Exon skipping in Duchenne Muscle dystrophy due to a silent p.Ser443= mutation in the DMD gene

Duchenne Muscle dystrophy (DMD) is a X-linked inherited disease predominantly caused by severe mutations in DMD gene leading to absence of dystrophin protein. Here we report a 14-year-old Mongolian boy suffering from proximal muscle weakness, pseudohypertrophic deltoid and gastrocnemius muscles since early childhood. Lactate dehydrogenase (LDH) and creatine kinase (CK) levels were elevated. Mutation analysis including MLPA and sequencing of the DMD gene revealed a hemizygous silent variant, c.1329C>T (p.Ser443=) in exon 11. This silent mutation, listed in the SNP database (rs1060502631), was described as a variant of unknown significance (VUS) in ClinVar database. cDNA analysis demonstrated partial skipping of exon 11 due to this mutation. Although silent mutations are usually considered non-pathogenic, our case emphasizes that silent mutations can be potentially pathogenic. Hence, if silent variants are not annotated in database or not known to be benign, they should be analysed further at cDNA level.     

Hypomyelinating spastic dyskinesia and ichthyosis caused by a homozygous splice site mutation leading to exon skipping in ELOVL1

IntroductionNext generation sequencing technologies allow detection of very rare pathogenic gene variants and uncover cerebral palsy. Herein, we describe two siblings with cerebral palsy due to ELOVL1 splice site mutation in autosomal recessive manner. ELOVL1 catalyzes fatty acid elongation to produce very long-chain fatty acids (VLCFAs; ≥C21), most of which are components of sphingolipids such as ceramides and sphingomyelins. Ichthyotic keratoderma, spasticity, hypomyelination, and dysmorphic facies (MIM: 618527) stem from ELOVL1 gene deficiency in human.MethodsWe have studied a consanguineous family with whole exome sequencing (WES) and performed in depth analysis of cryptic splicing on the molecular level using RNA. Comprehensive analysis of ceramides in the skin stratum corneum of patients using liquid chromatography-tandem mass spectrometry (LC–MS/MS). ELOVL1 protein structure was computationally modelled.ResultsThe novel c.376-2A > G (ENST00000372458.8) homozygous variant in the affected siblings causes exon skipping. Comprehensive analysis of ceramides in the skin stratum corneum of patients using LC–MS/MS demonstrated significant shortening of fatty acid moieties and severe reduction in the levels of acylceramides.DiscussionIt has recently been shown that disease associated variants of ELOVL1 segregate in an autosomal dominant manner. However, our study for the first time demonstrates an alternative autosomal recessive inheritance model for ELOVL1. In conclusion, we suggest that in ultra-rare diseases, being able to identify the inheritance patterns of the disease-associated gene or genes can be an important guide to identifying the molecular mechanism of genetic cerebral palsy.     

ANO5 mutations in the Dutch limb girdle muscular dystrophy population

A Dutch cohort of 105 limb girdle muscular dystrophy (LGMD) patients were subject to subsequent genetic investigations. In half the families a causative mutation was found. Recently mutations were identified in ANO5 causing LGMD2L and Miyoshi-like myopathy (MMD3), but could also be found in patients with hyperCKemia only. Therefore, we analysed the index cases of the remaining 31 as yet undiagnosed families from our previously described cohort of LGMD patients for the presence of ANO5 mutations. Detailed history and neurological examination were available for all patients. Serum creatine kinase (CK) activity, skeletal muscle computed tomography (CT) and cardiological investigations were performed. Mutations in ANO5 were found in 16% of the families: 11 index patients and two sibs, eight males and five females. The founder mutation c.191dupA was present in 8 out of 13 patients. Ten different pathogenic mutations were identified of which seven were novel: five missense and two splice site mutations. The age of these patients ranged from 26 to 69 years and the age of onset varied from 21 to 57 years. Symptoms at onset were related to proximal leg weakness. The weakness was slowly progressive. Calf hypertrophy was present in three patients. Males were more severely affected than females. Serum CK activity was highly elevated in the early stage of disease and moderately increased in later stages. Muscle biopsy showed predominantly dystrophic changes. One patient had hypertrophic cardiomyopathy, two others had intraventricular septum thickening.     

