Premature Termination Codon In Exon Research Articles

Severe Microcytic Anemia Caused by Complex Hereditary Spherocytosis and X-Linked Sideroblastic Anemia with Mutations in SPTB and ALAS2 Genes

We report a case of severe anemia caused by complex hereditary spherocytosis (HS) and X-linked sideroblastic anemia (XLSA) with two mutations in the spectrin beta (SPTB) and 5-aminolevulinic acid synthase (ALAS2) genes. The proband was a 16-year-old male with severe jaundice and microcytic hypochromic anemia since his childhood. He had more severe anemia requiring erythrocyte transfusion, and had no response to vitamin B6 treatment. Next-generation sequencing (NGS) revealed double heterozygous mutations, one in exon 19 (c.3936G > A:p.W1312X) of the SPTB gene and another in exon 2 (c.37A > G:p.K13E) of the ALAS2 gene, and confirmed again by Sanger sequencing. The mutation of ALAS2 (c.37A > G) is inherited from his asymptomatic heterozygous mother, causing amino acid p.K13E, and the mutation has not yet been reported. The mutation of SPTB (c.3936G > A) is a nonsense mutation, leading to a premature termination codon in exon 19, and the mutation in the SPTB gene is not found in any of his relatives, which indicates a de novo monoallelic mutation. Conclusions: The double heterozygous mutations in the SPTB and ALAS2 genes lead to the joint occurrence of HS and XLSA in this patient, and are implicated in the more severe clinical phenotypes.

A missense mutation in TTC8/BBS8 affecting mRNA splicing in patients with non-syndromic retinitis pigmentosa.

Splicing disruption is one type of mutation mechanism for disease-predisposing alleles. To date, less than 30 mutations in TTC8/BBS8 have been reported; however, mutations affecting the splice site are rare. Generally missense mutations are assumed to alter protein function; however, reports have shown that mutations in protein coding exons can disrupt splicing by altering exonic splicing silencer or enhancer motifs. Hence, a missense mutation c.1347G > C (p.Q449H) involving final base of the exon 13 in the TTC8, previously identified by us to be linked with non-syndromic autosomal recessive retinitis pigmentosa (arRP), in an Indian family, that might deleteriously affect splicing has been functionally characterized. RNA was isolated, cDNA prepared and amplified using region-specific primers. PCR products were purified and sequenced bi-directionally by Sanger sequencing. Effect of mutation (c.1347G > C) on mRNA splicing has been predicted using bioinformatics tools. We reported that missense mutation (c.1347G > C) at the last base of exon 13 of TTC8 disrupted the canonical donor splice-site resulting in aberrant RNA splicing. A cryptic donor splice-site got activated 77 bases downstream of the authentic splice donor site in intron 13, resulting in the retention of 77 bases of intron 13, and a frameshift leading to pre-mature termination codon in exon 14 at codon 486. Further, duplication of exon 15 and fusion of its duplicated copy occurred with exon 13. The binding site for SC35 protein, normally involved in splicing, also got disrupted (as predicted by SpliceAid2 software), hence, leading to alternative splicing. Our findings strongly suggest that a missense mutation c.1347G > C in TTC8 disrupted the splice donor site causing retention of 77 bases of intron 13, resulting in a frameshift and subsequently introduced a pre-mature termination codon into exon 14, hence creating an altered mRNA transcript. These findings emphasize the significance of examining missense mutations especially in TTC8, to determine their pathogenic role through alternative splicing. Present findings also reiterate the notion that mutations in the TTC8/BBS8 cause phenotypic heterogeneity and does not always follow Mendelian genetics in this ciliopathy.

Mutations in the ribosome biogenesis factor gene LTV1 are linked to LIPHAK syndrome, a novel poikiloderma-like disorder.

