Clinical and genetic investigation of a large Tunisian family with complete achromatopsia: identification of a new nonsense mutation in GNAT2 gene.

Complete achromatopsia is a rare autosomal recessive disease associated with CNGA3, CNGB3, GNAT2 and PDE6C mutations. This retinal disorder is characterized by complete loss of color discrimination due to the absence or alteration of the cones function. The purpose of the present study was the clinical and the genetic characterization of achromatopsia in a large consanguineous Tunisian family. Ophthalmic evaluation included a full clinical examination, color vision testing and electroretinography. Linkage analysis using microsatellite markers flanking CNGA3, CNGB3, GNAT2 and PDE6C genes was performed. Mutations were screened by direct sequencing. A total of 12 individuals were diagnosed with congenital complete achromatopsia. They are members of six nuclear consanguineous families belonging to the same large consanguineous family. Linkage analysis revealed linkage to GNAT2. Mutational screening of GNAT2 revealed three intronic variations c.119-69G>C, c.161+66A>T and c.875-31G>C that co-segregated with a novel mutation p.R313X. An identical GNAT2 haplotype segregating with this mutation was identified, indicating a founder mutation. All patients were homozygous for the p.R313X mutation. This is the first report of the clinical and genetic investigation of complete achromatopsia in North Africa and the largest family with recessive achromatopsia involving GNAT2; thus, providing a unique opportunity for genotype-phenotype correlation for this extremely rare condition.

Mutation prevalence of cerebral cavernous malformation genes in Spanish patients.

OBJECTIVE To study the molecular genetic and clinical features of cerebral cavernous malformations (CCM) in a cohort of Spanish patients. METHODS We analyzed the CCM1, CCM2, and CCM3 genes by MLPA and direct sequencing of exons and intronic boundaries in 94 familial forms and 41 sporadic cases of CCM patients of Spanish extraction. When available, RNA studies were performed seeking for alternative or cryptic splicing. RESULTS A total of 26 pathogenic mutations, 22 of which predict truncated proteins, were identified in 29 familial forms and in three sporadic cases. The repertoire includes six novel non-sense and frameshift mutations in CCM1 and CCM3. We also found four missense mutations, one of them located at the third NPXY motif of CCM1 and another one that leads to cryptic splicing of CCM1 exon 6. We found four genomic deletions with the loss of the whole CCM2 gene in one patient and a partial loss of CCM1and CCM2 genes in three other patients. Four families had mutations in CCM3. The results include a high frequency of intronic variants, although most of them localize out of consensus splicing sequences. The main symptoms associated to clinical debut consisted of cerebral haemorrhage, migraines and epileptic seizures. The rare co-occurrence of CCM with Noonan and Chiari syndromes and delayed menarche is reported. CONCLUSIONS Analysis of CCM genes by sequencing and MLPA has detected mutations in almost 35% of a Spanish cohort (36% of familial cases and 10% of sporadic patients). The results include 13 new mutations of CCM genes and the main clinical symptoms that deserves consideration in molecular diagnosis and genetic counselling of cerebral cavernous malformations.

Lethal skeletal dysplasia in mice and humans lacking the golgin GMAP-210.

BACKGROUND: Establishing the genetic basis of phenotypes such as skeletal dysplasia in model organisms can provide insights into biologic processes and their role in human disease. METHODS: We screened mutagenized mice and observed a neonatal lethal skeletal dysplasia with an autosomal recessive pattern of inheritance. Through genetic mapping and positional cloning, we identified the causative mutation. RESULTS: Affected mice had a nonsense mutation in the thyroid hormone receptor interactor 11 gene (Trip11), which encodes the Golgi microtubule-associated protein 210 (GMAP-210); the affected mice lacked this protein. Golgi architecture was disturbed in multiple tissues, including cartilage. Skeletal development was severely impaired, with chondrocytes showing swelling and stress in the endoplasmic reticulum, abnormal cellular differentiation, and increased cell death. Golgi-mediated glycosylation events were altered in fibroblasts and chondrocytes lacking GMAP-210, and these chondrocytes had intracellular accumulation of perlecan, an extracellular matrix protein, but not of type II collagen or aggrecan, two other extracellular matrix proteins. The similarities between the skeletal and cellular phenotypes in these mice and those in patients with achondrogenesis type 1A, a neonatal lethal form of skeletal dysplasia in humans, suggested that achondrogenesis type 1A may be caused by GMAP-210 deficiency. Sequence analysis revealed loss-of-function mutations in the 10 unrelated patients with achondrogenesis type 1A whom we studied. CONCLUSIONS: GMAP-210 is required for the efficient glycosylation and cellular transport of multiple proteins. The identification of a mutation affecting GMAP-210 in mice, and then in humans, as the cause of a lethal skeletal dysplasia underscores the value of screening for abnormal phenotypes in model organisms and identifying the causative mutations.

Evolutionary origin and functions of retrogene introns.

