Dataset Information

Exon skipping causes atypical phenotypes associated with a loss-of-function mutation in FLNA by restoring its protein function.

ABSTRACT: Loss-of-function mutations in filamin A (FLNA) cause an X-linked dominant disorder with multiple organ involvement. Affected females present with periventricular nodular heterotopia (PVNH), cardiovascular complications, thrombocytopenia and Ehlers-Danlos syndrome. These mutations are typically lethal to males, and rare male survivors suffer from failure to thrive, PVNH, and severe cardiovascular and gastrointestinal complications. Here we report two surviving male siblings with a loss-of-function mutation in FLNA. They presented with multiple complications, including valvulopathy, intestinal malrotation and chronic intestinal pseudo-obstruction (CIPO). However, these siblings had atypical clinical courses, such as a lack of PVNH and a spontaneous improvement of CIPO. Trio-based whole-exome sequencing revealed a 4-bp deletion in exon 40 that was predicted to cause a lethal premature protein truncation. However, molecular investigations revealed that the mutation induced in-frame skipping of the mutated exon, which led to the translation of a mutant FLNA missing an internal region of 41 amino acids. Functional analyses of the mutant protein suggested that its binding affinity to integrin, as well as its capacity to induce focal adhesions, were comparable to those of the wild-type protein. These results suggested that exon skipping of FLNA partially restored its protein function, which could contribute to amelioration of the siblings' clinical courses. This study expands the diversity of the phenotypes associated with loss-of-function mutations in FLNA.

SUBMITTER: Oda H

PROVIDER: S-EPMC4755370 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Exon skipping causes atypical phenotypes associated with a loss-of-function mutation in FLNA by restoring its protein function.

Oda Hirotsugu H Sato Tatsuhiro T Kunishima Shinji S Nakagawa Kenji K Izawa Kazushi K Hiejima Eitaro E Kawai Tomoki T Yasumi Takahiro T Doi Hiraku H Katamura Kenji K Numabe Hironao H Okamoto Shinya S Nakase Hiroshi H Hijikata Atsushi A Ohara Osamu O Suzuki Hidenori H Morisaki Hiroko H Morisaki Takayuki T Nunoi Hiroyuki H Hattori Seisuke S Nishikomori Ryuta R Heike Toshio T

European journal of human genetics : EJHG 20150610 3

Loss-of-function mutations in filamin A (FLNA) cause an X-linked dominant disorder with multiple organ involvement. Affected females present with periventricular nodular heterotopia (PVNH), cardiovascular complications, thrombocytopenia and Ehlers-Danlos syndrome. These mutations are typically lethal to males, and rare male survivors suffer from failure to thrive, PVNH, and severe cardiovascular and gastrointestinal complications. Here we report two surviving male siblings with a loss-of-functio ...[more]

PMID: 26059841

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Project description:Schizophrenia is a heterogeneous psychiatric disorder characterized by positive symptoms such as hallucinations and delusions, negative symptoms such as anhedonia and flat affect, and cognitive impairment. Recently, glucuronate (GlucA) levels were reported to be significantly higher in serum of patients with schizophrenia than those in healthy controls. The accumulation of GlucA is known to be related to treatment-resistant schizophrenia, since GlucA is known to promote drug excretion by forming conjugates with drugs. However, the cause of GlucA accumulation remains unclear. Aldo-keto reductase family one member A1 (AKR1A1) is an oxidoreductase that catalyzes the reduction of GlucA. Genetic loss of AKR1A1 function is known to result in the accumulation of GlucA in rodents. Here, we aimed to explore genetic defects in AKR1A1 in patients with schizophrenia, which may result in the accumulation of GlucA. We identified 28 variants of AKR1A1 in patients with schizophrenia and control subjects. In particular, we identified a silent c.753G > A (rs745484618, p. Arg251Arg) variant located at the first position of exon 8 to be associated with schizophrenia. Using a minigene assay, we found that the c.753G > A variant induced exon 8 skipping in AKR1A1, resulting in a frameshift mutation, which in turn led to truncation of the AKR1A1 protein. Using the recombinant protein, we demonstrated that the truncated AKR1A1 completely lost its activity. Furthermore, we showed that AKR1A1 mRNA expression in the whole blood cells of individuals with the c.753G > A variant tended to be lower than that in those without the variants, leading to lower AKR activity. Our findings suggest that AKR1A1 carrying the c.753G > A variant induces exon skipping, leading to a loss of gene expression and enzymatic activity. Thus, GlucA patients with schizophrenia with the c.753G > A variant may show higher GlucA levels, leading to drug-resistant schizophrenia, since drug excretion by GlucA is enhanced.

Project description:The molecular basis of the skipping of constitutive exons in many messenger RNAs is not fully understood. A well-studied example is exon 9 of the human cystic fibrosis transmembrane conductance regulator gene (CFTR), in which an abbreviated polypyrimidine tract between the branch point A and the 3' splice site is associated with increased exon skipping and disease. However, many exons, both in CFTR and in other genes and have short polypyrimidine tracts in their 3' splice sites, yet they are not skipped. Inspection of the 5' splice sites immediately up- and downstream of exon 9 revealed deviations from consensus sequence, so we hypothesized that this exon may be inherently vulnerable to skipping. To test this idea, we constructed a CFTR minigene and replicated exon 9 skipping associated with the length of the polypyrimidine tract upstream of exon 9. We then mutated the flanking 5' splice sites and determined the effect on exon skipping. Conversion of the upstream 5' splice site to consensus by replacing a pyrimidine at position +3 with a purine resulted in increased exon skipping. In contrast, conversion of the downstream 5' splice site to consensus by insertion of an adenine at position +4 resulted in a substantial reduction in exon 9 skipping, regardless of whether the upstream 5' splice site was consensus or not. These results suggested that the native downstream 5' splice site plays an important role in CFTR exon 9 skipping, a hypothesis that was supported by data from sheep and mouse genomes. Although CFTR exon 9 in sheep is preceded by a long polypyrimidine tract (Y(14)), it skips exon 9 in vivo and has a nonconsensus downstream 5' splice site identical to that in humans. On the other hand, CFTR exon 9 in mice is preceded by a short polypyrimidine tract (Y(5)) but is not skipped in vivo. Its downstream 5' splice site differs from that in humans by a 2-nt insertion, which, when introduced into the human CFTR minigene, abolished exon 9 skipping. Taken together, these observations place renewed emphasis on deviations at 5' splice sites in nucleotides other than the invariant GT, particularly when such changes are found in conjunction with other altered splicing sequences, such as a shortened polypyrimidine tract. Thus, careful inspection of entire 5' splice sites may identify constitutive exons that are vulnerable to skipping.

Dataset Information

Exon skipping causes atypical phenotypes associated with a loss-of-function mutation in FLNA by restoring its protein function.

Publications

Exon skipping causes atypical phenotypes associated with a loss-of-function mutation in FLNA by restoring its protein function.

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