scholarly journals Clinical Characteristics of POC1B-Associated Retinopathy and Assignment of Pathogenicity to Novel Deep Intronic and Non-Canonical Splice Site Variants

2021 ◽  
Vol 22 (10) ◽  
pp. 5396
Author(s):  
Nicole Weisschuh ◽  
Pascale Mazzola ◽  
Miriam Bertrand ◽  
Tobias B. Haack ◽  
Bernd Wissinger ◽  
...  
Keyword(s):  
Genome Sequencing ◽  
Splice Site ◽  
Age Of Onset ◽  
Splice Variants ◽  
Exon Skipping ◽  
Cone Dystrophy ◽  
Compound Heterozygous ◽  
Pathogenic Variants ◽  
Splice Site Variant

Mutations in POC1B are a rare cause of inherited retinal degeneration. In this study, we present a thorough phenotypic and genotypic characterization of three individuals harboring putatively pathogenic variants in the POC1B gene. All patients displayed a similar, slowly progressive retinopathy (cone dystrophy or cone-rod dystrophy) with normal funduscopy but disrupted outer retinal layers on optical coherence tomography and variable age of onset. Other symptoms were decreased visual acuity and photophobia. Whole genome sequencing revealed a novel homozygous frameshift variant in one patient. Another patient was shown to harbor a novel deep intronic variant in compound heterozygous state with a previously reported canonical splice site variant. The third patient showed a novel nonsense variant and a novel non-canonical splice site variant. We aimed to validate the effect of the deep intronic variant and the non-canonical splice site variant by means of in vitro splice assays. In addition, direct RNA analysis was performed in one patient. Splicing analysis revealed that the non-canonical splice site variant c.561-3T>C leads to exon skipping while the novel deep intronic variant c.1033-327T>A causes pseudoexon activation. Our data expand the genetic landscape of POC1B mutations and confirm the benefit of genome sequencing in combination with downstream functional validation using minigene assays for the analysis of putative splice variants. In addition, we provide clinical multimodal phenotyping of the affected individuals.

scholarly journals Clear evidence of LAMA5 gene biallelic truncating variants causing infantile nephrotic syndrome

Kidney360 ◽  
2021 ◽  
pp. 10.34067/KID.0004952021
Author(s):  
Yukimasa Taniguchi ◽  
China Nagano ◽  
Kiyotoshi Sekiguchi ◽  
Atsushi Tashiro ◽  
Noriko Sugawara ◽  
...  
Keyword(s):  
Nephrotic Syndrome ◽  
Splice Site ◽  
Japanese Patients ◽  
Compound Heterozygous ◽  
Missense Variants ◽  
Targeted Next Generation Sequencing ◽  
Pathogenic Variants ◽  
Infantile Nephrotic Syndrome ◽  
Splice Site Variant

Background: Pathogenic variants in single genes encoding podocyte-associated proteins have been implicated in about 30% of steroid resistant nephrotic syndrome (SRNS) patients in children. However, LAMA5 gene biallelic variants have been identified in only 7 patients so far, and most are missense variants of unknown significance. Furthermore, no functional analysis had been conducted for all but one of these variants. Here, we report three patients with LAMA5 gene biallelic truncating variants manifesting infantile nephrotic syndrome and one SRNS case with biallelic LAMA5 missense variants. Methods: We conducted comprehensive gene screening of Japanese patients with severe proteinuria. Using targeted next-generation sequencing, 62 podocyte-related genes were screened in 407 unrelated patients with proteinuria. For the newly discovered LAMA5 variants, we conducted in vitro heterotrimer formation assays. Results: Biallelic truncating variants in the LAMA5 gene (NM_005560) were detected in 3 patients from 2 families. All patients presented with proteinuria within 6 months of age. Patients 1 and 2 were siblings possessing a nonsense variant (c.9232C>T, p.(Arg3078*)) and a splice site variant (c.1282+1G>A) that led to exon 9 skipping and a frameshift. Patient 3 had a remarkable irregular contour of the glomerular basement membrane. She was subsequently found to have a nonsense variant (c.8185C>T, p.(Arg2720*)) and the same splice site variant in patients 1 and 2. By in vitro heterotrimer formation assays, both truncating variants produced smaller laminin α5 proteins that nevertheless formed trimers with laminin β1 and γ1 chains. Patient 4 showed SRNS at the age of eight and carried compound heterozygous missense variants (c.1493C>T, p.(Ala498Val) and c.8399G>A, p.(Arg2800His)). Conclusions: Our patients showed clear evidence of biallelic LAMA5 truncating variants causing infantile nephrotic syndrome. We also discerned the clinical and pathological characteristics observed in LAMA5-related nephropathy. LAMA5 variant screening should be performed in congenital/infantile nephrotic syndrome patients.

