Splicing Mutation In Intron Research Articles

Further evidence for GRIN2B mutation as the cause of severe epileptic encephalopathy

Epileptic encephalopathies (EE) include a range of severe epilepsies in which intractable seizures or severe sub-clinical epileptiform activity are accompanied by impairment of motor and cognitive functions. Mutations in several genes including ion channels and other genes whose function is not completely understood have been associated to some EE. In this report, we provide a detailed clinical description of a sporadic male patient with early-onset epilepsy and epileptic encephalopathy in whom we performed complete exome sequencing (WES) and identified a GRIN2B mutation. The GRIN2B splicing mutation in intron 10 (c.2011-1G>A) was revealed in a WES study. The result was confirmed by Sanger sequencing. No mutation was found in both parents. Our finding confirms that early-onset EE may be caused not only by gain-of-function variants but also by splice site mutations-in particular those affecting the splice acceptor site of the 10th intron of the GRIN2B gene. © 2016 Wiley Periodicals, Inc.

Next-generation sequencing of hereditary hemochromatosis-related genes: Novel likely pathogenic variants found in the Portuguese population

Hereditary hemochromatosis (HH) is an autosomal recessive disorder characterized by excessive iron absorption resulting in pathologically increased body iron stores. It is typically associated with common HFE gene mutation (p.Cys282Tyr and p.His63Asp). However, in Southern European populations up to one third of HH patients do not carry the risk genotypes.This study aimed to explore the use of next-generation sequencing (NGS) technology to analyse a panel of iron metabolism-related genes (HFE, TFR2, HJV, HAMP, SLC40A1, and FTL) in 87 non-classic HH Portuguese patients.A total of 1241 genetic alterations were detected corresponding to 53 different variants, 13 of which were not described in the available public databases. Among them, five were predicted to be potentially pathogenic: three novel mutations in TFR2 [two missense (p.Leu750Pro and p.Ala777Val) and one intronic splicing mutation (c.967-1G>C)], one missense mutation in HFE (p.Tyr230Cys), and one mutation in the 5′-UTR of HAMP gene (c.-25G>A).The results reported here illustrate the usefulness of NGS for targeted iron metabolism-related gene panels, as a likely cost-effective approach for molecular genetics diagnosis of non-classic HH patients. Simultaneously, it has contributed to the knowledge of the pathophysiology of those rare iron metabolism-related disorders.

Variable clinical phenotypes of X-linked lymphoproliferative syndrome in China: Report of five cases with three novel mutations and review of the literature

BackgroundX-linked lymphoproliferative disease (XLP) is a rare life-threatening syndrome. Rapid recognition and definitive diagnosis are critical to improve the prognosis and survival of patients with XLP. Nowadays, little is known about patients with XLP in China. MethodsWe report the characterization of five Chinese XLP patients with three novel mutations and review the literature related to this syndrome. Male patients with fulminant infectious mononucleosis (FIM), Epstein-Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) or persistent EBV viraemia were enrolled in this study. The patients’ clinical features were assessed by retrieval of data from medical records. Immunological function included analysis of lymphocyte subsets and the detection of immunoglobulins G, A, M and/or E were evaluated by flow cytometry and nephelometry. Direct sequencing was used to detect SH2D1A/XIAP gene mutations. ResultsTwenty-two male patients with FIM, EBV-associated HLH or persistent EBV viraemia were evaluated among 421 PID patients in our centre. Four patients had SH2D1A mutations, and one patient had an XIAP mutation. The onset age of the 5 patients range from 1month to 4years which was earlier than that in the western world. The diagnosis age was between 16months and 9years with a long diagnosis lag (1–97months). Two of them had positive family history. The clinical phenotypes varied in different patients among which two patients with FHLH and hypogammaglobulinaemia, one with hypogammaglobulinaemia, lymphoma and aplastic anaemia (AA) which is the first case with AA in China, one with hypogammaglobulinaemia only and the other one with FHLH. For immunological function, three exhibited reduced CD4/CD8 ratios. Arg55stop mutations as well as splice mutation in intron 1 were most frequently found and exon 2 was the hottest exon in China. Two patients died at the time of diagnosis for severe infection or hepatic coma. Three were alive and waiting for haematopoietic stem cell transplantation (HSCT). ConclusionFor patients with severe EBV-associated HLH, hypogammaglobulinaemia, lymphoma and aplastic anaemia, possibility of XLP should be considered and if confirmed, HSCT should be performed as soon as possible.

