POGLUT1 biallelic mutations cause myopathy with reduced satellite cells, α-dystroglycan hypoglycosylation and a distinctive radiological pattern.

Protein O-glucosyltransferase 1 (POGLUT1) activity is critical for the Notch signaling pathway, being one of the main enzymes responsible for the glycosylation of the extracellular domain of Notch receptors. A biallelic mutation in the POGLUT1 gene has been reported in one family as the cause of an adult-onset limb-girdle muscular dystrophy (LGMD R21; OMIM# 617232). As the result of a collaborative international effort, we have identified the first cohort of 15 patients with LGMD R21, from nine unrelated families coming from different countries, providing a reliable phenotype-genotype and mechanistic insight. Patients carrying novel mutations in POGLUT1 all displayed a clinical picture of limb-girdle muscle weakness. However, the age at onset was broadened from adult to congenital and infantile onset. Moreover, we now report that the unique muscle imaging pattern of "inside-to-outside" fatty degeneration observed in the original cases is indeed a defining feature of POGLUT1 muscular dystrophy. Experiments on muscle biopsies from patients revealed a remarkable and consistent decrease in the level of the NOTCH1 intracellular domain, reduction of the pool of satellite cells (SC), and evidence of α-dystroglycan hypoglycosylation. In vitro biochemical and cell-based assays suggested a pathogenic role of the novel POGLUT1 mutations, leading to reduced enzymatic activity and/or protein stability. The association between the POGLUT1 variants and the muscular phenotype was established by in vivo experiments analyzing the indirect flight muscle development in transgenic Drosophila, showing that the human POGLUT1 mutations reduced its myogenic activity. In line with the well-known role of the Notch pathway in the homeostasis of SC and muscle regeneration, SC-derived myoblasts from patients' muscle samples showed decreased proliferation and facilitated differentiation. Together, these observations suggest that alterations in SC biology caused by reduced Notch1 signaling result in muscular dystrophy in LGMD R21 patients, likely with additional contribution from α-dystroglycan hypoglycosylation. This study settles the muscular clinical phenotype linked to POGLUT1 mutations and establishes the pathogenic mechanism underlying this muscle disorder. The description of a specific imaging pattern of fatty degeneration and muscle pathology with a decrease of α-dystroglycan glycosylation provides excellent tools which will help diagnose and follow up LGMD R21 patients.

Phenotype-genotype analysis of dystrophinopathy caused by duplication mutation in Dystrophin gene in an African patient.

BACKGROUND: The dystrophinopathies, duchenne muscular dystrophy (DMD) and Becker muscular dystrophy are common X-linked genetic myopathies resulting from mutations in the dystrophin gene. Duplication is an uncommon mechanism of mutation occurring in about 5% of DMD cases. The global prevalence of DMD is reported as 1/18,000 males. There is little clinical or epidemiological data on African patients. OBJECTIVE: To present the genotype-phenotype analysis of dystrophinopathy with an exon 8 through 9 duplication mutation in a patient of African/Ghanaian descent and his asymptomatic mother. METHODS: Investigations including a biopsy of the vastus lateralis muscle and genetic testing of the patient and his mother. RESULTS: Genetic testing demonstrated a duplication of exons 8 through 9 of the dystrophin gene in both the patient and his mother. The muscle biopsy of the patient showed partial expression of the dystrophin protein. In the absence of a family history of dystrophinopathy, we hypothesize that this is a sporadic mutation occurring in the grand maternal lineage. CONCLUSION: This case extends the world wide epidemiology of this disease to include the African/Ghanaian population and confirms the vulnerability of the dystrophin gene to recurrent spontaneous mutations at the exon 8 and 9 site.

The G1138A mutation rate in the fibroblast growth factor receptor 3 (FGFR3) gene is increased in cells carrying the t (4; 14) translocation.