scholarly journals X-Linked Retinitis Pigmentosa Caused by Non-Canonical Splice Site Variants in RPGR

2021 ◽  
Vol 22 (2) ◽  
pp. 850
Author(s):  
Friederike Kortüm ◽  
Sinja Kieninger ◽  
Pascale Mazzola ◽  
Susanne Kohl ◽  
Bernd Wissinger ◽  
...  
Keyword(s):  
Retinitis Pigmentosa ◽  
Splice Site ◽  
Exon Skipping ◽  
Open Reading Frame ◽  
Aberrant Splicing ◽  
Reading Frame ◽  
Pathogenic Variants ◽  
Nucleotide Addition ◽  
Intron 2

We aimed to validate the effect of non-canonical splice site variants in the RPGR gene in five patients from four families diagnosed with retinitis pigmentosa. Four variants located in intron 2 (c.154 + 3_154 + 6del), intron 3 (c.247 + 5G>A), intron 7 (c.779-5T>G), and intron 13 (c.1573-12A>G), respectively, were analyzed by means of in vitro splice assays. Splicing analysis revealed different aberrant splicing events, including exon skipping and intronic nucleotide addition, which are predicted to lead either to an in-frame deletion affecting relevant protein domains or to a frameshift of the open reading frame. Our data expand the landscape of pathogenic variants in RPGR, thereby increasing the genetic diagnostic rate in retinitis pigmentosa and allowing patients harboring the analyzed variants to be enrolled in clinical trials.

scholarly journals Novel Splice-Site and Missense Variant of PNPLA6 in an Austrian Family Causing Spastic Paraplegia-39 with Cerebellar Oculomotor Disorder

2022 ◽  
Author(s):  
Sebastian Viertauer ◽  
Ingo Kurth ◽  
Katja Eggermann ◽  
Christian Eggers
Keyword(s):  
Splice Site ◽  
Lower Limb ◽  
Clinical Symptoms ◽  
Hypogonadotropic Hypogonadism ◽  
Missense Variant ◽  
Compound Heterozygous ◽  
Spastic Paraplegia ◽  
Meaningful Improvement ◽  
Pathogenic Variants ◽  
Splice Site Variant

Abstract Background The term hereditary spastic paraplegia comprises an ever-expanding array of neurological disorders with distinct aetiologies. Spastic paraplegia 39 is one of the many variants with additional features of other organs and neurological systems. We describe a large kindred with two hitherto undescribed mutations of PNPLA6 and a novel clinical phenotype as, in addition to spastic paraplegia, affected subjects suffered from a prominent cerebellar oculomotor dysfunction. Methods Three of five genetically tested family members of a large kindred were affected by spastic gait and cerebellar oculomotor dysfunction. Clinical, imaging, laboratory and electrophysiological data were analyzed. Genetic analysis was done using next-generation sequencing. Segregation analyses were performed by Sanger sequencing. To assess the pathogenicity of genetic variants on the encoded protein, in silico assessments were carried out. Results Two hitherto unknown sequence variants in the PNPLA6 gene, a splice-site variant c.1635+3G>T and a missense variant c.3401A>T, p.(Asp1134Val), were detected. Compound-heterozygous siblings presented with lower limb spasticity and a marked cerebellar oculomotor disorder accompanied by moderate hypogonadotropic hypogonadism in the female. A paternal uncle being homozygous for the splice-site variant of PNPLA6 presented with increased lower limb reflexes, an unstable gait and cerebellar oculomotor dysfunction. Treatment with 4-aminopyridin, a potassium channel blocker, lead to meaningful improvement of clinical symptoms. Conclusion PNPLA6 gene variants are associated with a broad phenotypic spectrum to which we add cerebellar oculomotor dysfunction. In our kindred, the full clinical manifestation only occurred in compound-heterozygous subjects indicating that biallelic pathogenic variants lead to more serious and earlier onset of symptoms. Our findings emphasize the role of PNPLA6 in different neurodegenerative disorders.