329. Using CRISPR/Cas9 as a Therapeutic Approach for Leber Congenital Amaurosis 10 (LCA10)

Introduction: Leber congenital amaurosis (LCA) is the most severe form of inherited retinal diseases with early onset of symptoms in the first year of life. The most frequent genetic cause of LCA, accounting for approximately 15% of all LCA cases in western countries, is a deep-intronic mutation c. 2991+1655A>G located in the intron 26 of human CEP290 gene. LCA caused by CEP290 mutation is known as LCA10. The intronic mutation of CEP290 generates a cryptic splice donor site, resulting in the inclusion of a pseudoexon that leads to a premature stop codon and a truncated protein. The size of human CEP290 cDNA (~7.4 kb) exceeds the cargo size (~4.8 kb) of recombinant adeno-associated viral vectors (rAAVs), which makes this gene challenging for gene replacement therapy. In this regard, we tested if we can use the new genome editing technology CRISPR/Cas9 as an alternative strategy for LCA10 by removing the intronic mutation of CEP290 and preventing the cryptic splicing.Methods: CRISPR/SpCas9 was employed to introduce the intronic splice mutation c. 2991+1655 A>G into HEK 293FT cells to create a cellular model for LCA10/CEP290. In this cellular model, we used guide RNA pairs coupled with SpCas9 to delete the intronic region flanking the intronic mutation.Results: The deep-intronic mutation c. 2991+1655 A>G was successfully introduced into 293FT cells through the homology directed repair (HDR) pathway of CRISPR/spCas9. CEP290 expression levels were markedly reduced in the mutant cell line. Using this cellular model, we identified three sgRNA pairs that could efficiently (>50%) delete the intronic mutation, significantly rescue wild-type CEP290 expression levels and reduce mutant CEP290 expression.Conclusions: Our results demonstrate that the paired sgRNAs coupled with SpCas9 are capable of removing the c. 2991+1655A>G mutation and are highly efficient in preventing the splicing of the mutant cryptic exon and restoring wild-type CEP290 expression.

Mutations in ATP6V1B1 and ATP6V0A4 genes cause recessive distal renal tubular acidosis in Mexican families.

BackgroundAutosomal recessive distal renal tubular acidosis (dRTA) is a rare disease characterized by a hyperchloremic metabolic acidosis with normal anion gap, hypokalemia, hypercalciuria, hypocitraturia, nephrocalcinosis, and conserved glomerular filtration rate. In some cases, neurosensorial deafness is associated. dRTA is developed during the first months of life and the main manifestations are failure to thrive, vomiting, dehydration, and anorexia.MethodsNine unrelated families were studied: seven children, a teenager, and an adult with dRTA. Hearing was preserved in four children. Coding regions of the genes responsible for recessive dRTA were analysed by Sanger sequencing.ResultsMolecular defects were found in the genes ATP6V1B1 and ATP6V0A4. We identified three homozygous variants in ATP6V1B: a frameshift mutation (p.Ile386Hisfs*56), a nucleotide substitution in exon 10 (p.Pro346Arg), and a new splicing mutation in intron 5. Three patients were homozygous for one novel (p.Arg743Trp) and one known (p.Asp411Tyr) missense mutations in the ATP6V0A4 gene. Three patients were compound heterozygous: one proband displayed two novel mutations, the frameshift mutation p.Val52Metfs*25, and a large deletion of exons 18–21; two probands showed the missense mutation p.Asp411Tyr and as a second mutation, p.Arg194Ter and c.1691+2dup, respectively.Conclusion ATP6V0A4 and ATP6V1B1 genes were involved in recessive dRTA of Mexican families. All ATP6V1B1 mutations detected were homozygous and all patients developed sensorineural hearing loss (SNHL) early in infancy. ATP6V0A4 mutations were found in one infant and three children without SNHL, and in one teenager and one adult with SNHL confirming the phenotypic variability in this trait. The mutation p.Asp411Tyr detected in four Mexican families was due to a founder effect. Screening of these mutations could provide a rapid and valuable tool for diagnosis of dRTA in this population.