In the framework of the UK 100 000 Genomes Project, we investigated the genetic origin of a previously undescribed recessive dermatological condition, which we named LIPHAK (LTV1-associated Inflammatory Poikiloderma with Hair abnormalities and Acral Keratoses), in four affected individuals from two UK families of Pakistani and Indian origins, respectively. Our analysis showed that only one gene, LTV1, carried rare biallelic variants that were shared in all affected individuals, and specifically they bore the NM_032860.5:c.503A > G, p.(Asn168Ser) change, found homozygously in all of them. In addition, high-resolution homozygosity mapping revealed the presence of a small 652-kb stretch on chromosome 6, encompassing LTV1, that was haploidentical and common to all affected individuals. The c.503A > G variant was predicted by in silico tools to affect the correct splicing of LTV1’s exon 5. Minigene-driven splicing assays in HEK293T cells and in a skin sample from one of the patients confirmed that this variant was indeed responsible for the creation of a new donor splice site, resulting in aberrant splicing and in a premature termination codon in exon 6 of this gene. LTV1 encodes one of the ribosome biogenesis factors that promote the assembly of the small (40S) ribosomal subunit. In yeast, defects in LTV1 alter the export of nascent ribosomal subunits to the cytoplasm; however, the role of this gene in human pathology is unknown to date. Our data suggest that LIPHAK could be a previously unrecognized ribosomopathy.

Biallelic variants in MESD, which encodes a WNT-signaling-related protein, in four new families with recessively inherited osteogenesis imperfecta.

SummaryThe bone disorder osteogenesis imperfecta (OI) is genetically heterogeneous. Most affected individuals have an autosomal dominant disorder caused by heterozygous variants in either of the type I collagen genes (COL1A1 or COL1A2). To date, two reports have linked Mesoderm Development LRP Chaperone (MESD) to autosomal recessive OI type XX. Four different biallelic pathogenic variants in MESD were shown to cause a progressively deforming phenotype, associated with recurrent fractures and oligodontia in five individuals in five families. Recently, compound heterozygosity for a frameshift predicted to lead to a premature termination codon in exon 2 of the 3-exon gene and a second frameshift in the terminal exon in MESD were detected in three stillbirths in one family with severe OI consistent with the neonatal lethal phenotype. We have identified four additional individuals from four independent families with biallelic variants in MESD: the earlier reported c.632dupA (p.Lys212Glufs∗19) and c.676C>T (p.Arg226∗)—which are associated with a severe form of OI—and one new pathogenic variant, c.603-606delTAAA (p.Asn201Lysfs∗15), which causes a neonatal lethal form of OI. MESD acts in the WNT signaling pathway, where it is thought to play a role in the folding of the WNT co-receptors low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/LRP6) and in chaperoning their transit to the cell surface. Our report broadens the phenotypic and genetic spectrum of MESD-related OI, provides additional insight into the pathogenic pathways, and underscores the necessity of MESD for normal WNT signaling in bone formation.

Identification of a novel heterozygous SPTB mutation by whole genome sequencing in a Chinese patient with hereditary spherocytosis and atrial septal defect: a case report

BackgroundHereditary spherocytosis (HS) is a common inherited red blood cell membrane disorder characterized by an abnormal increase of spherocytes in peripheral blood. SPTB gene mutation is one of the most common causes of HS; however, few cases of HS resulting from SPTB mutation in the Chinese population have been reported so far.Case presentationA 3-year-old Chinese girl presented to Qingdao Women and Children’s Hospital, Qingdao University, with atrial septal defect (ASD). Meanwhile, she was clinically diagnosed with HS. Whole genome sequencing (WGS) was performed for the proband and her parents for genetic molecular analysis. A novel SPTB mutation (c.1756delG) was detected by WGS and confirmed by Sanger sequencing in the proband. This mutation results in a frameshift with a premature termination codon in exon 12, leading to a nonsense mutation (p.Ala586Profs*7). Her parents had no similar symptoms, and blood routine and serum biochemical tests showed no significant abnormalities. The patient’s mother did not know of any relatives with HS-like symptoms. Percutaneous transcatheter closure was successfully performed for treating the ASD.ConclusionIn this study, we identified a novel SPTB frameshift mutation in a Chinese girl with HS. This finding would expand the spectrum of SPTB mutations, provide a valuable insight into the genotyping of HS in the Chinese population, and contribute to the clinical management and genetic counseling in HS.

CRISPR/Cas9 induces exon skipping that facilitates development of fragrant rice.

Splicing of precursor-mRNAs (pre-mRNAs) is a critical biological process of gene expression. Normally, pre-mRNAs are processed by spliceosomes to produce mature mRNAs by removing introns at 5'-(donor) and 3'-(acceptor) splice sites based on the canonical GU-AG rule (Reddy et al., 2013). Mutation at either the intron donor or acceptor sites should cause mRNA mis-splicing. Recently, an exon skipping method has been developed to generate loss of gene function in mammalian cells using base editors to mutate nucleotides at the acceptor sites (Gapinske et al., 2018).