Segawa disease with a novel heterozygous mutation in exon 5 of the GCH-1 gene (E183K)

We report a novel missense mutation in the GCH-1 gene resulting in Segawa disease. The patient, a 6-year-old girl, presented with dystonia. Her CSF biopterin and neopterin levels were reduced, suggesting Segawa disease. L-dopa administration led to clinical improvement. Genetic analysis revealed a missense mutation in exon 5 of the GCH-1 gene (E183K). Although dystonia or other movement disorders were not identified in her family, this may be explained by the low penetrance of Segawa disease.     

A novel LMNA gene mutation Leu162Pro and the associated clinical characteristics in a family with autosomal-dominant emery-dreifuss muscular dystrophy

We report the clinical characteristics, genetic analysis, and muscle biopsy findings of a family with Emery-Dreifuss muscular dystrophy and a novel mutation (Leu162Pro) in the LMNA gene. Within this single family, the age of onset and disease severity varied among the family members. In addition, focal defects of nuclear membranes with chromatin blebs in endothelial cells was shown via electron microscopy.     

A mitochondrial DNA mutation in a patient with an extensive family history of Duchenne muscular dystrophy

One challenge in the molecular diagnosis of mitochondrial DNA (mtDNA) disorders is detection of a low percentage of mutant heteroplasmy. We report a patient who had a delayed molecular diagnosis of mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome due to the complication of an extensive family history of another neuromuscular disease, Duchenne muscular dystrophy, and the failure to detect a low proportion of mutant A3243G mtDNA with a polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP)/ethidium bromide detection method. Using an improved, more sensitive allele-specific oligonucleotide (ASO) radioactive dot-blot hybridization method, a low degree of A3243G heteroplasmy was detected in several tissues from this patient. This case underscores the importance of a sensitive mutation detection method and the need for a search for mtDNA mutations if the patient's clinical symptoms suggest a mitochondrial disorder despite the family background of another neuromuscular disease.     

Asymptomatic Becker muscular dystrophy in a family with a multiexon deletion

We report a Becker muscular dystrophy (BMD) family with one 5‐year‐old affected patient and a 69‐year‐old asymptomatic grandfather. Dystrophin gene multiplex polymerase chain reaction and multiplex ligation‐dependant probe amplification analysis showed that both males carried an in‐frame deletion of exons 45–55. Segregation analysis revealed two additional asymptomatic boys in this family. Our finding supports previous predictions that exons 45–55 are the optimal multiexon skipping target in antisense gene therapy to transform the severe Duchenne muscular dystrophy into the milder BMD, or even asymptomatic, phenotype. Muscle Nerve, 2008     

阿尔茨海默病患者早老素-1基因5号外显子新突变点的研究

目的探讨早老素－１基因突变在散发性阿尔茨海默病（ＳｐｏｒａｄｉｃＡｌｚｈｅｉｍｅｒ′ｓｄｉｓｅａｓｅ,ＳＡＤ）患者发病机制中的作用。方法应用聚合酶链反应－单链构像多态性（ＰＣＲ－ＳＳＣＰ）及ＤＮＡ直接测序技术检测６８名ＳＡＤ患者、１３名血管性痴呆（ＶＤ）患者和６５名正常老年人的早老素－１基因第５号外显子。结果发现４名ＳＡＤ患者的ＰＣＲ产物ＳＳＣＰ分析发生泳动异常,ＤＮＡ序列分析发现：这４名ＳＡＤ患者的１３０号密码子发生了ＣＴＧ→ＡＴＧ错义突变（３８８位点发生Ｃ→Ａ突变）,使编码的氨基酸由亮氨酸变为蛋氨酸（Ｌｅｕ１３０Ｍｅｔ）;１５７号密码子发生了ＧＴＧ→ＣＴＧ错义突变（４６９位点发生Ｇ→Ｃ突变）,使编码的氨基酸由缬氨酸变为亮氨酸（Ｖａｌ１５７Ｌｅｕ）,另有１１名患者ＳＳＣＰ表现为一条单链增快,其性质待定。结论ＳＡＤ患者也存在早老素－１基因第５号外显子突变,该突变点可能为中国人ＳＡＤ患者早老素基因突变点之一。     

