Splice Site Point Research Articles

318P DMD splice site point mutation in intron 68 maintains the reading frame and almost full-length dystrophin expression with cell-specific pattern

318P DMD splice site point mutation in intron 68 maintains the reading frame and almost full-length dystrophin expression with cell-specific pattern

Spectrum of Neurodegeneration with Brain Iron Accumulation with PLA2G6 Variation: A Report of Three Cases from Two Families

AbstractNeurodegeneration with brain iron accumulation consists of disorders characterized by progressive neuronal degeneration, cognitive decline, brain iron accumulation in extrapyramidal system, dentate nucleus, and gray and white matters boundary. We present a case series of PLA2G6-associated neurodegeneration (PLAN), with definite PLA2G6 gene mutations in two cases and suspected in one case. Diagnosis was based on clinical presentations, brain magnetic resonance imaging (MRI) findings, and detection of PLA2G6 mutations. Case 1: An 8-year-old boy presented with weakness of lower limbs, subnormal intelligence, scanning speech, spasticity, dysdiadochokinesia, pendular knee jerk, and extensor plantar reflex. MRI of the brain showed diffuse cerebellar atrophy and white matter T2 hyperintensity with iron deposition in bilateral globus pallidi. Case 2: Elder sister of Case 1, who developed ataxia at the age of 6 years and became bedridden at 14 years. She had nocturnal enuresis, seizures, cervical dystonia, dysphagia, and died at 23 years of age. MRI showed cerebral and cerebellar atrophies and iron deposition in basal ganglia and substantia nigra. Case 3 had infantile onset with quadriparesis, optic atrophy, developmental delay, cerebral and cerebellar atrophies, and brain iron accumulation in basal ganglia. Case 1 revealed two heterozygous mutations of PLA2G6 gene in exons 16 (c.2264G > A, p. Arg755Gln) and 12 (c.1637G > A, p. Arg546Gln), classified as likely pathogenic. Elder sister (Case 2) could not be tested for this mutation. Case 3 showed homozygous silent splice site point variation in exon 7 (c.1077 G > A; p. Ser 359 Ser) of PLA2G6 gene. Thus, in patients presenting with neurodegeneration and imaging findings of brain iron accumulation, diagnosis can be established by PLA2G6 gene mutation analysis.

Generation of genomic-integration-free human induced pluripotent stem cells and the derived cardiomyocytes of X-linked dilated cardiomyopathy from DMD gene mutation

We derived an integration-free induced pluripotent stem cell (iPSC) line from the peripheral blood mononuclear cells (PBMCs) of a 23-year-old male patient. This patient carries a 5' splice site point mutation in intron 1 (c.31+1G>A) of the dystrophin gene, a mutation associated with X-linked dilated cardiomyopathy (XLDCM). Sendai virus was used to reprogram the PBMCs and deliver OCT3/4, SOX2, c-MYC, and KLF4 factors. The iPSC line (HKUi002-A) generated preserved the mutation, expressed common pluripotency markers, differentiated into three germ layers in vivo, and exhibited a normal karyotype. Further differentiation into cardiomyocytes enables the study of the disease mechanisms of XLDCM.

Pathogenic/likely pathogenic variants in the SHOX, GHR and IGFALS genes among Indian children with idiopathic short stature.

Background Our objective was to estimate the prevalence of pathogenic/likely pathogenic variants in the SHOX, GHR, and IGFALS genes among Indian children with idiopathic short stature (ISS), and assess the genotype-phenotype correlation. Methods We recruited 61 children with short stature, who were born appropriate for gestational age, had no obvious dysmorphism or disproportion, and in whom step-wise investigative work-up (including provocative growth hormone test) was normal. Multiplex ligation-dependent probe amplification was undertaken for identifying deletions/duplications in the SHOX gene. Bidirectional sequencing was performed for identifying variants in the SHOX and GHR genes in all, and for the IGFALS gene in those with serum insulin-like growth factor-1 (IGF-1) <-1 standard deviation. The genotype-phenotype correlation was studied. Results Four children (6.5%) had pathogenic heterozygous variants in the SHOX gene, with one child each having duplication of exon 5, splice site point variant c.278-1G > C in exon 3, partial deletion and complete deletion. None of the patients had pathogenic variants in the GHR gene. Of the 39 patients in whom the IGFALS gene was sequenced, novel heterozygous likely pathogenic variants were found in two children. One had the frameshift variant c.764_765insT, p.A265Gfs*114. The second had the missense variant c.1793G > A, p.R598H predicted by MutationTaster as 'disease causing', and indicated by the protein-modelling study as having compromised binding with IGF-1 and insulin-like growth factor binding protein-3 (IGFBP-3) due to altered conformation of the interacting loop. Conclusions Pathogenic variants in the SHOX and IGFALS genes account for a significant proportion of Indian children with ISS. Further molecular studies using next generation sequencing are needed to gain insight into pathophysiological mechanisms and effective treatment strategies for ISS.