A rare deep intronic mutation of PKHD1 gene, c.8798-459 C > A, causes autosomal recessive polycystic kidney disease by pseudoexon activation.

Autosomal recessive polycystic kidney disease (ARPKD), is a rare hepatorenal fibrocystic disorder primarily associated with progressive growth of multiple cysts in the kidneys causing progressive loss of renal function. The disease is linked to mutations in the PKHD1 gene. In this study, we describe the gene diagnosis and prenatal diagnosis for a consanguineous family with two fetuses diagnosed with polycystic kidney disease by fetal sonography during the pregnancy. Sequence analysis of cDNA synthesized from the PKHD1 mRNA of the second induced fetus identified a 111-nucleotide insert at the junction of exon 56 and 57 that originated from intervening sequence (IVS) 56. Further genomic sequencing of IVS 56 of the PKHD1 gene identified a rare homozygous deep intronic mutation (c.8798-459 C > A), which was inherited from the parents and not detectable in 100 unrelated control subjects. Moreover, we explored the pathogenicity of this deep intronic mutation by conducting a minigene splicing assay experiment, which demonstrated that the mutation causes a pseudoexon insertion, which results in a frameshift followed by a premature termination codon in exon 57. Eventually, the parents had a healthy baby by undergoing prenatal genetic diagnosis based on the targeted detection of the intron mutation. The newly identified deep intronic mutation is associated with a rare mechanism of abnormal splicing that expands the spectrum of known PKHD1 gene mutations. It can be used in evidence-based genetic and reproductive counseling for families with ARPKD.

Homozygous KIDINS220 loss-of-function variants in fetuses with cerebral ventriculomegaly and limb contractures.

Heterozygous mutations in KIDINS220 were recently suggested a cause of spastic paraplegia, intellectual disability, nystagmus and obesity. All patients carried terminal nonsense de novo mutations that seemed to escape nonsense-mediated mRNA decay. The mechanism for pathogenicity is yet unexplained, as it seems that heterozygous loss-of-function variants of KIDINS220 are generally well tolerated. We present a consanguineous couple who experienced four pregnancy terminations due to repeated findings in the fetuses comprising enlarged cerebral ventricles and limb contractures. Exome sequencing in two of the aborted fetuses revealed a shared homozygous frameshift variant in exon 24 in KIDINS220. Sanger sequencing of the variant in available family members showed complete segregation with the affection status, resulting in a LOD score of 2.5 under an autozygous inheritance model. mRNA studies revealed destruction of the original splice site, resulting in an out-of-frame transcript and introduction of a premature termination codon in exon 25. Premature termination codons in this position are likely to cause activation of nonsense-mediated mRNA decay and result in complete absence of KIDINS220 protein in individuals homozygous for the variant. The phenotype of the presented fetuses overlaps with findings in functional studies of knockout Kidins220 mice embryos that are non-viable with enlarged cerebral ventricles. The human fetuses also exhibit several similarities to the milder phenotype described in patients with heterozygous KIDINS220 mutations. We hence propose that the identified homozygous loss-of-function variant in KIDINS220 causes the phenotype in the presented fetuses, and that this represents a hitherto undescribed severe autosomal recessive neurodevelopmental disorder.

Position of premature termination codons determines susceptibility of hERG mutations to nonsense-mediated mRNA decay in long QT syndrome

The degradation of human ether-a-go-go-related gene (hERG, KCNH2) transcripts containing premature termination codon (PTC) mutations by nonsense-mediated mRNA decay (NMD) is an important mechanism of long QT syndrome type 2 (LQT2). The mechanisms governing the recognition of PTC-containing hERG transcripts as NMD substrates have not been established. We used a minigene system to study two frameshift mutations, R1032Gfs*25 and D1037Rfs*82. R1032Gfs*25 introduces a PTC in exon 14, whereas D1037Rfs*82 causes a PTC in the last exon (exon 15). We showed that R1032Gfs*25, but not D1037Rfs*82, reduced the level of mutant mRNA compared to the wild-type minigene in an NMD-dependent manner. The deletion of intron 14 prevented degradation of R1032Gfs*25 mRNA indicating that a downstream intron is required for NMD. The recognition and elimination of PTC-containing transcripts by NMD required that the mutation be positioned >54–60nt upstream of the 3′-most exon–exon junction. Finally, we used a full-length hERG splicing-competent construct to show that inhibition of downstream intron splicing by antisense morpholino oligonucleotides inhibited NMD and rescued the functional expression of a third LQT2 mutation, Y1078*. The present study defines the positional requirements for the susceptibility of LQT2 mutations to NMD and posits that the majority of reported LQT2 nonsense and frameshift mutations are potential targets of NMD.