Sensory and autonomic deficits in a new humanized mouse model of familial dysautonomia.

Familial dysautonomia (FD) is an autosomal recessive neurodegenerative disease that affects the development and survival of sensory and autonomic neurons. FD is caused by an mRNA splicing mutation in intron 20 of the IKBKAP gene that results in a tissue-specific skipping of exon 20 and a corresponding reduction of the inhibitor of kappaB kinase complex-associated protein (IKAP), also known as Elongator complex protein 1. To date, several promising therapeutic candidates for FD have been identified that target the underlying mRNA splicing defect, and increase functional IKAP protein. Despite these remarkable advances in drug discovery for FD, we lacked a phenotypic mouse model in which we could manipulate IKBKAP mRNA splicing to evaluate potential efficacy. We have, therefore, engineered a new mouse model that, for the first time, will permit to evaluate the phenotypic effects of splicing modulators and provide a crucial platform for preclinical testing of new therapies. This new mouse model, TgFD9; Ikbkap(Δ20/flox) was created by introducing the complete human IKBKAP transgene with the major FD splice mutation (TgFD9) into a mouse that expresses extremely low levels of endogenous Ikbkap (Ikbkap(Δ20/flox)). The TgFD9; Ikbkap(Δ20/flox) mouse recapitulates many phenotypic features of the human disease, including reduced growth rate, reduced number of fungiform papillae, spinal abnormalities, and sensory and sympathetic impairments, and recreates the same tissue-specific mis-splicing defect seen in FD patients. This is the first mouse model that can be used to evaluate in vivo the therapeutic effect of increasing IKAP levels by correcting the underlying FD splicing defect.

The Spectrum of Alpha-Spectrin Associated Hereditary Spherocytosis

▪Hereditary spherocytosis (HS) is a genetically and phenotypically heterogeneous hemolytic anemia caused by deficiency in red blood cell (RBC) cytoskeleton proteins leading to disruptions in the vertical association of the cytoskeleton with the RBC lipid bilayer. Monoallelic mutations in the genes encoding ankyrin (ANK1), beta-spectrin (SPTB) and band 3 (SLC4A1) or biallelic mutations in the genes encoding alpha-spectrin (SPTA1), ankyrin, and protein 4.2 (EPB42) result in HS. Autosomal recessive HS due to compound heterozygous defects in SPTA1 is typically severe and diagnosis based on phenotypic assays like RBC morphology, osmotic fragility or ektacytometry is complicated by transfusion dependence resulting in most of the circulating RBCs to be of donor origin. We have developed a rapid comprehensive next-generation sequencing-based assay that evaluates 27 genes with published disease-causing mutations for RBC cytoskeletal disorders, enzymopathies, and CDAs. We describe here patients with hemolytic anemia due to SPTA1 mutations, identified utilizing this assay, and their phenotype-genotype correlation. Each of these cases, when possible, has been also evaluated with ektacytometry and immunoblotting of RBC ghosts for alpha-spectrin quantitation.Wichterle et al in 1996 had estimated that alphaLEPRA(Low Expression PRAgue) mutation (c.4339-99C>T) occurs in SPTA1 gene in about 5% of Caucasians. This mutation leads to activation of an alternate acceptor splice site at position -70 of intron 30, causing frame shift and premature termination, thereby leading to decrease in alpha-spectrin production in this allele to about 16% of normal. We have found a cohort of three transfusion-dependent hereditary hemolytic anemia cases where a nonsense mutation in SPTA1 gene has occurred in trans to alphaLEPRA mutation, resulting in premature termination (see Table 1). Transfusion dependence was alleviated in two of these patients after splenectomy; the third one did not have splenectomy yet. RBC phenotype explored after splenectomy revealed an ektacytometry curve indicating spherocytosis (Figure 1A) and severely decreased alpha-spectrin on immunoblotting along with significant decrease of the associated beta-spectrin (Figure 1B).A patient with moderately severe form of HS, maintaining a hemoglobin value greater than 7 g/dL and requiring only occasional transfusions during periods of illness or stress, was found to have alphaLEPRA occurring in trans to an intronic splicing mutation c.1351-1G>TG where there is substitution at nucleotide-1 of intron positioned between nucleotides 1350 and 1351 of the SPTA1 mRNA. This splicing mutation may allow for some expression of functional alpha-spectrin protein from this allele in contrast to no protein expression in the previous cases of premature termination. Alternatively, other gene mutations, not identified by the next-generation sequencing panel we used, may contribute to this patient's milder phenotype.A couple with history of two fetal losses associated with hydrops fetalis seeked genetic counseling and gave consent to have diagnostic evaluation of genes associated with non-immune hemolytic anemia using targeted next-generation sequencing. Results of the panel revealed a heterozygous frameshift SPTA1 mutation in each of the parents (c.4206delG in the father and c.4180delT in the mother). These mutations in compound heterozygous state in the offspring likely caused total absence of alpha spectrin and fatal hemolytic anemia by the time of birth.Hereditary Spherocytosis is characterized by wide phenotypic variability that will be better understood with studies of genotype-phenotype association. While complete absence of alpha-spectrin expression due to null mutations of both SPTA1 alleles is incompatible with life, a nonsense or splicing SPTA1 mutation in trans to an alphaLEPRA low expression allele causes severe or moderately severe recessive HS, respectively. Targeted next-generation sequencing can be an effective diagnostic tool particularly for patients requiring frequent transfusions that preclude meaningful phenotypical testing of their red blood cells. [Display omitted] [Display omitted] DisclosuresBegtrup:GeneDx: Employment.

