Judith Armstrong

Effects of Formalin Fixation, Paraffin Embedding, and Time of Storage on DNA Preservation in Brain Tissue: A BrainNet Europe Study

There is a large amount of tissue stored in brain collections and brain banks, but little is known about whether formalin‐fixed tissues and paraffin blocks stored for years in brain banks are suitable for the retrospective genetic studies. The study was carried out in order to: (i) compare DNA preservation in frozen, formalin‐fixed and paraffin‐embedded tissues stored for different periods; (ii) study point mutations and triplet expansions in frozen, formalin‐fixed and paraffin‐embedded material stored for variable periods, and using different fixative solutions; (iii) compare different methods to optimize DNA extraction and DNA amplification from suboptimally preserved brain tissue. DNA preservation is suitable for genetic studies in samples stored at −80°C for several years. Formalin‐fixed, paraffin‐embedded tissue was inferior to frozen tissue, but did yield adequate results in many cases depending on the type of fixative solution and time of fixation before embedding. Prolonged fixation in formalin rarely yielded useful DNA. Similar results were obtained in samples from prion diseases. The best results were obtained by using the Qiagen kits (QIAmp DNA Micro) in frozen material, paraffin blocks and formalin‐fixed tissue. Genomiphi™ and TaKaRa Ex Taq™ methods were also assayed in paraffin blocks and in formalin‐fixed samples with limited success.

Novel FOXG1 mutations associated with the congenital variant of Rett syndrome

Background Rett syndrome is a severe neurodevelopmental disorder representing one of the most common genetic causes of mental retardation in girls. The classic form is caused by MECP2 mutations. In two patients affected by the congenital variant of Rett we have recently identified mutations in the FOXG1 gene encoding a brain specific transcriptional repressor, essential for early development of the telencephalon. Methods 60 MECP2/CDKL5 mutation negative European Rett patients (classic and variants), 43 patients with encephalopathy with early onset seizures, and four atypical Rett patients were analysed for mutations in FOXG1. Results and conclusions Mutations have been identified in four patients, independently classified as congenital Rett variants from France, Spain and Latvia. Clinical data have been compared with the two previously reported patients with mutations in FOXG1. In all cases hypotonia, irresponsiveness and irritability were present in the neonatal period. At birth, head circumference was normal while a deceleration of growth was recognised soon afterwards, leading to severe microcephaly. Motor development was severely impaired and voluntary hand use was absent. In contrast with classic Rett, patients showed poor eye contact. Typical stereotypic hand movements with hand washing and hand mouthing activities were present continuously. Some patients showed abnormal movements of the tongue and jerky movements of the limbs. Brain magnetic resonance imaging showed corpus callosum hypoplasia in most cases, while epilepsy was a variable sign. Scoliosis was present and severe in the older patients. Neurovegetative symptoms typical of Rett were frequently present.

Rett syndrome in Spain: mutation analysis and clinical correlations

Rett syndrome (RTT) is an X-linked neurodevelopmental disease that affects girls almost exclusively. In a high proportion of patients the disease is caused by de novo mutations at the MECP2 gene, encoding methyl-CpG-binding protein 2. With the aim to characterize the spectrum of mutations in a series of sporadic RTT patients, including an affected male, and to relate the genetic results to the clinical features of the disease, a clinical checklist and a score system were elaborated to evaluate the clinical severity of the disease. Mutation analysis of the MECP2 coding region was done by direct sequencing. De novo mutations were found in 60% of the patients, including both classic and atypical forms. The change R133H was identified in a 13-year-old boy showing a classic RTT phenotype and normal karyotype. Significant differences were observed among missense and truncating mutations regarding disease severity, age of onset of stereotypies, and the ability of the patients to sit alone and to walk.