O.14 Optimization and evaluation of new read-through compounds (RTCs) for the treatment of Duchenne Muscular Dystrophy (DMD)

Approximately 13% of mutations causing DMD are due to premature termination codons (PTCs) that lead to degradation of the mRNA template and to the production of a non-functional, truncated polypeptide. Using a luciferase-independent high-throughput screening assay we have identified novel RTCs capable of suppressing PTCs and demonstrated their ability to suppress the mdx UAA premature termination codon in exon 23 of the dystrophin gene. Among those, RTC13 was shown to restore dystrophin expression into skeletal muscles of mdx mice more efficiently than gentamicin or PTC124 suggesting that this compound may have great therapeutic potential for the treatment of DMD. When administered systemically, RTC13 was shown to restore significant amounts of dystrophin in different muscle groups, including diaphragm and heart. Improved muscle strength was detected in all treated animals and was accompanied by a significant decrease in creatine kinase (CK) levels. Structure activity relationship (SAR) studies were used to optimize the molecular structure of RTC13 and to identify a derivative that meets optimal safety profiles while still maintaining maximal read-through activity. Pharmacokinetics, biodistribution and safety profiles were assessed after oral delivery of the compound and to determine the effective dose to be administered to achieve sustained effects. Dystrophin expression was assessed in mdx mice subjected to a 4-week oral regimen and correlated to functional and morphological analyses of muscle. No signs of toxicity were detected in mdx after prolonged administration of RTC13 demonstrating that the compound was well tolerated in mice. These results demonstrate that RTC13 is an ideal candidate for the treatment of DMD due to nonsense mutations as well as other forms of muscular dystrophies characterized by PTCs.

In Vitro and Ex Vivo Investigation of Aberrant and Alternative Splicing of Von Willebrand Factor.

AbstractAbstract 2179Von Willebrand disease (VWD) is the most commonly inherited bleeding disorder with a symptomatic prevalence of 1:1000. Type 1 VWD comprises ∼80% of VWD cases and is defined by mild to moderate deficiencies of the large multimeric glycoprotein von Willebrand factor (VWF). Sequence variation in consensus splice sites is a common culprit in the induction of aberrant splicing, and occurs in approximately 10% of type 1 VWD cases. Investigation of the VWF mRNA is required in order to determine whether these sequence variants are causative mutations and the manner in which they affect splicing. In this study, aberrant VWF splicing from a consensus splice site mutation was investigated using mRNA from patient platelets and patient-derived blood outgrowth endothelial cells (BOEC). Both aberrant and alternative VWF splice forms were identified and their effects were further characterized through expression studies in HEK293T cells as well as further investigation of the patient BOEC.Patient T152 presented with a type 1 VWD phenotype: VWF:Ag 0.49IU/mL, VWF:RCo 0.42IU/mL, FVIII:C 1.16IU/mL and an abnormal bleeding score of 8. Direct sequencing of the patient‘s VWF gene revealed c.5842+1G>C in intron 34. This variation was thought to cause the type 1 VWD phenotype through aberrant splicing. Reverse transcription of the VWF mRNA detected different splice variants within the platelet and BOEC RNA. Wildtype VWF mRNA as well as transcripts encoding in-frame double exon skipping of exons 33 and 34 were found in both RNA sources. An additional VWF transcript which skipped exon 33 and introduced a premature termination codon in exon 34 was present in the BOEC RNA. Sequencing of this transcript determined it was produced from the patient’s normal allele and may be a product of alternative splicing. qRT-PCR quantification determined the patient VWF mRNA to be 51±5% wildtype, 28±4% exon33–34 skipped and 21±9% exon 33 skipped transcripts. The skipping of exon 33 was confirmed as an alternative transcript comprising 12±8% of VWF mRNA in 4 normal individuals.VWF expression vectors were created through site-directed mutagenesis for all three of these transcripts. Calcium-phosphate mediated transfections of these mutant vectors into HEK293T cells with varying wildtype:mutant ratios (100:0, 90:10, 75:25, 50:50, 0:100) for both mutant transcripts, as well as in the biological ratio (51:28:21) determined as above from the patient qRT-PCR. The alternative exon 33 skipping transcript does not decrease VWF secretion compared to wildtype (p>0.5). The presence of even 10% of the double exon skipping mutant transcript decreased secretion of VWF to 62± 1%(p<0.05) of wildtype, demonstrating a dominant negative effect. Similar to the plasma VWF:Ag level of 0.49 IU/mL, VWF secretion decreased to 46± 8% (p<0.01) when transfected at biological mRNA ratios. When stimulated with PMA, the transfections mimicking the patient biological VWF mRNA ratio increased VWF secretion 22±10% which approximates the patient’s clinical DDAVP response. A similar degree of increase was seen in PMA stimulated wildtype transfections. Multimer analysis of concentrated transfection media showed that the in-frame exon 33–34 is normally multimerized, which is consistent with the patient plasma multimers.BOEC isolated from the patient enabled analysis of these VWF mutants ex vivo. Patient BOEC expressed 48±10% (p<0.01) less VWF relative to BOEC from normal individuals. Upon stimulus by thrombin and PMA for 24 hours, normal BOEC showed a 28±13% and 93±70% increase in VWF secretion respectively. Similar increases in secretion (35±11% and 120±32% respectively) were seen with T152’s stimulated BOEC. Immunofluorescent staining of these BOEC showed abnormal morphology of Weibel-Palade bodies (WPBs). WPBs are characteristically cigar shaped; however, the patient’s WPBs appear shortened and rounded relative to their normal counterparts.We therefore conclude that the consensus splice site mutation c.5842+1G>C causes aberrant double exon skipping of exons 33 and 34 in this individual and is responsible for type 1 VWD through a dominant negative decrease in secretion. Additionally, the presence of VWF lacking exons 33 and 34 also affects the formation of WPBs. The alternative transcript which skips exon 33 was found to have no effect on VWF secretion and may be degraded by nonsense mediated decay. Disclosures:James:CSL-Behring, Baxter, Bayer: Honoraria, Research Funding.