X-linked Dilated Cardiomyopathy with Mutation in the 5′ Splice Site Intron 1 of Dystrophin Gene with Utrophin Upregulation

AbstractWe report a teenage boy presented with dilated cardiomyopathy (DCM) with no initial skeletal involvement and initial normal creatine kinases. One year after the heart transplantation, he had exercise-induced transient muscle weakness with elevated creatine kinases (CKs). Muscle biopsy showed normal structures and normal dystrophin immunohistochemical labeling, but utrophin, which is an autosomal homologue of dystrophin, was overexpressed at sarcolemma. Sanger sequencing confirmed a heterozygous c.31 + 1G &gt; A, 5′ splice site point mutation at the first intron of dystrophin gene. A review of previous reports of patients with different point mutations in the same region, the first exon–intron boundary that involved the muscle promotor to exon 1, confirmed a high correlation of cardiospecific phenotype sparing the muscles with this specific site of mutations. The confirmation of upregulation of brain and Purkinje isoforms of dystrophin protein in the skeletal muscles but not in the heart in past studies help to explain the skeletal sparing presentation. X-linked DCM (XLDCM) is an important cause of isolated cardiomyopathy. Routine immunohistochemical staining study including dystrophin in cardiac muscle biopsy, and dystrophin and utrophin labelling on skeletal muscle biopsy for patients with subsequent muscle symptoms or raised creatine kinases, help in the early diagnosis of the XLDCM. Future experimental study to determine the aberrant pre-mRNA splicing of this specific splice site mutation involving exon 1 and intron 1 will help to understand better the underlying complex mechanism of the splicing regulation.

Diagnostic validation of a familial hypercholesterolaemia cohort provides a model for using targeted next generation DNA sequencing in the clinical setting

Our aim was to assess the sensitivity and specificity of a next generation DNA sequencing (NGS) platform using a capture based DNA library preparation method. Data and experience gained from this diagnostic validation can be used to progress the applications of NGS in the wider molecular diagnostic setting. A technical cross-validation comparing the current molecular diagnostic gold standard methods of Sanger DNA sequencing and multiplex ligation-dependant probe amplification (MLPA) versus a customised capture based targeted re-sequencing method on a SOLiD 5500 sequencing platform was carried out using a cohort of 96 familial hypercholesterolaemia (FH) samples. We compared a total of 595 DNA variations (488 common single nucleotide polymorphisms, 73 missense mutations, 9 nonsense mutations, 3 splice site point mutations, 13 small indels, 2 multi-exonic duplications and 7 multi-exonic deletions) found previously in the 96 FH samples. DNA variation detection sensitivity and specificity were both 100% for the SOLiD 5500 NGS platform compared with Sanger sequencing and MLPA only when both LifeScope and Integrative Genomics Viewer softwares were utilised. The methods described here offer a high-quality strategy for the detection of a wide range of DNA mutations in diseases with a moderate number of well described causative genes. However, there are important issues related to the bioinformatic algorithms employed to detect small indels.

LGI1mutations in autosomal dominant and sporadic lateral temporal epilepsy

Autosomal dominant lateral temporal epilepsy (ADLTE) or autosomal dominant partial epilepsy with auditory features (ADPEAF) is an inherited epileptic syndrome with onset in childhood/adolescence and benign evolution. The hallmark of the syndrome consists of typical auditory auras or ictal aphasia in most affected family members. ADTLE/ADPEAF is associated in about half of the families with mutations of the leucine-rich, glioma-inactivated 1 (LGI1) gene. In addition, de novo LGI1 mutations are found in about 2% of sporadic cases with idiopathic partial epilepsy with auditory features, who are clinically similar to the majority of patients with ADLTE/ADPEAF but have no family history. Twenty-five LGI1 mutations have been described in familial and sporadic lateral temporal epilepsy patients. The mutations are distributed throughout the gene and are mostly missense mutations occurring in both the N-terminal leucine rich repeat (LRR) and C-terminal EPTP (beta propeller) protein domains. We show a tridimensional model of the LRR protein region that allows missense mutations of this region to be divided into two distinct groups: structural and functional mutations. Frameshift, nonsense and splice site point mutations have also been reported that result in protein truncation or internal deletion. The various types of mutations are associated with a rather homogeneous phenotype, and no obvious genotype-phenotype correlation can be identified. Both truncating and missense mutations appear to prevent secretion of mutant proteins, suggesting a loss of function effect of mutations. The function of LGI1 is unclear. Several molecular mechanisms possibly leading to lateral temporal epilepsy are illustrated and briefly discussed.