Prevalence of mutations nt656g and V281L in indigenous children of Russian Far North

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders which is most often caused by deficiency of steroid 21-hydroxylase (CYP-21). High level of inbreeding in the populations of indigenous people of Russian Far North was the reason to assess the prevalence of CYP21-inactivating mutations nt656g (classical sodium wasting phenotype of CAH) and V281L (non-classical CAH) and the genotype-phenotype relationship in children-inhabitants of Yakutia and Yamal-Nenets Autonomous Region (YNAR). We examined children living in 9 settlements of Yakutia and in 4 settlements of YNAR: 65 Russians, 206 Sakha, 344 Evens, 88 Evenks, 34 Yukagirs, 7 Chukchi, 99 Dolgans, 41 Nenets, 137 Selkups, 21 others. Blood samples were drawn from 315 children living in Yakutia and from 176 children living in YNAR. Allele specific PCR was used (primers In2ns, In2ms, In2cs for mutation in intron 2 (656) A/C → G and primer Ex7ma for mutation in exon 7 (V281L)) with positive control DNA. In cases of positive reaction the procedure was repeated and if it was again positive sequencing was performed. We didn’t find homozygotes. Twelve heterozygotes have been revealed with mutations at CYP21 gene. Of them splicing mutation in intron 2 (nucleotide 656) was revealed on 1 chromosome, mutation in exon 7 (V281L) - on 11 chromosomes. Prevalence of mutation nt656g in Evens was 1:41 (2,4 : 100). In other ethnic groups it was not revealed. Hence on average for the whole studied group of children the prevalence of mutation nt656g was 0,4 : 100. The prevalence of mutation V281L was the highest in Chukchi (5,1 : 100) and Yukagirs (5 : 100) with average prevalence for the whole studied group 2,4 : 100. The prevalence of heterozygotes for the V281L mutation in whole studied group was found to be almost similar to the prevalence of heterozygotes for non-classical CAH in non-Ashkenazi population (1 : 60, Speiser P.W. et al., 1985). In Nenets and Selkup children living in YNAR the prevalence was half as high as the average for Native children in Yakutia.