Human Adipose-Derived Mesenchymal Stromal Cells Injected Systemically into GRMD Dogs without Immunosuppression are Able to Reach the Host Muscle and Express Human Dystrophin

Duchenne muscular dystrophy (DMD), a lethal X-linked disorder, is the most common and severe form of muscular dystrophies, affecting 1 in 3,500 male births. Mutations in the DMD gene lead to the absence of muscle dystrophin and a progressive degeneration of skeletal muscle. The possibility to treat DMD through cell therapy has been widely investigated. We have previously shown that human adipose-derived stromal cells (hASCs) injected systemically in SJL mice are able to reach and engraft in the host muscle, express human muscle proteins, and ameliorate the functional performance of injected animals without any immunosuppression. However, before starting clinical trials in humans many questions still need to be addressed in preclinical studies, in particular in larger animal models, when available. The best animal model to address these questions is the golden retriever muscular dystrophy (GRMD) dog that reproduces the full spectrum of human DMD. Affected animals carry a mutation that predicts a premature termination codon in exon 8 and a peptide that is 5% the size of normal dystrophin. These dogs present clinical signs within the first weeks and most of them do not survive beyond age two. Here we show the results of local and intravenous injections of hASCs into GRMD dogs, without immunosuppression. We observed that hASCs injected systemically into the dog cephalic vein are able to reach, engraft, and express human dystrophin in the host GRMD dystrophic muscle up to 6 months after transplantation. Most importantly, we demonstrated that injecting a huge quantity of human mesenchymal cells in a large-animal model, without immunosuppression, is a safe procedure, which may have important applications for future therapy in patients with different forms of muscular dystrophies.

A splicing variant leads to complete loss of function of betaine–homocysteine methyltransferase (BHMT) gene in hepatocellular carcinoma