Dysferlin mutation analysis in a group of Italian patients with limb-girdle muscular dystrophy and Miyoshi myopathy

Mutations in the dysferlin gene (DYSF) on chromosome 2p13 cause distinct phenotypes of muscular dystrophy: limb-girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy (MM), and distal anterior compartment myopathy, which are known by the term 'dysferlinopathy'. We performed mutation analyses of DYSF in 14 Italian patients from 10 unrelated families with a deficiency of dysferlin protein below 20% of the value in normal controls by immunoblotting analysis. We identified 11 different mutations, including eight missense and three deletion mutations. Nine of them were novel mutations. We also identified a unique 6-bp insertion polymorphism within the coding region of DYSF in 15% of Italian population, which was not observed in East Asian populations. The correlation between clinical phenotype and the gene mutations was unclear, which suggested the role of additional genetic and epigenetic factors in modifying clinical symptoms.     

Two cases of oculopharyngeal muscular dystrophy (OPMD) with the rare PABPN1 c.35G>C; p.Gly12Ala point mutation

Oculopharyngeal muscular dystrophy is a neuromuscular disease usually presenting in the 5th or 6th decades of life with a dominant inheritance pattern. In almost all cases the cause of the disease is the expansion of a DNA repeat sequence containing GCG and GCA codons in exon 1 of the PABPN1 gene from 10 to between 12 and 17 repeats. However one case has been previously reported without the gene expansion but instead with a c.35G > C missense mutation converting a glycine codon to an alanine and resulting in a sequence of 13 contiguous alanine codons, thus mimicking the effect of the common expansion mutation. Here we report two further cases of OPMD caused by the c.35G > C point mutation. Clinical and pedigree data indicate the usual OPMD dominant inheritance pattern.     

Genotype-phenotype analysis in X-linked Emery-Dreifuss muscular dystrophy and identification of a missense mutation associated with a milder phenotype.

Direct sequencing of the emerin gene in 22 families with Emery-Dreifuss muscular dystrophy (EMD) revealed mutations in 21 (95%), confirming that emerin mutations can be identified in the majority of families with X-linked EMD. Most emerin mutations result in absence of the protein. In this study three mutations (a missense mutation Pro183Thr and two in-frame deletions removing residues 95-99 and 236-241, respectively) were unusual in being associated with expression of mutant protein. The phenotype in these families was compared in detail with the clinical features in cases with typical null mutations. For the in-frame deletions there were no significant differences. In the family with the missense mutation the phenotype was milder. Age at onset was later for first symptoms and for development of ankle contractures and muscle weakness. These findings have diagnostic implications as well as pointing to functionally important regions of the emerin protein.     

Identification of a novel mutation in a Korean patient with oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset muscle disorder characterized by progressive dysphagia and bilateral ptosis. Mutations in the polyadenylate binding protein nuclear 1 (PABPN1) gene have been found to cause OPMD. The typical mutation is a stable trinucleotide repeat expansion in the first exon of the PABPN1 gene, in which (GCG)(6) is the normal repeat length. We investigated a Korean patient with OPMD and identified a novel mutation: a heterozygous insertion of a 9-bp sequence [(GCG)(GCA)(GCA); c.27_28insGCGGCAGCA] instead of the (GCG) repeat expansion, resulting in an in-frame insertion of three alanines (p.A10insAAA). To the best of our knowledge, this is the first report of a genetically confirmed case of OPMD in Korea.     