10 Mild cystic fibrosis phenotype in adult patients with novel 3272–16T>A mutation

Objective Patients who have milder symptoms or atypical cystic fibrosis (CF) may have at least one so called class V mutation. This class includes mutations that leave residual levels of normal CFTR transcripts and protein. Recently by sequencing of the coding region of the CFTR gene and exon-intron junctions we've revealed 3272–16T>A mutation in Russian adult CF patients. As far as we know this mutation hasn't been described (before December 2014) by any other geneticists and it is not in the CFTR1 and CFTR2 lists. 3272–16T>A mutation is a splicing mutation in intron 19 of CFTR gene (thymine to adenine substitution). Aim To reveal the clinical characteristics of 3272–16T>A mutation in CF adult patients. Methods Adult CF patients carrying 3272–16T>A mutation were included in the study. The following data were evaluated: the age at CF diagnosis, pancreatic status, the presence of the following complications: meconium ileus or DIOS, diabetes and liver cirrhosis. Results We've found 10 patients (6 men) carried 3272–16T>A mutation and the following mutations in the other CFTR allele: F508del – 6 patients, 394delTT – 2 patients, 2184insA – 1 patient, not identified – 1 patient. The average age of patients was 27.4±5.5 years (from 18.4 to 35.0 years). Age at diagnosis ranged from 3.5 to 26.9 years (12.3±7.0 yrs). All 10 were pancreatic sufficient. None of them had meconium ileus in anamnesis or DIOS, diabetes and liver cirrhosis. Conclusion 3272–16T>A mutation is a mild mutation associated with pancreatic sufficiency and lack of development of some complications specific to the classical CF genotypes.

Congenital hypofibrinogenemia associated with novel homozygous fibrinogen Aα and heterozygous Bβ chain mutations

We report the molecular characterisation of two novel cases of inherited hypofibrinogenemia. After sequencing all coding regions and intron-exon boundaries of the three fibrinogen genes (FGA, FGB, and FGG), two different novel mutations were found, one homozygous and one heterozygous. The first patient, with a mild bleeding history and mild discrepancy between functional and immunological fibrinogen, showed a novel homozygous nonsense mutation in exon 5 of FGA (p.Trp373*, p.Trp354* according to the mature protein) caused by a G>A transition at nucleotide position 1,119. The resulting truncation in the Aα chain is likely to reduce the efficiency of fibrinogen assembly and secretion. The second patient, referred after ischemic stroke (functional fibrinogen 77mg/dL), had a novel heterozygous splicing mutation in intron 5 of FGB (IVS5+2T>A or c.832+2T>A), which we demonstrated to cause either exon 5 skipping or the inclusion of 75bp belonging to intron 5. Neither splicing defect alters the reading frame: one results in a 38-residue deletion and the other in a 25-residue insertion in the D domain of fibrinogen Bβ chain. This report confirms that genetically determined partial deficiencies of fibrinogen with levels greater than 50mg/dL are rarely associated with significant bleeding symptoms and that homozygous null mutations removing a significant portion of the Aα chain may be associated with mild fibrinogen deficiency.

A Novel Deep Intronic Mutation Introducing a Cryptic Exon Causing Neurofibromatosis Type 1 in a Family with Highly Variable Phenotypes: A Case Study

Neurofibromatosis type 1 (NF1) is a common dominant inherited disorder with highly variable expressivity. Genetic testing for this condition has been more available the last decade. Here we present a case report of a NF1 family including seven affected family members, some of them with a very severe phenotype. When searching for a causative mutation in the NF1 gene, no mutation was found at DNA level. However, a misspliced transcript including a subsequence of intron 3 appeared when screening RNA. The underlying cause at DNA level was determined to be a deep intronic variant (c.288+1137C>T). This intronic point mutation creates a new splice site causing the insertion of a cryptic exon (r.288_289ins288+1018_1135), leading to reading frameshift at the protein level. Deep intronic mutations introducing a cryptic exon are known to be a cause of NF1, and we reviewed the literature to evaluate how common this mutation is in NF1 syndrome. We found 20 different deep intronic NF1 splice mutations, including the one found in the present study. In conclusion, this case illustrates the value of RNA analysis to detect the cause of genetic diseases, and we decided to use RNA based mutation screening as standard procedure for NF1 genetic testing in our laboratory.

A novel ABCD1 gene mutation in a Chinese patient with X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) (OMIM: 300100) is a recessive neurodegenerative disorder caused by defects in the ABCD1 gene on chromosome Xq28. Childhood cerebral ALD (CCALD) is the most frequent phenotype. We describe an affected boy who developed normally until he was 8 years old then suffered progressive neurological deficits that ultimately led to death. Diagnosis was based on clinical symptoms, an abnormal very long chain fatty acid profile in plasma, typical CCALD MRI pattern, and molecular analysis. Direct sequencing of the ABCD1 gene in this patient identified a novel splicing mutation (IVS1+1G>A) in intron 1, which is considered to be the pathogenic mutation. We have identified a novel ABCD1 mutation as the likely cause of CCALD in a Chinese patient.