The remethylation of homocyteine into methionine is catalyzed either by methionine synthase (MTR) or by betaine–homocysteine methyltransferase (BHMT), in the liver. Choline/betaine deficiency and impaired BHMT pathway have been associated with hepatocellular carcinogenesis, in animal models. The molecular mechanisms that impair the BHMT pathway are unknown. We aimed to investigate BHMT, BHMT2, and MTR expression in HepG2 cells and human hepatocarcinoma tissues. Transcripts were quantified by RT-qPCR and splicing was assessed by analysis of exon junctions and sequencing of variants. Protein expression was studied by Western Blot, immunohistochemistry and enzyme activity. Tumor tissue was compared with surrounding healthy tissue. RT-qPCR of HepG2 cells and of tumor samples showed a strong decrease of transcripts of BHMT and BHMT2, compared to normal. MTR transcript levels were not different. The decreased BHMT expression resulted from the transcription of a splicing variant that produced a frameshift in exon 4, with a premature termination codon in exon 5 and a loss of function of the gene. This splicing variant did not fit with any mechanism resulting from known splicing consensus sequences and was not detected in normal adult and fetal liver. Consistently, BHMT activity was abolished in HepG2 and protein expression was not detectable in HepG2 and in 5 of the 6 tumor samples, compared to normal tissues. In conclusion, a transcription variant of exon 4 produces a loss of function of BHMT in human hepatocarcinoma. Whether this abnormal transcription of BHMT is part or consequence of liver carcinogenesis should deserve further investigations.

Luminal expression of cubilin is impaired in Imerslund-Grasbeck syndrome with compound AMN mutations in intron 3 and exon 7

Juvenile megaloblastic anaemia 1 (OMIM # 261100) is a rare autosomic disorder characterized by selective cobalamin mal-absorption and inconstant proteinuria produced by mutations in either CUBN or AMN genes. Amnionless, the gene product of AMN, is a transmembrane protein that binds tightly to the N-terminal end of cubilin, the gene product of CUBN. Cubilin binds to intrinsic factor-cobalamin complex and is expressed in the distal intestine and the proximal renal tubule. We report a compound AMN heterozygosity with c.742C>T, p.Gln248X and c.208-2A>G mutations in 2 siblings that led to premature termination codon in exon 7 and exon 6, respectively. It produced a dramatic decrease in receptor activity in urine, despite absence of CUBN mutation and normal affinity of the receptor for intrinsic factor binding. Heterozygous carriers for c.742T and c.208-2G had no pathological signs. These results indicate that amnionless is essential for the correct luminal expression of cubilin in humans.

Identification and Characterization of L1-Mediated Large Tandem Duplication, Spanning Exon 4 to 10 of the F13A1, In a Patient with Congenital Factor XIII Deficiency.

AbstractAbstract 3659Blood coagulation factor XIII (FXIII) is a plasma glycoprotein that plays an important role in the stabilization of fibrin clot in the final stage of blood coagulation. The FXIII circulates as a heterotetramer composed of two A and two B subunits in plasma. The A subunit (FXIIIA) possesses catalytic activity and this catalytic subunit is carried and protected by the B subunit (FXIIIB). Inherited deficiency of FXIII is a rare autosomal recessive bleeding disorder. Based on the genotype, it is classified into two types: FXIIIA deficiency (>95% of all cases), characterized by mutations in the F13A1 gene, and FXIIIB deficiency, characterized by mutations in the F13B gene. At the time of writing, at least 86 different mutations, most of which are point mutations, have been identified and registered in the F13-database. Here we show a novel, large tandem duplication in the F13A1 gene of a patient with congenital factor XIII deficiency. A female patient, born to consanguineous parents, suffered from severe bleeding diathesis, including menorrhagia, intracranial hemorrhage and ovarian hemorrhage, from childhood. Bleeding manifestations had been successfully controlled by monthly prophylactic replacement therapy using factor XIII concentrate (Fibrogammin). Trough levels of both factor XIII activity and antigen were 19% (Berichrom FXIII) and <10% (ELISA method), respectively. No pathogenic mutations associated with FXIII deficiency were detected from nucleotide sequencing of the coding region, 5′-UTR and 3′-UTR of both F13A1 and F13B. However, from an observation of the RT-PCR amplification state, the F13A1 mRNA level of the patient was apparently lower than that of healthy individuals. This result suggested the existence of abnormalities in the patient's F13A1. Relative exon copy number analysis using real-time PCR revealed two times as many of the continuing 7 exons (exon 4–10) as in the remaining region of the F13A1. This is likely due to some genomic rearrangement, probably homologous recombination in the F13A1 gene. IVS-3 and IVS-10 of F13A1 were very large and contained many repetitive sequences. The existence of an almost full-length (≂f6kb) L1 element, a well known long interspersed repetitive element (LINE) in humans, in both introns suggests that the recombination might arise from the L1 element. The provision of the PCR product (amplified by an IVS-10 specific forward primer and an IVS-3 specific reverse primer) confirmed that IVS-10 connected to IVS-3 with L1 as the boundary. Furthermore, sequencing this PCR product identified the 15bp sequence in the L1 element as an actual breakpoint. Taken together with the results of the analysis of the genomic DNA, this confirmed that the L1-mediated large (≂f109kb) tandem duplication was located in the patient's F13A1. In order to quantify the F13A1 mRNA, a relative real-time PCR quantification was performed using TaqMan Gene Expression Assays. Three different positions: one upstream of duplication (Exon2-3), one at duplication (Exon5-6) and one downstream of the duplication (Exon13-14) were used. The mRNA level of the patient was a markedly low compared to the normal control and the duplicated region (28% of normal) was clearly higher than both the upstream and downstream positions (9% of normal for each). This result reflected that the mRNA was probably maintaining the duplication. In order to analyze the mRNA splicing of the joint between the two duplicated regions, RT-PCR using a forward primer in exon 9 and a reverse primer in exon 4 of F13A1 was performed. Two major transcripts were amplified. The larger transcript was the product that keeps the genomic exon order and connects exon 10 to 4. However, the transcript is thought to lead to frameshift and to generate premature termination codon in exon 4. The other was the product that was skipping exon 10 and connected exon 9 to 4. This transcript is thought to escape frameshift and may translate to the unusual extra large FXIIIA. However, it is unlikely that the protein translated from the extra large mRNA circulates in blood. In conclusion, we identified an L1-mediated large tandem duplication, spanning exon 4 to 10 of the F13A1 gene, as an etiology of the congenital FXIII deficiency. Disclosures:No relevant conflicts of interest to declare.