Spontaneous mutations are a common phenomenon, occurring in both germ-line and somatic genomes. They may have deleterious consequences including the development of genetic disorders or, when occurring in somatic tissues, may participate in the process of carcinogenesis. Similar to many mutational hotspots, the G1138A mutation in the fibroblast growth factor receptor 3 (FGFR3) gene occurs at a CpG site. In germ-line tissues, the G1138A mutation results in achondroplasia and has one of the highest spontaneous mutation rates in the human genome. Although not at the G1138A site, there are increased rates of other somatic mutations in the FGFR3 gene that have been reported in multiple myeloma cases associated with a translocation, t (4; 14). The chromosome-4 break points in this translocation are clustered in a 70-kb region centromeric to the FGFR3 gene. We hypothesized that this translocation may impact the mutation rate at the G1138A site. We employed a semi-quantitative polymerase chain reaction-based assay to measure the frequency of this mutation in multiple myeloma cell lines carrying t (4; 14) translocation. Analysis of these cell lines varied from no change to a 10-fold increase in the mutation frequency compared with normal controls. In general, there was an increase in the G1138A mutational frequency suggesting that chromosomal rearrangement can affect the stability of the CpG hotspots.

The intron 4c allele of the NOS3 gene is associated with ischemic stroke in African Americans.

BACKGROUND: Ischemic stroke is the most common cause of disability in North America and in addition to the generally accepted risk factors, there is increasing evidence for the potential pathophysiological role of genes. One of these genes, the endothelial nitric oxide synthase gene (NOS3) has been reported as a genetic risk factor for ischemic stroke. To independently confirm and extend the results of these previous reports, we investigated this gene as a risk factor for stroke in an ethnically diverse study population. METHODS: Using the TOAST classification, we characterized and studied 377 patients with ischemic stroke. We genotyped two common variants in the NOS3 gene, the intron 4 insertion/deletion and an exonic single nucleotide polymorphism (SNP), G894T, in these patients and compared them with 502 controls. Chi-square or Fisher's exact tests were used to examine allele effects on stroke and stroke subtypes. Logistic regression analysis was used to adjust for confounding covariate effects. RESULTS: All genotypes are in Hardy-Weinberg equilibrium except for intron 4c, which is overrepresented in ischemic stroke patients. In pooled analysis of all patients, intron 4c, but not intron 4a, intron 4b or G894T alleles are associated with stroke (p < 0.01). In subgroup analysis by race, the intron 4c allele is most strongly associated with large artery ischemic stroke in African Americans (p < 0.01). CONCLUSION: We are unable to confirm previous reports of an association of the intron 4a or the G894T alleles with ischemic stroke. However, although limited by a relatively small sample size, our study suggests a potentially important role of the intron 4c allele as a genetic marker of ischemic stroke in African Americans.

A simple and rapid quantitative method of detection of the common achondroplasia mutation: analysis in mismatch repair deficient cells.

Achondroplasia is the most common form of dwarfism and has an incidence of approximately 1/7500. In more than 97% of cases, it is caused by a recurrent point mutation, a G to A substitution at nucleotide position 1138 (G1138A) of the fibroblast growth factor receptor 3 gene. Although this is an autosomal dominant condition, more than 90% of all mutations occur sporadically making this one of the most mutagenic sites in the human genome. The reasons for the high spontaneous G1138A mutation rate are not known. This investigation was performed by developing a simple and rapid semi-quantitative allele specific PCR based assay capable of reliably detecting more than 25 mutant G1138A copies in a pool of 300 000 wild type molecules. Using this assay, the G1138A mutation frequency was measured in cell lines deficient in mismatch repair (LoVo, SW48) and comparing it with controls. No differences were found in the frequency of this point mutation between the mismatch repair deficient and wild type cell lines.

Clinical and genetic analysis of CMT1B in a Nigerian family.

We report a Nigerian family with a late-onset autosomal dominant neuropathy consistent with Charcot-Marie-Tooth disease. Electrophysiological examination of the index patient confirmed a severe demyelinating neuropathy with secondary axonal features. Sequence analysis of the myelin protein zero (MPZ) gene identified a C-to-G transversion at nucleotide position 234, resulting in a serine-to-tryptophan mutation in codon 78 (S78W) of the translated protein. The presence of this novel missense mutation suggests a diagnosis of Charcot-Marie-Tooth disease type 1B. Our study confirms the worldwide distribution of this disorder and extends the genetic spectrum of mutations in the MPZ gene.