New description of a family with an autosomal recessive cathecholergic ventricular tachycardia due to Triadin gene

We describe a family with suspicion of genetic arrhythmia that has benefited from a wide genetic exploration. The eldest of the siblings presented syncope at age 5.5 years and cardiac explorations were normal. A few months later, her elder sister presented a sudden death at age 4.5 years, while she was playing in the garden. The cardiac explorations showed a heart of normal structure but presence of polymorphic premature ventricular complexes. Isoprenaline test was positive. Treatment with beta-blockers (nadolol 50 mg/m 2 ) was introduced. There was no family history of sudden death or other cardiac defects. Because of these two serious rhythmic events occurring in two young children, a genetic study was initiated by next generation sequencing of 42 genes involved in cardiac arrhythmias (long QT, Brugada, cathecholaminergic ventricular tachycardia). Two heterozygous mutations (c.613C > T/p.Gln205* and c.22 + 29 A > G) were identified in the Triadin gene, coding for a protein of the calcium release complex, recently involved in cathecholaminergic ventricular tachycardia in two families (Roux-Buisson et al., 2012). The parents of our two cases were each carriers of a heterozygous mutation and had no cardiac symptoms. Their cardiac assessment did not show any abnormality (ECG Holter, exercise test, Isoprenaline test). The nonsense p.Gln205* mutation was present in one of the published families; however the splicing mutation in intron 1 had never been identified. Minigene experiments helped to confirm its pathogenicity. Presymptomatic testing was then proposed to the third child of the family (age 3), finding the two pathogenic mutations. She was therefore put under the same treatment as her sisters. This is the second report of an autosomal recessive cathecholaminergic ventricular tachycardia due to the Triadin gene. This case illustrates the interest of Next Generation Sequencing exploring simultaneously several candidate genes, in cases of sudden death of unknown origin.

Intronic splicing mutations in PTCH1 cause Gorlin syndrome

Gorlin syndrome is an autosomal dominant disorder characterized by multiple early-onset basal cell carcinoma, odontogenic keratocysts and skeletal abnormalities. It is caused by heterozygous mutations in the tumour suppressor PTCH1. Routine clinical genetic testing, by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) to confirm a clinical diagnosis of Gorlin syndrome, identifies a mutation in 60-90% of cases. We undertook RNA analysis on lymphocytes from ten individuals diagnosed with Gorlin syndrome, but without known PTCH1 mutations by exonic sequencing or MLPA. Two altered PTCH1 transcripts were identified. Genomic DNA sequence analysis identified an intron 7 mutation c.1068-10T>A, which created a strong cryptic splice acceptor site, leading to an intronic insertion of eight bases; this is predicted to create a frameshift p.(His358Alafs*12). Secondly, a deep intronic mutation c.2561-2057A>G caused an inframe insertion of 78 intronic bases in the cDNA transcript, leading to a premature stop codon p.(Gly854fs*3). The mutations are predicted to cause loss of function of PTCH1, consistent with its tumour suppressor function. The findings indicate the importance of RNA analysis to detect intronic mutations in PTCH1 not identified by routine screening techniques.

Tafazzin splice variants and mutations in Barth syndrome

Barth syndrome is caused by mutations in the TAZ (tafazzin) gene on human chromosome Xq28. The human tafazzin gene produces four major mRNA splice variants; two of which have been shown to be functional (TAZ lacking exon 5 and full-length) in complementation studies with yeast and Drosophila. This study characterizes the multiple alternative splice variants of TAZ mRNA and their proportions in blood samples from a cohort of individuals with Barth syndrome (BTHS). Because it has been reported that collection and processing methods can affect the expression of various genes, we tested and chose a stabilizing medium for collecting, shipping and processing of the blood samples of these individuals. In both healthy controls and in BTHS individuals, we found a greater variety of alternatively spliced forms than previously described, with a sizeable proportion of minor splice variants besides the four dominant isoforms. Individuals with certain exonic and intronic splice mutations produce additional mutant mRNAs that could be translated into two or more proteins with different amino acid substitutions in a single individual. A fraction of the minor splice variants is predicted to be non-productive.