Frontotemporal dementia in a large Swedish family is caused by a progranulin null mutation

Mutations in the progranulin (PGRN) gene have recently been identified in families with frontotemporal lobar degeneration and ubiquitin-positive brain inclusions linked to chromosome 17q21. We have previously described a Swedish family displaying frontotemporal dementia with rapid progression and linkage to chromosome 17q21. In this study, we performed an extended clinical and neuropathological investigation of affected members of the family and a genetic analysis of the PGRN gene. There was a large variation of the initial presenting symptoms in this family, but common clinical features were non-fluent aphasia and loss of spontaneous speech as well as personality and behavioural changes. Mean age at onset was 54 years with disease duration of close to 4 years. Neuropathological examination revealed frontotemporal neurodegeneration with ubiquitin and TAR DNA binding protein-43 immunoreactive intraneuronal inclusions. Mutation screening of the PGRN gene identified a 1 bp deletion in exon 1 causing a frameshift of the coding sequence and introducing a premature termination codon in exon 2 (Gly35GlufsX19). Analysis of PGRN messenger RNA (mRNA) levels revealed a considerable decrease in lymphoblasts from mutation carriers and fragment size separation, and sequence analysis confirmed that the mutated mRNA allele was almost absent in these samples. In conclusion, the PGRN Gly35fs mutation causes frontotemporal dementia with variable clinical presentation in a large Swedish family, most likely through nonsense-mediated decay of mutant PGRN mRNA and resulting haploinsufficiency.

Sustained Dystrophin Expression Induced by Peptide-conjugated Morpholino Oligomers in the Muscles of mdx Mice

Cell-penetrating peptides (CPPs), containing arginine (R), 6-aminohexanoic acid (X), and/or beta-alanine (B) conjugated to phosphorodiamidate morpholino oligomers (PMOs), enhance their delivery in cell culture. In this study, the potency, functional biodistribution, and toxicity of these conjugates were evaluated in vivo, in EGFP-654 transgenic mice that ubiquitously express the aberrantly spliced EGFP-654 pre-mRNA reporter. Correct splicing and enhanced green fluorescence protein (EGFP) upregulation serve as a positive readout for peptide-PMO (PPMO) entry into cells and access to EGFP-654 pre-mRNA in the nucleus. Intraperitoneal injections of a series of PPMOs, A-N (12 mg/kg), administered once a day for four successive days resulted in splicing correction in numerous tissues. PPMO-B was highly potent in the heart, diaphragm, and quadriceps, which are key muscles in the treatment of Duchenne muscular dystrophy. We therefore investigated PPMO M23D-B, designed to force skipping of stop-codon containing dystrophin exon 23, in an mdx mouse model of the disease. Systemic delivery of M23D-B yielded persistent exon 23 skipping, yielding high and sustained dystrophin protein expression in body-wide muscles, including cardiac muscle, without detectable toxicity. The rescued dystrophin reduced serum creatinine kinase to near-wild-type levels, indicating improvement in muscle integrity. This is the first report of oligonucleotide-mediated exon skipping and dystrophin protein induction in the heart of treated animals.