Familial arachnoid cysts associated with oculopharyngeal muscular dystrophy.

Cerebral arachnoid cysts that occur in more than one member of a family have been rarely reported. These familial cases are important because they imply a genetic component in the pathophysiology of these arachnoid cysts. We present an unusual family in which two conditions, a genetic myopathy, oculopharyngeal muscular dystrophy (OPMD), and arachnoid cysts occur together. OPMD is caused by a mutation in the PAPB2 gene that localizes to chromosome 14. In this family, two siblings with genetically confirmed OPMD both have left hemispheric intracranial arachnoid cysts unassociated with other cerebral abnormalities. The association of these two disorders suggests that in this family, a chromosome 14 gene may play a role in the development of arachnoid cysts.

Castleman's disease-associated neuropathy: no evidence of human herpesvirus type 8 infection.

Castleman's disease (CD) is a rare lymphoproliferative disorder that may be associated with a neuropathy. In a recent report, the presence of human herpesvirus type 8 (HHV-8) DNA sequences were detected in an HIV-negative patient with polyneuropathy, organomegaly, endocrinopathy, M-protein and skin changes (POEMS) associated with the multicentric hyaline vascular variant of CD. It was proposed that the presence of these sequences may have a role in the pathophysiology of the neuropathy. We describe an HIV-negative woman with the multicentric plasma cell form of CD who presented with a disabling neuropathy. In addition to a severe demyelinating polyneuropathy, she had some of the other features of POEMS including an IgA lambda gammopathy and lymphadenopathy. We were unable to detect the presence of either HHV-8 DNA or proteins in this patient. The significance of our results and the relationship between CD, POEMS and neuropathy are discussed.

X-linked Charcot-Marie-Tooth disease caused by a novel point mutation in the connexin-32 gene.

We report the clinical and electrophysiological findings of a patient with X-linked Charcot-Marie-Tooth disease and a novel point mutation in the connexin-32 gene. A 31-year-old man presented with a 5 year history of progressive imbalance and distal weakness in his legs. Electrophysiological studies confirmed an asymmetric, predominantly axonal sensorimotor neuropathy with some demyelinating features. Genetic testing revealed a G/A transition (Ala40Thr) in a conserved transmembrane region of the connexin-32 gene.

Large expansion of the ATTCT pentanucleotide repeat in spinocerebellar ataxia type 10.

Spinocerebellar ataxia type 10 (SCA10; MIM 603516; refs 1,2) is an autosomal dominant disorder characterized by cerebellar ataxia and seizures. The gene SCA10 maps to a 3.8-cM interval on human chromosome 22q13-qter (refs 1,2). Because several other SCA subtypes show trinucleotide repeat expansions, we examined microsatellites in this region. We found an expansion of a pentanucleotide (ATTCT) repeat in intron 9 of SCA10 in all patients in five Mexican SCA10 families. There was an inverse correlation between the expansion size, up to 22.5 kb larger than the normal allele, and the age of onset (r2=0.34, P=0.018). Analysis of 562 chromosomes from unaffected individuals of various ethnic origins (including 242 chromosomes from Mexican persons) showed a range of 10 to 22 ATTCT repeats with no evidence of expansions. Our data indicate that the new SCA10 intronic ATTCT pentanucleotide repeat in SCA10 patients is unstable and represents the largest microsatellite expansion found so far in the human genome.

Mitochondrial DNA deletion mutation levels are elevated in ALS brains.