Effect of 5' splice site mutations on splicing of the preceding intron

1990 ◽  
Vol 10 (12) ◽  
pp. 6299-6305
Author(s):  
M Talerico ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Exon Skipping ◽  
Splicing Factors ◽  
Exon 2 ◽  
Intron 1 ◽  
Direct Assembly ◽  
Internal Exon ◽  
Exon 1 ◽  
Lariat Rna

Three exon constructs containing identical intron and exon sequences were mutated at the 5' splice site beginning intron 2 and assayed for the effect of the mutation on splicing of the upstream intron in vitro. Alteration of two or six bases within the 5' splice site reduced removal of intron 1 at least 20-fold, as determined by quantitation of either spliced product or released lariat RNA. The prominent product was skip splicing of exon 1 to exon 3. Examination of complex formation indicated that mutation of the 5' splice site terminating exon 2 depressed the ability of precursor RNAs containing just the affected exon to direct assembly in vitro. These results suggest that mutation at the end of an internal exon inhibits the ability of the exon to be recognized by splicing factors. A comparison of the known vertebrate 5' splice site mutations in which the mutation resides at the end of an internal exon indicated that exon skipping is the preferred phenotype for this type of mutation, in agreement with the in vitro observation reported here. Inhibition of splicing by mutation at the distal and of the exon supports the suggestion that exons, rather than splice sites, are the recognition units for assembly of the spliceosome.

scholarly journals In vivo recognition of a vertebrate mini-exon as an exon-intron-exon unit.

1993 ◽  
Vol 13 (5) ◽  
pp. 2677-2687 ◽  
Author(s):  
D A Sterner ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Troponin I ◽  
Exon Skipping ◽  
Splice Sites ◽  
Small Vertebrate ◽  
Upstream Exon ◽  
I Gene

Very small vertebrate exons are problematic for RNA splicing because of the proximity of their 3' and 5' splice sites. In this study, we investigated the recognition of a constitutive 7-nucleotide mini-exon from the troponin I gene that resides quite close to the adjacent upstream exon. The mini-exon failed to be included in spliced RNA when placed in a heterologous gene unless accompanied by the upstream exon. The requirement for the upstream exon disappeared when the mini-exon was internally expanded, suggesting that the splice sites bordering the mini-exon are compatible with those of other constitutive vertebrate exons and that the small size of the exon impaired inclusion. Mutation of the 5' splice site of the natural upstream exon did not result in either exon skipping or activation of a cryptic 5' splice site, the normal vertebrate phenotypes for such mutants. Instead, a spliced RNA accumulated that still contained the upstream intron. In vitro, the mini-exon failed to assemble into spliceosome complexes unless either internally expanded or accompanied by the upstream exon. Thus, impaired usage of the mini-exon in vivo was accompanied by impaired recognition in vitro, and recognition of the mini-exon was facilitated by the presence of the upstream exon in vivo and in vitro. Cumulatively, the atypical in vivo and in vitro properties of the troponin exons suggest a mechanism for the recognition of this mini-exon in which initial recognition of an exon-intron-exon unit is followed by subsequent recognition of the intron.

scholarly journals Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF

1993 ◽  
Vol 13 (5) ◽  
pp. 2993-3001
Author(s):  
A Mayeda ◽  
D M Helfman ◽  
A R Krainer
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Exon Skipping ◽  
Splicing Factor ◽  
Alternative Exon ◽  
Hnrnp A1 ◽  
Exon Inclusion ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Nuclear Ribonucleoprotein