The impact of MECP2 mutations in the expression patterns of Rett syndrome patients

Rett syndrome (RTT), the second most common cause of mental retardation in females, has been associated with mutations in MeCP2, the archetypical member of the methyl-CpG binding domain (MBD) family of proteins. MeCP2 additionally possesses a transcriptional repression domain (TRD). We have compared the gene expression profiles of RTT- and normal female-derived lymphoblastoid cells by using cDNA microarrays. Clustering analysis allowed the classification of RTT patients according to the localization of the MeCP2 mutation (MBD or TRD) and those with clinically diagnosed RTT but without detectable MeCP2 mutations. Numerous genes were observed to be overexpressed in RTT patients compared with control samples, including excellent candidate genes for neurodevelopmental disease. Chromatin immunoprecipitation analysis confirmed that binding of MeCP2 to corresponding promoter CpG islands was lost in RTT-derived cells harboring a mutation in the region of the MECP2 gene encoding the MBD. Bisulfite genomic sequencing demonstrated that the majority of MeCP2 binding occurred in DNA sequences with methylation-associated silencing. Most importantly, the finding that these genes are also methylated and bound by MeCP2 in neuron-related cells suggests a role in this neurodevelopmental disease. Our results provide new data of the underlying mechanisms of RTT and unveil novel targets of MeCP2-mediated gene repression.

Clinical and myopathological evaluation of early- and late-onset subtypes of myofibrillar myopathy

Myofibrillar myopathies (MFM) are a group of disorders associated with mutations in DES, CRYAB, MYOT, ZASP, FLNC, or BAG3 genes and characterized by disintegration of myofibrils and accumulation of degradation products into intracellular inclusions. We retrospectively evaluated 53 MFM patients from 35 Spanish families. Studies included neurologic exam, muscle imaging, light and electron microscopic analysis of muscle biopsy, respiratory function testing and cardiologic work-up. Search for pathogenic mutations was accomplished by sequencing of coding regions of the six genes known to cause MFM. Mutations in MYOT were the predominant cause of MFM in Spain affecting 18 of 35 families, followed by DES in 11 and ZASP in 3; in 3 families the cause of MFM remains undetermined. Comparative analysis of DES, MYOT and ZASP associated phenotypes demonstrates substantial phenotypic distinctions that should be considered in studies of disease pathogenesis, for optimization of subtype-specific treatments and management, and directing molecular analysis.

Classic rett syndrome in a boy as a result of somatic mosaicism for a mecp2 mutation

Rett syndrome (RTT) is a neurodevelopmental disorder affecting females in a sporadic manner. In a high proportion of patients the disease is caused by de novo dominant mutations at MECP2 gene (Xq28). The existence of RTT males has been discussed extensively, and less restrictive diagnostic criteria have been proposed to include this variant, which should be considered when evaluating boys with idiopathic developmental regression, autistic features, and loss of hand function. Nevertheless, no MECP2 analysis has been reported from Rett-like males to date. Analysis of 2 familial cases showed that boys carrying the same MECP2 mutation that caused RTT in their sisters suffered from severe–fatal neonatal encephalopathy. Recent data, however, demonstrate that the clinical spectrum of MECP2 mutations is wider than previously expected. With a frequency comparable to that of fragile X syndrome, recessive nonspecific X-linked mental retardation can be caused by missense mutations at MECP2, different than those causing RTT. Mutations have also been described in patients with congenital nonprogressive encephalopathy and in some cases of Angelman syndrome (AS), the only reported AS boy being a somatic mosaic for a MECP2 truncating mutation. We document the first MECP2 analysis of a boy with classic RTT and a normal 46,XY karyotype. The patient is 14 years of age and fulfils eight of nine necessary criteria, seven of eight supportive criteria, and no exclusion criteria, according to the Rett Syndrome Diagnostic Criteria Work Group. Genetic informed consent was obtained and the study was approved by the Ethical and Investigation Commissions of our hospital. MECP2 sequencing of two independent patients’ DNA samples from peripheral lymphocytes revealed the presence of a heterozygous change 398G3A, causing an R133H substitution. The mutation had been previously described in 2 RTT female patients. As the boy had a normal karyotype, heterozygosity could be explained by (1) a low frequency mosaicism for a Klineffelter syndrome, discarded by FISH on prophasic nuclei; (2) a MECP2 locus duplication, rejected by high-resolution karyotype and observation of hemizygosity for X-linked markers and two intragenic MECP2 polymorphisms; or (3) somatic mosaicism for the mutation. To test this last hypothesis, DNA was prepared from the patient’s oral mucosa and sequenced. The normal sequence, with only a small amount of the mutated allele, was observed. These results demonstrated that the RTT boy is a somatic mosaic for the R133H mutation and seemed to indicate that the mutation is present in a high proportion of lymphocytes but at a lower frequency in oral mucous. To specifically test and semiquantify the heterozygous status of the mutation, an amplification–refractory mutation system (ARMS) experiment was designed. Both the normal and mutated alleles were amplified in the patient’s lymphocytes and oral mucosa (Fig). Relative differences in band intensities corroborated the different degree of mosaicism between the two tissues. The infrequent clinical picture of this patient is thus explained by the existence of somatic mosaicism for an RTTcausing MECP2 mutation. Concerning X-linked diseases, this phenomenon in males mimics the result of lyonization naturally occurring in females and may explain the classic Rett syndrome phenotype of the boy.