This study was performed to explore the potential role of mitochondrial DNA mutations in the neurodegenerative process in amyotrophic lateral sclerosis (ALS). Using a semi-quantitative assay, a common mitochondrial DNA deletion mutation (mt DNA4977) was assayed in brain tissue obtained from six sporadic ALS patients and compared to four controls. In each brain, levels of this mutation were measured in a brain region affected by neurodegeneration, the motor cortex (Brodmann area 4), and compared to the temporal cortex (Brodmann area 17). In the ALS but not control brains, levels of mt DNA4977 were an average of more than 30-fold (range 15-250) higher in Brodmann area 4. These results support and extend those of previous studies implying that mitochondria may participate in the neurodegenerative process in ALS.

Mutation analysis of oculopharyngeal muscular dystrophy in Hispanic American families.

BACKGROUND: Oculopharyngeal muscular dystrophy (OPMD) is a late-onset autosomal dominant muscular dystrophy characterized by progressive ptosis, swallowing difficulties, and proximal limb weakness. Recently, the genetic basis of this disease has been characterized by mutations in the PABP2 gene that involve short expansions of the trinucleotide repeat GCG. OBJECTIVES: To independently confirm the presence and study the meiotic stability of the GCG expansion mutations in a distinct ethnic population with OPMD. SETTINGS: Hospital and university research laboratories in Los Angeles, Calif. SUBJECTS AND METHODS: Three unrelated families of Hispanic American descent were identified in whom OPMD was transmitted in an autosomal dominant pattern. All of these families can trace affected ancestors to the southwestern United States or to the bordering states of Mexico. In these families, 14 persons with OPMD were identified and studied. RESULTS: Our results confirm that in these families, expansion mutations characterized by a gain of 3 GCG repeats in the wild-type allele result in an abnormal nucleotide length of 9 GCG repeats in the PABP2 gene. In these families, these mutations are associated with the OPMD phenotype. The identical repeat mutation ([GCG]9) is found in all affected members of these unrelated families and shows relative meiotic stability. CONCLUSIONS: These results support and extend our study of haplotype analysis and suggest that a founder effect may have occurred for OPMD in this Hispanic American population.

C1qB and clusterin mRNA increase in association with neurodegeneration in sporadic amyotrophic lateral sclerosis.

We analyzed postmortem tissues of sporadic amyotrophic lateral sclerosis (SALS) for mRNA levels of two inflammatory proteins, complement C1qB and clusterin (apoJ). By Northern blot hybridization, SALS was associated with increased mRNA for C1qB and clusterin in the motor cortex (Brodmann area A4), but not in superior temporal cortex (A17), relative to neurologically normal controls. By in situ hybridization, SALS spinal cords showed increased C1qB and clusterin mRNA in areas undergoing neurodegeneration. This evidence implicates inflammatory mechanisms during neurodegenerative processes in SALS.

French Machado-Joseph disease patients do not exhibit gametic segregation distortion: a sperm typing analysis.

Segregation distortion has been reported to occur in a number of the trinucleotide repeat disorders. On the basis of a sperm typing study performed in patients of Japanese descent with Machado-Joseph disease (MJD), it was reported that disease alleles are preferentially transmitted during meiosis. We performed a sperm typing study of five MJD patients of French descent and analysis of the pooled data shows a ratio of mutant to normal alleles of 379:436 (46.5:53.5%), which does not support meiotic segregation distortion. To confirm these results, sperm typing analysis was also performed using a polymorphic marker, D14S1050, closely linked to the MJD1 gene. Among 910 sperm analyzed, the allele linked to the disease chromosome was detected in 50.3% of the samples and the allele linked to the normal chromosome was found in 49.6% of the sperm. The difference in frequency of these two alleles is not significant ( P = 0.8423). Likelihood-based analysis of segregation distortion in the single sperm data using the SPERMSEG program also showed no support for segregation distortion at the gamete level in this patient population. The previous report on the Japanese patients also suggested that disease allele stability may be influenced by a trans effect of an intragenic polymorphism (987 G/C) in the wild-type allele. All of the French patients were heterozygous for this polymorphism. However, analysis of the variance in repeat number in sperm from the French MJD patients overlapped significantly with the variance in repeat number observed in the C/C homozygous Japanese patients.

Neurodegeneration in Xeroderma Pigmentosum: a trinucleotide repeat mutation analysis.