The essential splicing factor SF2/ASF and the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) modulate alternative splicing in vitro of pre-mRNAs that contain 5' splice sites of comparable strengths competing for a common 3' splice site. Using natural and model pre-mRNAs, we have examined whether the ratio of SF2/ASF to hnRNP A1 also regulates other modes of alternative splicing in vitro. We found that an excess of SF2/ASF effectively prevents inappropriate exon skipping and also influences the selection of mutually exclusive tissue-specific exons in natural beta-tropomyosin pre-mRNA. In contrast, an excess of hnRNP A1 does not cause inappropriate exon skipping in natural constitutively or alternatively spliced pre-mRNAs. Although hnRNP A1 can promote alternative exon skipping, this effect is not universal and is dependent, e.g., on the size of the internal alternative exon and on the strength of the polypyrimidine tract in the preceding intron. With appropriate alternative exons, an excess of SF2/ASF promotes exon inclusion, whereas an excess of hnRNP A1 causes exon skipping. We propose that in some cases the ratio of SF2/ASF to hnRNP A1 may play a role in regulating alternative splicing by exon inclusion or skipping through the antagonistic effects of these proteins on alternative splice site selection.

scholarly journals Biallelic DAB1 Variants Are Associated With Mild Lissencephaly and Cerebellar Hypoplasia

2021 ◽  
Vol 7 (2) ◽  
pp. e558
Author(s):  
Daphne J. Smits ◽  
Rachel Schot ◽  
Martina Wilke ◽  
Marjon van Slegtenhorst ◽  
Marie Claire Y. de Wit ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Splice Variants ◽  
Brain Mri ◽  
Cerebellar Vermis ◽  
Compound Heterozygous ◽  
Loss Of Function ◽  
Functional Studies ◽  
Pathogenic Variants ◽  
Whole Exome

ObjectiveWe aimed to identify pathogenic variants in a girl with epilepsy, developmental delay, cerebellar ataxia, oral motor difficulty, and structural brain abnormalities with the use of whole-exome sequencing.MethodsWhole-exome trio analysis and molecular functional studies were performed in addition to the clinical findings and neuroimaging studies.ResultsBrain MRI showed mild pachygyria, hypoplasia of the cerebellar vermis, and abnormal foliation of the cerebellar vermis, suspected for a variant in one of the genes of the Reelin pathway. Trio whole-exome sequencing and additional functional studies were performed to identify the pathogenic variants. Trio whole-exome sequencing revealed compound heterozygous splice variants in DAB1, both affecting the highly conserved functional phosphotyrosine-binding domain. Expression studies in patient-derived cells showed loss of normal transcripts, confirming pathogenicity.ConclusionsWe conclude that these variants are very likely causally related to the cerebral phenotype and propose to consider loss-of-function DAB1 variants in patients with RELN-like cortical malformations.

scholarly journals Biallelic ERBB3 loss-of-function variants are associated with a novel multisystem syndrome without congenital contracture

2019 ◽  
Vol 14 (1) ◽  
Author(s):  
Niu Li ◽  
Yufei Xu ◽  
Yi Zhang ◽  
Guoqiang Li ◽  
Tingting Yu ◽  
...  
Keyword(s):  
Postnatal Growth ◽  
Compound Heterozygous ◽  
Loss Of Function ◽  
Atrioventricular Canal ◽  
Multiple Systems ◽  
Feeding Difficulties ◽  
Functional Studies ◽  
Pathogenic Variants ◽  
Compound Heterozygous Variants

Abstract Background Gain-of-function pathogenic variants of the Erb-B2 receptor tyrosine kinase 3 (ERBB3) gene contribute to the occurrence and development of a variety of human carcinomas through activation of phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase (ERK) signaling. ERBB3 gene homozygous germline variants, whose loss of function may cause autosomal recessive congenital contractural syndrome, were recently identified. This study aims to identify the disease-causing gene in a Chinese pedigree with variable phenotypes involving multiple systems, including developmental delay, postnatal growth retardation, transient lower limb asymmetry, facial malformations, atrioventricular canal malformation, bilateral nystagmus and amblyopia, feeding difficulties, immunodeficiency, anemia, and liver damage, but without congenital contracture. Methods Trio-whole exome sequencing (WES) was performed to identify the disease-causing gene in a 24-month-old Chinese female patient. The pathogenicity of the identified variants was evaluated using in silico tools and in vitro functional studies. Results Trio-WES revealed compound heterozygous variants of c.1253 T > C (p.I418T) and c.3182dupA (p.N1061Kfs*16) in the ERBB3 gene. Functional studies showed that p.I418T resulted in normal expression of ERBB3, which was capable of interacting with ERBB2. However, the variant impaired ERBB3 phosphorylation, consequently blocking ERBB2 phosphorylation and AKT and ERK activation. The truncated protein resulting from the c.3182dupA variant also lacked the capacity to activate downstream signaling pathways. Conclusions We report the first patient with a novel multisystem syndrome disorder without congenital contracture resulting from biallelic loss-of-function variants of ERBB3.