Phenotypic patterns of desminopathy associated with three novel mutations in the desmin gene

Desminopathy represents a subgroup of myofibrillar myopathies caused by mutations in the desmin gene. Three novel disease-associated mutations in the desmin gene were identified in unrelated Spanish families affected by cardioskeletal myopathy. A selective pattern of muscle involvement, which differed from that observed in myofibrillar myopathy resulting from mutations in the myotilin gene, was observed in each of the three families with novel mutations and each of three desminopathy patients with known desmin mutations. Prominent joint retractions at the ankles and characteristic nasal speech were observed early in the course of illness. These findings suggest that muscle imaging in combination with routine clinical and pathological examination may be helpful in distinguishing desminopathy from other forms of myofibrillar myopathy and ordering appropriate molecular investigations.

Assessment of a targeted resequencing assay as a support tool in the diagnosis of lysosomal storage disorders

BackgroundWith over 50 different disorders and a combined incidence of up to 1/3000 births, lysosomal storage diseases (LSDs) constitute a major public health problem and place an enormous burden on affected individuals and their families. Many factors make LSD diagnosis difficult, including phenotype and penetrance variability, shared signs and symptoms, and problems inherent to biochemical diagnosis. Developing a powerful diagnostic tool could mitigate the protracted diagnostic process for these families, lead to better outcomes for current and proposed therapies, and provide the basis for more appropriate genetic counseling.MethodsWe have designed a targeted resequencing assay for the simultaneous testing of 57 lysosomal genes, using in-solution capture as the enrichment method and two different sequencing platforms. A total of 84 patients with high to moderate-or low suspicion index for LSD were enrolled in different centers in Spain and Portugal, including 18 positive controls.ResultsWe correctly diagnosed 18 positive blinded controls, provided genetic diagnosis to 25 potential LSD patients, and ended with 18 diagnostic odysseys.ConclusionWe report the assessment of a next–generation-sequencing-based approach as an accessory tool in the diagnosis of LSDs, a group of disorders which have overlapping clinical profiles and genetic heterogeneity. We have also identified and quantified the strengths and limitations of next generation sequencing (NGS) technology applied to diagnosis.

Giant SCA8 alleles in nine children whose mother has two moderately large ones.

We report here a family in which each of nine children has inherited giant SCA8 CTG expansions from a homozygous mother who has two moderately large SCA8 CTG alleles. In contrast, three homozygous male individuals and a case of coexistence of two expansions of the FRDA gene and one of SCA8, all of them with moderately large alleles, have transmitted their respective SCA8 expanded alleles with minor changes, as usually occurs in heterozygous male transmissions. Ann Neurol 2005;57:549–553

Epilepsy in Rett syndrome—Lessons from the Rett networked database

Rett syndrome is an X‐linked dominant neurodevelopmental disorder caused by mutations in the MECP2 gene, and characterized by cognitive and communicative regression, loss of hand use, and midline hand stereotypies. Epilepsy is a core symptom, but literature is controversial regarding genotype–phenotype correlation. Analysis of data from a large cohort should overcome this shortcoming.