The Biflavonoid Isoginkgetin Is a General Inhibitor of Pre-mRNA Splicing

Membrane-permeable compounds that reversibly inhibit a particular step in gene expression are highly useful tools for cell biological and biochemical/structural studies. In comparison with other gene expression steps where multiple small molecule effectors are available, very few compounds have been described that act as general inhibitors of pre-mRNA splicing. Here we report construction and validation of a set of mammalian cell lines suitable for the identification of small molecule inhibitors of pre-mRNA splicing. Using these cell lines, we identified the natural product isoginkgetin as a general inhibitor of both the major and minor spliceosomes. Isoginkgetin inhibits splicing both in vivo and in vitro at similar micromolar concentrations. It appears to do so by preventing stable recruitment of the U4/U5/U6 tri-small nuclear ribonucleoprotein, resulting in accumulation of the prespliceosomal A complex. Like two other recently reported general pre-mRNA splicing inhibitors, isoginkgetin has been previously described as an anti-tumor agent. Our results suggest that splicing inhibition is the mechanistic basis of the anti-tumor activity of isoginkgetin. Thus, pre-mRNA splicing inhibitors may represent a novel avenue for development of new anti-cancer agents.

Thirty-four novel mutations of the GLA gene in 121 patients with Fabry disease

Fabry disease (FD) is an X-chromosomal disorder caused by mutations in the GLA gene encoding the lysosomal enzyme alpha-galactosidase A. We performed mutation screening on a cohort of 121 patients including 84 male and 37 female index cases and identified a total of 90 different mutations, 34 of which are reported for the first time here. Both point mutations (74.4%) and 'short length' rearrangements (25.6%) were found, including missense (54.4%), nonsense (14.4%), and splice site point mutations (5.6%), deletions (17.8%) or insertions/duplications (5.6%) of a few nucleotides, and complex rearrangements including larger deletions (2.2%). GLA mutations were identified in 82 (97.6%) of the 84 unrelated male patients.

Isolated growth hormone deficiency in Chilean patients: clinical and molecular analysis.

Isolated growth hormone deficiency (IGHD) is a disorder that leads to short stature. It has been classified into types IA, IB, II and III. GH gene mutations and growth hormone releasing hormone (GHRH) receptor gene mutations have been described in patients with IGHD. We report here a clinical and molecular study of 27 Chilean patients with IGHD. We performed GH stimulation tests with GHRH and GHRP, and segregation and molecular analysis of the GH, GHRH and GHRH receptor genes. We describe four patients with IGHD IA bearing a 7 kb mutation (13%), and two IGHD II patients who showed two different splice site point mutations (6.8%). In 21 patients, we did not find a mutation in any of the three genes examined. These results led us to conclude that the molecular causes of IGHD involve other genes besides those analyzed in this report, as has been reported previously in patients of different ethnic origins.

Spectrum of SPG4 mutations in autosomal dominant spastic paraplegia.

Autosomal dominant hereditary spastic paraplegia (AD-HSP) is a group of genetically heterogeneous neurodegenerative disorders characterized by pro- gressive spasticity of the lower limbs. Five AD-HSP loci have been mapped to chromosomes 14q, 2p, 15q, 8q and 12q. The SPG4 locus at 2p21-p22 has been shown to account for approximately 40% of all AD-HSP families. SPG4 encoding spastin, a putative nuclear AAA protein, has recently been identified. Here, sequence analysis of the 17 exons of SPG4 in 87 unrelated AD-HSP patients has resulted in the detection of 34 novel mutations. These SPG4 mutations are scattered along the coding region of the gene and include all types of DNA modification including missense (28%), nonsense (15%) and splice site point (26.5%) mutations as well as deletions (23%) and insertions (7.5%). The clinical analysis of the 238 mutation carriers revealed a high proportion of both asymptomatic carriers (14/238) and patients unaware of symptoms (45/238), and permitted the redefinition of this frequent form of AD-HSP.