Characterization Of Von Willebrand Factor Splice Variants From Exonic & Intronic Splicing Mutations

von 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 von Willebrand factor (VWF). Aberrant splicing causes 10-15% of type 1 VWD cases and the mutations are primarily found in consensus splice sites. Investigation of the VWF mRNA is required in order to determine whether the sequence variants outside of consensus splice sites affect splicing. In this study, aberrant splicing of VWF was studied using platelet and/or blood outgrowth endothelial cell (BOEC) mRNA from 3 patients from two type 1 VWD families with putative exonic and intronic splicing mutations. Variant splice forms were identified and their effects were further characterized through expression studies in HEK293(T) cells, as well as further investigation of the patient-derived BOEC. Table 1 displays the patient phenotype, mutation status, and the VWF splice forms identified through reverse transcription of the patient mRNA.Table 1Patient phenotype, mutation, and identified splice variantsPatient IDBleeding ScoreVWF: Ag (IU/mL)VWF:RCo (IU/mL)FVIII:C (IU/mL)Nucleotide ChangeVWF Splice VariantsV70110.270.170.45c.3538G>A heterozygouswildtype (wt) VWF exon 23 skipping exon 26 skipping exons 23&26 skippingV447110.400.380.51c.6599-20A>T heterozygouswt VWF exon 38 skippingV44940.440.440.75c.6599-20A>T heterozygouswt VWF exon 38 skippingThe skipping of exon 23, 26, or the combined skipping of these two exons are in-frame changes to the VWF mRNA and therefore truncated VWF would be produced from these transcripts. Skipping exon 38 however, introduces a premature termination codon (PTC) which may induce nonsense mediated decay (NMD) of the VWF mRNA. Site-directed mutagenesis was used to create expression vectors for each of the aberrant VWF splice variants, which were then transfected into HEK293T cells at varying ratios mutant:wt VWF (100:0, 50:50, 25:75, 10:90, 0:100). All 4 of these VWF variants displayed a significant dose dependent impairment of VWF secretion relative to the wt transfections (Table 2). This decrease in VWF secretion was only matched by a corresponding increase in VWF in the cell lysates for the exon 26 skipping transfections (p<0.05) indicating intracellular retention of the VWF. Where intracellular retention was not observed, proteasomal degradation of the mutant VWF was proposed as the pathogenic mechanism. Proteasomes were inhibited by MG-132; however, intracellular VWF did not increase with treatment and therefore an alternate pathway may be responsible for decreased expression of these proteins. Additionally, calcium ionophore did not appear to stimulate secretion of the VWF mutants lacking either exon 26, exons 23&26, or exon 38 in the absence of wt VWF (Table 2).Table 2VWF secretion (media/total VWF) relative to wt and fold change (fc) with calcium ionophore stimulus (*** p<0.001; ** p<0.01; *p<0.05)Exon 23 SkippingExon 26 SkippingExons 23&26 SkippingExon 38 Skipping% mutant vectorwt(%)fcwt(%)fcwt(%)fcwt(%)fc1005±3.1 ***4.7±0.27±2.3 ***0.4±0.414±5.1 ***0±0.022±0.6***0.96±0.15029±20*2.3±0.1*11±4.5 ***14.1±2.264±12*2.3±1.7*58±17*1.3±0.12552±21 *53±15 *79±4168±19 *1079±2180±28113±5284±10Pseudo Weibel-Palade body (WPB) localization of the VWF was observed through confocal immunofluorescence of HEK293 cells transfected with wt VWF and VWF skipping exon 26. VWF skipping exon 23 and exons 23&26 show some localization of the VWF in rounded organelles; however this is less organized than was seen with wt VWF. Staining of VWF lacking exon 38 appears to resemble retention in the ER, and co-localization with PDI will be investigated.Overall, we have found that the exonic mutation c.3538G>A causes skipping of exons 23 and/or 26 which cause type 1 VWD in the heterozygous patient V70 through the combined action of intracellular retention of VWF lacking exon 26, and decreased secretion of VWF skipping exon 23 and VWF skipping exons 23&26. Additionally, the skipping of exon 38 from the intronic mutation c.6599-20A>T causes type 1 VWD in patients V447 and V449 likely due to NMD of the PTC containing transcript and potentially ER degradation of the protein product of any residual mutant mRNA. Therefore these mutations lead to pathogenic VWF splicing variants which cause type 1 VWD through multiple mechanisms. Disclosures:James:CSL Behring: Honoraria, Research Funding; Octapharma: Honoraria, Research Funding; Baxter: Honoraria; Bayer: Honoraria.