Myoclonus-dystonia syndrome with severe depression is caused by an exon-skipping mutation in the epsilon-sarcoglycan gene.

We describe two affected individuals in a family with myoclonus-dystonia syndrome complicated with severe depression. One individual committed suicide. Molecular genetic analysis revealed a heterozygous point mutation in the epsilon-sarcoglycan gene, which we show leads to skipping of exon 5. This report suggests that the psychiatric spectrum of MDS includes more severe depression.     

1例CADASIL家系的临床特征与基因突变分析

目的 分析1例皖江地区CADASIL家系的临床表现及基因突变特征.方法 随访记录1例CADASIL家系先证者的临床表现、实验室检查及影像学检查,对其NOTCH3基因突变热点区行基因检测.结果 先证者为1例53岁男性患者,病程中反复发作脑卒中并有家族发病史,另表现假性球麻痹、情绪障碍、冷漠和轻度认知功能障碍,无偏头痛史.其头颅核磁共振显示对称性双侧颞极及外囊区白质高信号病变.患者NOTCH3基因突变分析发现4号外显子区已知致病突变（c.580T〉C）和常见核苷酸多态性（rs1043994）.结论 c.580T〉C突变是中国人CADASIL病例首次报道,基因检测是确诊CADASIL的金标准.     

Congenital muscular dystrophy phenotype with neuromuscular spindles excess in a 5-year-old girl caused by HRAS mutation

We report on a 5-year-old girl who presented with an association of symptoms reminiscent of an Ullrich-like congenital muscular dystrophy including congenital hypotonia, proximal joint contractures, hyperlaxity of distal joints, normal cognitive development, and kyphoscoliosis. There was an excess of neuromuscular spindles on the skeletal muscle biopsy. This very peculiar feature on muscle biopsy has been reported only in patients with mutations in the HRAS gene. Sequence analysis of the subject’s HRAS gene from blood leukocytes and skeletal muscle revealed a previously described heterozygous missense mutation (c.187G>A, p. Glu63Lys). The present report thus extends the differential diagnosis of congenital muscular dystrophy with major “retractile” phenotypes and adds congenital muscular dystrophy to the clinical spectrum of HRAS-related disorders.     

DYSF mutation analysis in a group of Chinese patients with dysferlinopathy

Objective

Dysferlinopathies belong to heterogeneous group of autosomal recessive muscular disorders caused by mutations in the gene encoding dysferlin. The classifications of the dysferlinopathies mainly include limb-girdle muscular dystrophy 2B (LGMD2B) with predominantly proximal weakness, Miyoshi myopathy (MM) with calf muscle weakness and atrophy, and distal myopathy with anterior tibial onset (DMAT) with tibialis muscle atrophy. We describe the genetic character of dysferlinopathies in a group of Chinese patients.

Methods

DYSF mutations screening were done after muscle biopsy and immunohistochemical staining.

Results

Eight patients showed an absence or drastic decrease of dysferlin expression in biopsied muscle. We identified 6 different mutations, including one nonsense mutation, two insertion mutation, two deletion mutations and one splice site mutation. Five of them were novel mutations.

Conclusion

We described 8 Chinese patients with dysferlinopathy (four had a distal phenotype of MM; one had a phenotype of DMAT and three presented with LGMD2B). It is the first report of genetic confirmed DMAT in China. Mutations c.3112C>T and c.1045dup, may be recurrent mutations in China.     

Phenotypic heterogeneity in a family with FAP due to a TTR Leu58Arg mutation: a clinicopathologic study

A family with familial amyloid polyneuropathy (FAP) due to a transthyretin (TTR) Leu58Arg mutation was investigated clinicopathologically. The proband presented with sensorimotor-autonomic polyneuropathy and autopsy demonstrated massive amyloid deposition in the peripheral nerves and heart. However, the mother was characterized by carpal tunnel syndrome and ocular vitreous opacities. Thus, there was considerable phenotypic heterogeneity among family members despite the identical TTR genotype.