Retroposed genes (retrogenes) originate via the reverse transcription of mature messenger RNAs from parental source genes and are therefore usually devoid of introns. Here, we characterize a particular set of mammalian retrogenes that acquired introns upon their emergence and thus represent rare cases of intron gain in mammals. We find that although a few retrogenes evolved introns in their coding or 3' untranslated regions (untranslated region, UTR), most introns originated together with untranslated exons in the 5' flanking regions of the retrogene insertion site. They emerged either de novo or through fusions with 5' UTR exons of host genes into which the retrogenes inserted. Generally, retrogenes with introns display high transcription levels and show broader spatial expression patterns than other retrogenes. Our experimental expression analyses of individual intron-containing retrogenes show that 5' UTR introns may indeed promote higher expression levels, at least in part through encoded regulatory elements. By contrast, 3' UTR introns may lead to downregulation of expression levels via nonsense-mediated decay mechanisms. Notably, the majority of retrogenes with introns in their 5' flanks depend on distant, sometimes bidirectional CpG dinucleotide-enriched promoters for their expression that may be recruited from other genes in the genomic vicinity. We thus propose a scenario where the acquisition of new 5' exon-intron structures was directly linked to the recruitment of distant promoters by these retrogenes, a process potentially facilitated by the presence of proto-splice sites in the genomic vicinity of retrogene insertion sites. Thus, the primary role and selective benefit of new 5' introns (and UTR exons) was probably initially to span the often substantial distances to potent CpG promoters driving retrogene transcription. Later in evolution, these introns then obtained additional regulatory roles in fine tuning retrogene expression levels. Our study provides novel insights regarding mechanisms underlying the origin of new introns, the evolutionary relevance of intron gain, and the origin of new gene promoters.

Lack of the mitochondrial protein acylglycerol kinase causes Sengers syndrome.

Exome sequencing of an individual with congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, and lactic acidosis, all typical symptoms of Sengers syndrome, discovered two nonsense mutations in the gene encoding mitochondrial acylglycerol kinase (AGK). Mutation screening of AGK in further individuals with congenital cataracts and cardiomyopathy identified numerous loss-of-function mutations in an additional eight families, confirming the causal nature of AGK deficiency in Sengers syndrome. The loss of AGK led to a decrease of the adenine nucleotide translocator in the inner mitochondrial membrane in muscle, consistent with a role of AGK in driving the assembly of the translocator as a result of its effects on phospholipid metabolism in mitochondria.

Mutations in DNAH5 account for only 15% of a non-preselected cohort of patients with primary ciliary dyskinesia.

BACKGROUND: Primary ciliary dyskinesia (PCD) is characterised by recurrent infections of the upper respiratory airways (nose, bronchi, and frontal sinuses) and randomisation of left-right body asymmetry. To date, PCD is mainly described with autosomal recessive inheritance and mutations have been found in five genes: the dynein arm protein subunits DNAI1, DNAH5 and DNAH11, the kinase TXNDC3, and the X-linked retinitis pigmentosa GTPase regulator RPGR. METHODS: We screened 89 unrelated individuals with PCD for mutations in the coding and splice site regions of the gene DNAH5 by denaturing high performance liquid chromatography (DHPLC) and sequencing. Patients were mainly of European origin and were recruited without any phenotypic preselection. RESULTS: We identified 18 novel (nonsense, splicing, small deletion and missense) and six previously described mutations. Interestingly, these DNAH5 mutations were mainly associated with outer + inner dyneins arm ultrastructural defects (50%). CONCLUSION: Overall, mutations on both alleles of DNAH5 were identified in 15% of our clinically heterogeneous cohort of patients. Although genetic alterations remain to be identified in most patients, DNAH5 is to date the main PCD gene.

Transcriptional silencing of nonsense codon-containing immunoglobulin micro genes requires translation of its mRNA

Eukaryotes have evolved quality control mechanisms that prevent the expression of genes in which the protein coding potential is crippled by the presence of a premature translation-termination codon (PTC). In addition to nonsense-mediated mRNA decay (NMD), a well documented posttranscriptional consequence of the presence of a PTC in an mRNA, we recently reported the transcriptional silencing of PTC-containing immunoglobulin (Ig) mu and gamma minigenes when they are stably integrated into the genome of HeLa cells. Here we demonstrate that this transcriptional silencing of PTC-containing Ig-mu constructs requires active translation of the cognate mRNA, as it is not observed under conditions where translation of the PTC-containing mRNA is inhibited through an iron-responsive element in the 5'-untranslated region. Furthermore, RNA interference-mediated depletion of the essential NMD factor Upf1 not only abolishes NMD but also reduces the extent of nonsense-mediated transcriptional gene silencing (NMTGS). Collectively, our data indicate that NMTGS and NMD are linked, relying on the same mechanism for PTC recognition, and that the NMTGS pathway branches from the NMD pathway at a step after Upf1 function.