A novel ALMS1 splice mutation in a non-obese juvenile-onset insulin-dependent syndromic diabetic patient

Insulin-dependent juvenile-onset diabetes may occur in the context of rare syndromic presentations suggesting monogenic inheritance rather than common multifactorial autoimmune type 1 diabetes. Here, we report the case of a Lebanese patient diagnosed with juvenile-onset insulin-dependent diabetes presenting ketoacidosis, early-onset retinopathy with optic atrophy, hearing loss, diabetes insipidus, epilepsy, and normal weight and stature, who later developed insulin resistance. Despite similarities with Wolfram syndrome, we excluded the WFS1 gene as responsible for this disease. Using combined linkage and candidate gene study, we selected ALMS1, responsible for Alström syndrome, as a candidate gene. We identified a novel splice mutation in intron 18 located 3 bp before the intron-exon junction (IVS18-3T>G), resulting in exon 19 skipping and consequent frameshift generating a truncated protein (V3958fs3964X). The clinical presentation of the patient significantly differed from typical Alström syndrome by the absence of truncal obesity and short stature, and by the presence of ketoacidotic insulin-dependent diabetes, optic atrophy and diabetes insipidus. Our observation broadens the clinical spectrum of Alström syndrome and suggests that ALMS1 mutations may be considered in patients who initially present with an acute onset of insulin-dependent diabetes.

Recurrent porphyria attacks in a Chinese patient with a heterozygous PBGD mutation

We report here the case of a 32-year-old Chinese Han woman who presented with frequent severe abdominal pain, convulsion, numbness and confusion. She also had hypertension, hyponatremia, chronic renal failure, anemia and a high urinary δ-aminolevulinic acid concentration. We identified a heterozygous splicing mutation in intron 11 (IVS11-2A→G) of the porphobilinogen (PBG) deaminase gene (PBGD) in her genomic DNA. This mutation had previously been reported in a North American patient, but was absent from 50 healthy Chinese controls.

A case of SCNN1A splicing mutation presenting as mild systemic pseudohypoaldosteronism type 1

Systemic pseudohypoaldosteronism type 1 (PHA1) is characterized by excessive salt loss from the renal tubulus, colon, sweat and salivary glands. Here we present a case of systemic PHA1 whose genetic analysis revealed a homozygous splicing mutation in intron 4 of SCNN1A (c.684+2 T>A) and discuss with the patient's phenotype. Previously described systemic PHA cases show varying degrees of severity dependent on the mutation. Most of the SCNN1A gene mutations present with a severe phenotype. The long-term follow-up and phenotype of the two reported cases with splicing mutation of the SCNN1A gene are unknown. Our case, with a new splicing mutation of SCNN1A, presented with a severe phenotype in the neonatal period. Since then she has been well without any hospitalization and respiratory illness. Her requirement for medication also decreased gradually. After early infancy she presented a mild systemic PHA1 phenotype up to the age of 39 months. In conclusion, the mutation in the patient is located at the splicing site and is definitely a new and pathogenic one, and the phenotype of the patient was milder as observed in a patient with missense mutation.

TMEM165 Deficiency Causes a Congenital Disorder of Glycosylation

Protein glycosylation is a complex process that depends not only on the activities of several enzymes and transporters but also on a subtle balance between vesicular Golgi trafficking, compartmental pH, and ion homeostasis. Through a combination of autozygosity mapping and expression analysis in two siblings with an abnormal serum-transferrin isoelectric focusing test (type 2) and a peculiar skeletal phenotype with epiphyseal, metaphyseal, and diaphyseal dysplasia, we identified TMEM165 (also named TPARL) as a gene involved in congenital disorders of glycosylation (CDG). The affected individuals are homozygous for a deep intronic splice mutation in TMEM165. In our cohort of unsolved CDG-II cases, we found another individual with the same mutation and two unrelated individuals with missense mutations in TMEM165. TMEM165 encodes a putative transmembrane 324 amino acid protein whose cellular functions are unknown. Using a siRNA strategy, we showed that TMEM165 deficiency causes Golgi glycosylation defects in HEK cells.