Xeroderma Pigmentosum (XP) is a rare autosomal recessive disorder caused by defects in DNA repair. In some forms, it is clinically and pathologically characterized by neurological involvement and premature neuronal death. This study explores the hypothesis that defects in DNA repair in XP may contribute to neurological involvement by destabilizing trinucleotide repeats during replication causing expansion mutations into disease producing ranges. Trinucleotide repeat instability in each of the genes causing Machado-Joseph Disease, myotonic dystrophy, Kennedy's Disease and Huntington's Disease was analyzed by performing single genome PCR. The results of trinucleotide repeat analysis of 360 single genomes from three different forms of XP showed that the size of the repeats were in the normal range and that there was no mitotic instability. These results suggest that in XP, trinucleotide repeat expansion mutations are not involved in the pathophysiology of neurodegeneration.

Clinical and genetic analysis of a distinct autosomal dominant spinocerebellar ataxia.

OBJECTIVE: To characterize a distinct form of spinocerebellar ataxia (SCA) clinically and genetically. BACKGROUND: The SCAs are a genetically heterogeneous group of neurodegenerative disorders affecting the cerebellum and its connections. The mutations for SCA1, 2, 3, 6, and 7 have been identified and shown to be due to expansion of a CAG repeat in the coding region of these genes. Two additional SCA loci on chromosomes 16 and 11 have been designated SCA4 and SCA5. However, up to 20% of individuals with autosomal dominant forms of ataxias cannot be assigned any of these genotypes, implying the presence of other unidentified genes that may be involved in the development of ataxia. METHODS: We ascertained and clinically characterized a six-generation pedigree segregating an autosomal dominant trait for SCA. We performed direct mutation analysis and linkage analysis for all known SCA loci. RESULTS: The mutation analysis excludes SCA1, 2, 3, 6, and 7, and genetic linkage analysis excludes SCA4 and SCA5 (multipoint location scores < -2 across the candidate region). Clinical analysis of individuals in this family shows that all affected members have dysarthria, gait and limb ataxia, and nystagmus. No individuals have major brainstem or long-tract findings. Analysis of age at disease onset through multiple generations suggests anticipation. CONCLUSION: This pedigree represents a genetically distinct form of SCA with a phenotype characterized by predominantly cerebellar symptoms and signs.

Thymectomy for myasthenia gravis.

The pathophysiological role of the thymus in myasthenia gravis, and the mechanism of therapeutic effect of thymectomy, are incompletely understood. Nevertheless, thymectomy is a valuable treatment modality in selected patients with generalised myasthenia gravis. There are several types of thymectomy operation, but no one operative approach is clearly superior to the others. Total removal of the thymus gland is essential. Additional excision of associated mediastinal and cervical tissue, that may harbor ectopic thymic rests, is a controversial surgical issue. Surgeons that advocate thymectomy through small, cosmetically favourable, incisions usually believe that simple removal of the thymus gland is an adequate operation. Surgeons that emphasise the importance of removing extrathymic tissue, in addition to the thymus gland, usually favour greater operative exposure through a median sternotomy. To minimise operative morbidity, surgery for myasthenia gravis requires a multidisciplinary (neurology, surgery, anaesthesia) approach to peri-operative care.

Genetic mapping and haplotype analysis of oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant muscular dystrophy characterized by late onset ptosis, proximal muscle weakness and swallowing difficulties. This disease has been recently linked to chromosome 14q11.2-q13 in French-Canadian pedigrees. We studied three unrelated American families with OPMD of Hispanic descent and our results indicate that in this ethnic group, this disease also maps to chromosome 14q11.2-q13 (marker MYH7.24; Zmax = 3.98; theta max = 0). These results represent an independent demonstration of disease linkage in a second distinct ethnic group. Furthermore, our analysis demonstrates a unique haplotype that is shared by affected individuals from all three families suggesting a founder effect for OPMD in this population. Meiotic recombinants and radiation hybrid mapping permit the narrowing of the critical region to 1 Mb which will facilitate positional cloning of the OPMD disease gene.