Investigation of premature termination codon recognition in nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is best known for its role in quality control of mRNAs, where it recognizes premature translation termination codons (PTCs) and rapidly degrades the corresponding mRNA. The basic mechanism of NMD appears to be conserved among eukaryotes: aberrant translation termination triggers NMD. According to the current working model, correct termination requires the interaction of the ribosome with the poly(A)-binding protein (PABPC1) mediated through the eukaryotic release factors 1 (eRF1) and 3 (eRF3). The model predicts that in the absence of this interaction, the NMD core factor UPF1 binds to eRF3 instead and initiates the events ultimately leading to mRNA degradation. However, the exact mechanism of how the decision between proper and aberrant (i.e. NMD-inducing) translation termination occurs is not yet well understood. We address this question using a tethering approach in which proteins of interest are bound to a reporter transcript into the vicinity of a PTC. Subsequently, the ability of the tethered proteins to inhibit NMD and thus stabilize the reporter transcript is assessed. Our results revealed that the C-terminal domain interacting with eRF3 seems not to be necessary for tethered PABPC1 to suppress NMD. In contrast, the N-terminal part of PABPC1, consisting of 4 RNA recognition motifs (RRMs) and interacting with eukaryotic initiation factor 4G (eIF4G), retains the ability to inhibit NMD. We find that eIF4G is able to inhibit NMD in a similar manner as PABPC1 when tethered to the reporter mRNA. This stabilization by eIF4G depends on two key interactions. One of these interactions is to PABPC1, the other is to eukaryotic initiation factor 3 (eIF3). These results confirm the importance of PABPC1 in inhibiting NMD but additionally reveal a role of translation initiation factors in the distinction between bona fide termination codons and PTCs.

Infrequent Translation of a Nonsense Codon Is Sufficient to Decrease mRNA Level

In many organisms nonsense mutations decrease the level of mRNA. In the case of mammalian cells, it is still controversial whether translation is required for this nonsense-mediated RNA decrease (NMD). Although previous analyzes have shown that conditions that impede translation termination at nonsense codons also prevent NMD, the residual level of termination was unknown in these experiments. Moreover, the conditions used to impede termination might also have interfered with NMD in other ways. Because of these uncertainties, we have tested the effects of limiting translation of a nonsense codon in a different way, using two mutations in the immunoglobulin μ heavy chain gene. For this purpose we exploited an exceptional nonsense mutation at codon 3, which efficiently terminates translation but nonetheless maintains a high level of μ mRNA. We have shown 1) that translation of Ter462 in the double mutant occurs at only ∼4% the normal frequency, and 2) that Ter462 in cis with Ter3 can induce NMD. That is, translation of Ter462 at this low (4%) frequency is sufficient to induce NMD.

A novel WT1 heterozygous nonsense mutation (p.K248X) causing a mild and slightly progressive nephropathy in a 46,XY patient with Denys-Drash syndrome

WT1 mutations have been described in a variety of syndromes, including Denys-Drash syndrome (DDS), which is characterized by predisposition to Wilms` tumor, genital abnormalities and development of early nephropathy. The most frequent WT1 defects in DDS are missense mutations located in exons 8-9. Our aim is to report a novel WT1 mutation in a 46,XY patient with a DDS variant, who presented a mild nephropathy with a late onset diagnosed during adolescence. He had ambiguous genitalia at birth. At 4 months of age he underwent nephrectomy (Wilms` tumor) followed by chemotherapy. Ambiguous genitalia were corrected and bilateral gonadectomy was performed. Sequencing of WT1 identified a novel heterozygous mutation (c.742A > T) in exon 4 that generates a premature stop codon (p.K248X). Interestingly, this patient has an unusual DDS nephropathy progression, which reinforces that patients carrying WT1 mutations should have the renal function carefully monitored due to the possibility of late-onset nephropathy.

Analysis of the intranuclear life of nonsense transcripts

Dissertação para obtenção do Grau de Doutor em Biologia, Especialidade de Biologia Molecular

mRNA metabolism: nonsense mediated mRNA decay as a tool for gene therapy and the role of human DIS3L2 in transcript degradation

Tese de mestrado em Biologia Humana e Ambiente, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2015

Identification of the first nonsense CDSN mutation with expression of a truncated protein causing peeling skin syndrome type B.

BACKGROUND: Peeling skin disease (PSD), a generalized inflammatory form of peeling skin syndrome, is caused by autosomal recessive nonsense mutations in the corneodesmosin gene (CDSN). OBJECTIVES: To investigate a novel mutation in CDSN. METHODS: A 50-year-old white woman showed widespread peeling with erythema and elevated serum IgE. DNA sequencing, immunohistochemistry, Western blot and real-time polymerase chain reaction analyses of skin biopsies were performed in order to study the genetics and to characterize the molecular profile of the disease. RESULTS: Histology showed hyperkeratosis and acanthosis of the epidermis, and inflammatory infiltrates in the dermis. DNA sequencing revealed a homozygous mutation leading to a premature termination codon in CDSN: p.Gly142*. Protein analyses showed reduced expression of a 16-kDa corneodesmosin mutant in the upper epidermal layers, whereas the full-length protein was absent. CONCLUSIONS: These results are interesting regarding the genotype-phenotype correlations in diseases caused by CDSN mutations. The PSD-causing CDSN mutations identified heretofore result in total corneodesmosin loss, suggesting that PSD is due to full corneodesmosin deficiency. Here, we show for the first time that a mutant corneodesmosin can be stably expressed in some patients with PSD, and that this truncated protein is very probably nonfunctional.