the exon and the intron is sufficiently high (4NeC = 8.545) to explain the homogenization of intron sequences. Compatibility analyses estimate that these events primarily occur from the exon-intron boundary to about 20-30 bases into the intron. Additionally, the observation that divergent exon alleles share identical intron sequence supports the conclusion of disassociation of exon and intron evolutionary histories by recombination.     

Molecular characterization of two deletion events involving Alu-sequences, one novel base substitution and two tentative hotspot mutations in the hypoxanthine phosphoribosyltransferase (HPRT) gene in five patients with Lesch-Nyhan syndrome

Mutations identified in the hypoxanthine phosphoribosyltransferase (HPRT) gene of patients with Lesch-Nyhan (LN) syndrome are dominated by simple base substitutions. Few hotspot positions have been identified, and only three large genomic rearrangements have been characterized at the molecular level. We have identified one novel mutation, two tentative hot spot mutations, and two deletions by direct sequencing of HPRT cDNA or genomic DNA from fibroblasts or T-lymphocytes from LN patients in five unrelated families. One is a missense mutation caused by a 610C→T transition of the first base of HPRT exon 9. This mutation has not been described previously in an LN patient. A nonsense mutation caused by a 508C→T transition at a CpG site in HPRT exon 7 in the second patient and his younger brother is the fifth mutation of this kind among LN patients. Another tentative hotspot mutation in the third patient, a frame shift caused by a G nucleotide insertion in a monotonous repeat of six Gs in HPRT exon 3, has been reported previously in three other LN patients. The fourth patient had a tandem deletion: a 57-bp deletion in an internally repeated Alu-sequence of intron 1 was separated by 14 bp from a 627-bp deletion that included HPRT exon 2 and was flanked by a 4-bp repeat. This complex mutation is probably caused by a combination of homologous recombination and replication slippage. Another large genomic deletion of 2969 bp in the fifth patient extended from one Alu-sequence in the promoter region to another Alu-sequence of intron 1, deleting the whole of HPRT exon 1. The breakpoints were located within two 39-bp homologous sequences, one of which overlapped with a well-conserved 26-bp Alu-core sequence previously suggested as promoting recombination. These results contribute to the establishment of a molecular spectrum of LN mutations, support previous data indicating possible mutational hotspots, and provide evidence for the involvement of Alu-mediated recombination in HPRT deletion mutagenesis. Received: 21 April 1998 / Accepted: 16 July 1998     

Intron mobility in the T-even phages: high frequency inheritance of group I introns promoted by intron open reading frames

Intron mobility in the T-even phages has been demonstrated. Efficient nonreciprocal conversion of intron minus (In-) alleles to intron plus (In+) occurred for the td and sunY genes, but not for nrdB. Conversion to In+ was absolutely dependent on expression of the respective intron open reading frame (ORF). Introns were inserted at their cognate sites in an intronless phage genome via an RNA-independent, DNA-based, duplicative recombination event that was stimulated by exon homology. The td intron ORF product directs the endonucleolytic cleavage of DNA, targeting the site of intron integration. A 21 nucleotide deletion of the integration site abolished high frequency intron inheritance. These experiments provide a novel example of gene conversion in prokaryotes, while suggesting a molecular rationale for the inconsistent distribution of introns within highly conserved exon contexts of the T-even phage genomes.