Genotype-phenotype correlations in MYCN-related Feingold syndrome

Feingold syndrome (FS) is the most frequent cause of familial syndromic gastrointestinal atresia and follows autosomal dominant inheritance. FS is caused by germline mutations in or deletions of the MYCN gene. Previously, 12 different heterozygous MYCN mutations and two deletions containing multiple genes including MYCN were described. All these mutations result in haploinsufficiency of both the canonical MYCN protein and the shorter isoform, DeltaMYCN. We report 11 novel mutations including seven mutations in exon 2 that result in a premature termination codon (PTC) in the long MYCN transcript. Moreover, we have identified a PTC in exon 1 that only affects the DeltaMYCN isoform, without a phenotypic effect. This suggests that mutations in only DeltaMYCN do not contribute to the FS. Additionally, we found three novel deletions encompassing MYCN. Together with our previous report we now have a total of four missense mutations in the DNA binding domain, 19 PTCs of which six render the transcript subject to nonsense-mediated decay (NMD), and five larger deletions in a total of 77 patients. We have reviewed the clinical features of these patients, and found that digital anomalies, e.g., brachymesophalangy and toe syndactyly, are the most consistent features, present in 100% and 97% of the patients, respectively. Small head circumference was present in 89% of the cases. Gastrointestinal atresia remains the most important major congenital anomaly (55%), but cardiac and renal anomalies are also frequent. We suggest that the presence of brachymesophalangy and toe syndactyly in combination with microcephaly is enough to justify MYCN analysis.

Molecular dissection of ALS-associated toxicity of SOD1 in transgenic mice using an exon-fusion approach

Mutations in Cu,Zn superoxide dismutase (SOD1) are associated with amyotrophic lateral sclerosis (ALS). Among more than 100 ALS-associated SOD1 mutations, premature termination codon (PTC) mutations exclusively occur in exon 5, the last exon of SOD1. The molecular basis of ALS-associated toxicity of the mutant SOD1 is not fully understood. Here, we show that nonsense-mediated mRNA decay (NMD) underlies clearance of mutant mRNA with a PTC in the non-terminal exons. To further define the crucial ALS-associated SOD1 fragments, we designed and tested an exon-fusion approach using an artificial transgene SOD1T116X that harbors a PTC in exon 4. We found that the SOD1T116X transgene with a fused exon could escape NMD in cellular models. We generated a transgenic mouse model that overexpresses SOD1T116X. This mouse model developed ALS-like phenotype and pathology. Thus, our data have demonstrated that a ‘mini-SOD1’ of only 115 amino acids is sufficient to cause ALS. This is the smallest ALS-causing SOD1 molecule currently defined. This proof of principle result suggests that the exon-fusion approach may have potential not only to further define a shorter ALS-associated SOD1 fragment, thus providing a molecular target for designing rational therapy, but also to dissect toxicities of other proteins encoded by genes of multiple exons through a ‘gain of function’ mechanism.

Mutations in the hairless gene underlie APL in three families of Pakistani origin

Atrichia with papular lesions (APL) (OMIM#209500) is a rare autosomal recessively inherited form of irreversible alopecia characterized by papular lesions of keratin-filled cysts on various regions of the body. Males and females are equally affected and present with a distinct pattern of total hair loss on scalp, axilla and body. It begins shortly after birth with the development of hair loss, and patients are normally devoid of eyelashes and eyebrows. Mutations in the hairless (HR) gene have been previously shown to be responsible for APL. In this study, we studied the molecular basis of APL in three unrelated families of Pakistani origin. Molecular analysis of the HR genes was performed on genomic DNA from probands and family members. DNA sequencing of the HR gene in family A revealed a novel homozygous 2bp deletion in exon 6 leading to a frameshift and a downstream premature termination codon in exon 8 (1782-83delAG). In family B, we identified a novel homozygous deletion of a G nucleotide at the exon 15-intron 15 boundary, termed 3097delG. Family C carries a previously reported missense mutation consisting of an A-to-G transition at nucleotide 276 resulting in the mutation N970S in exon 14. Two mutations identified in this study are novel mutations in the HR gene and extend the body of evidence implicating the hairless gene family in the pathogenesis of human skin disorders. The one previously reported mutation suggests it may represent a recurrent mutation, or alternatively, an allele that is widely dispersed around the world.