scholarly journals Compound heterozygous inheritance of two novel COQ2 variants results in familial coenzyme Q deficiency

2020 ◽  
Vol 15 (1) ◽  
Author(s):  
Aliaa H. Abdelhakim ◽  
Avinash V. Dharmadhikari ◽  
Sara D. Ragi ◽  
Jose Ronaldo Lima de Carvalho ◽  
Christine L. Xu ◽  
...  
Keyword(s):  
Coenzyme Q ◽  
Clinical Manifestations ◽  
Missense Variant ◽  
Neurological Involvement ◽  
Cone Dystrophy ◽  
Compound Heterozygous ◽  
Pathogenic Variants ◽  
Whole Exome ◽  
Compound Heterozygous Variants ◽  
End Stage

Abstract Background Primary coenzyme Q10 deficiency is a rare disease that results in diverse and variable clinical manifestations. Nephropathy, myopathy and neurologic involvement are commonly associated, however retinopathy has also been observed with certain pathogenic variants of genes in the coenzyme Q biosynthesis pathway. In this report, we describe a novel presentation of the disease that includes nephropathy and retinopathy without neurological involvement, and which is the result of a compound heterozygous state arising from the inheritance of two recessive potentially pathogenic variants, previously not described. Materials and methods Retrospective report, with complete ophthalmic examination, multimodal imaging, electroretinography, and whole exome sequencing performed on a family with three affected siblings. Results We show that affected individuals in the described family inherited two heterozygous variants of the COQ2 gene, resulting in a frameshift variant in one allele, and a predicted deleterious missense variant in the second allele (c.288dupC,p.(Ala97Argfs*56) and c.376C > G,p.(Arg126Gly) respectively). Electroretinography results were consistent with rod-cone dystrophy in the affected individuals. All affected individuals in the family exhibited the characteristic retinopathy as well as end-stage nephropathy, without evidence of any neurological involvement. Conclusions We identified two novel compound heterozygous variants of the COQ2 gene that result in primary coenzyme Q deficiency. Targeted sequencing of coenzyme Q biosynthetic pathway genes may be useful in diagnosing oculorenal clinical presentations syndromes not explained by more well known syndromes (e.g., Senior-Loken and Bardet-Biedl syndromes).

Exon definition may facilitate splice site selection in RNAs with multiple exons

1990 ◽  
Vol 10 (1) ◽  
pp. 84-94 ◽  
Author(s):  
B L Robberson ◽  
G J Cote ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Exon Skipping ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Spliceosome Assembly ◽  
Exon Definition ◽  
Exon 2 ◽  
Correct Orientation

Interactions at the 3' end of the intron initiate spliceosome assembly and splice site selection in vertebrate pre-mRNAs. Multiple factors, including U1 small nuclear ribonucleoproteins (snRNPs), are involved in initial recognition at the 3' end of the intron. Experiments were designed to test the possibility that U1 snRNP interaction at the 3' end of the intron during early assembly functions to recognize and define the downstream exon and its resident 5' splice site. Splicing precursor RNAs constructed to have elongated second exons lacking 5' splice sites were deficient in spliceosome assembly and splicing activity in vitro. Similar substrates including a 5' splice site at the end of exon 2 assembled and spliced normally as long as the second exon was less than 300 nucleotides long. U2 snRNPs were required for protection of the 5' splice site terminating exon 2, suggesting direct communication during early assembly between factors binding the 3' and 5' splice sites bordering an exon. We suggest that exons are recognized and defined as units during early assembly by binding of factors to the 3' end of the intron, followed by a search for a downstream 5' splice site. In this view, only the presence of both a 3' and a 5' splice site in the correct orientation and within 300 nucleotides of one another will stable exon complexes be formed. Concerted recognition of exons may help explain the 300-nucleotide-length maximum of vertebrate internal exons, the mechanism whereby the splicing machinery ignores cryptic sites within introns, the mechanism whereby exon skipping is normally avoided, and the phenotypes of 5' splice site mutations that inhibit splicing of neighboring introns.

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