Peter Steinbach

Unusual mutations in high functioning fragile X males: apparent instability of expanded unmethylated CGG repeats.

We report on further cases of high functioning fragile X males showing decreased expression of FMR1 protein, absence of detectable methylation at the EagI site in the FMR1 gene promoter, and highly unusual patterns of fragile X mutations defined as smear of expansions extending from premutation to full mutation range. Very diffuse and therefore not easily detectable patterns of full mutations were also observed on prenatal testing using DNA from chorionic villi sampled at a time of development when full mutations were still unmethylated in this particular tissue. In the search for possible determinants of such unusual patterns, repeat expansions in the premutation and in the lower full mutation range were identified on genomic PstI blots previously prepared for fragile X DNA testing. Cases with 130 or more triplets, and a number of shorter repeats, were reinvestigated on EcoRI plus EagI digests. Among the 119 expansions, there were 22 in our sample showing either blurred bands or smears on PstI blots. This particular characteristic was strongly associated with the coincidence of a repeat size of more than 130 triplets and absence of EagI site methylation. Our data set also includes cases of mosaic patterns consisting of smears of unmethylated expansions to more than 130 CGGs and of clear bands of methylated expansions. We therefore suggest that in fragile X syndrome unusual smeared patterns of mutations result from somatic instability of larger repeats under circumstantial absence of repeat methylation.

Comparative analysis of brain structure, metabolism, and cognition in myotonic dystrophy 1 and 2

Objective: Myotonic dystrophy type 1 and 2 (DM1/DM2) are multisystemic diseases with common cognitive deficits beside the cardinal muscular symptoms. We performed a comprehensive analysis of cerebral abnormalities to compare the neuropsychological defects with findings in different imaging methods in the same cohort of patients. Methods: Neuropsychological investigations, structural cerebral MRI including brain parenchymal fraction (BPF) and voxel-based morphometry (VBM), and 18F-deoxy-glucose PET (FDG-PET) were performed in patients (20 DM1 and 9 DM2) and matched healthy controls, and analyzed using statistical parametric mapping (SPM2). Results: DM1 and DM2 patients showed typical neuropsychological deficits with a pronounced impairment of nonverbal episodic memory. Both patient groups showed a reduction of the global gray matter (measured by BPF), which could be localized to the frontal and parietal lobes by VBM. Interestingly, VBM revealed a bilateral hippocampal volume reduction that was correlated specifically to both a clinical score and episodic memory deficits. VBM also revealed a pronounced change of thalamic gray matter. White matter lesions were found in >50% of patients and their extent was correlated to psychomotor speed. FDG-PET revealed a frontotemporal hypometabolism, independent of the decrease in cortical gray matter. All abnormalities were similar in both patient groups but more pronounced for DM1. Conclusions: Our results suggest that 1) some of the characteristic cognitive deficits of these patients are linked to specific structural cerebral changes, 2) decreases in gray matter and metabolism are independent processes, and 3) the widespread brain abnormalities are more pronounced in DM1.

Four familial ALS pedigrees discordant for two SOD1 mutations : are all SOD1 mutations pathogenic?

Background 153 mutations in the Cu/Zn superoxide dismutase (SOD1) gene have been claimed to be associated with amyotrophic lateral sclerosis (ALS) in familial and sporadic ALS in an autosomal dominant or autosomal recessive pattern with complete or reduced penetrance. The authors now report four ALS pedigrees from Finland, France, Germany and Sweden with either the D90A or E100K SOD1 mutations in some but not all affected members. After re-collecting DNA, the non-segregation of the SOD1 mutations with disease was confirmed by three independent laboratories using different PCR primers: while some of the affected patients carry SOD1 mutations, other affected family members have two wildtype/normal SOD1 genes. In addition, some unaffected members within the same families are carriers of SOD1 gene mutations. To exclude other known genetic causes, the authors ruled out mutations within the genes coding for VAPB, ANG, TDP43, FUS and DCTN1 in affected individuals in the four pedigrees. Conclusions The authors find that the D90A and E100K SOD1 gene mutations found in some patients are not the exclusive cause of ALS in these pedigrees. Whether this is also the case for the other 151 SOD1 mutations reported in ALS pedigrees is unknown. The findings have consequences for genetic testing in clinical practice when diagnosing ALS and for genetic counselling in ALS. Some SOD1 mutations may be part of an oligo- or epigentic pattern of inheritance. Such a pattern of inheritance may model other oligo- or polygenetic traits responsible for other forms of ALS.

Characterization of FMR1 Promoter Elements by In Vivo-Footprinting Analysis

Fragile X syndrome is associated with silencing of the FMR1 gene. We studied the transcriptional regulation, by analysis of the FMR1 promoter region for the presence of in vivo protein/DNA interactions and for cytosine methylation at the single-nucleotide level. Four protein-binding sites were present in the unmethylated promoter of the active FMR1 gene. In the methylated promoter of inactive genes no footprints were detected, and no evidence of active repression was found in the region investigated. We propose that the silencing of FMR1 gene transcription results from a lack of transcription-factor binding.

Nonrandom distribution of methotrexate-induced aberrations on human chromosomes. Detection of further folic acid sensitive fragile sites

SummaryEleven folic acid sensitive fragile sites (3p14, 7p13, 7q31.1, 7q32, 9q32, 11p13, 14q23, 15q22, 16q23, Xp22.2, Xq22) were detected in one individual, eight of them previously unknown. These sites seem to bear each its specific sensitivity to folic acid deficiency. Six of the sites were observed simultaneously on both homologous chromosomes in at least one cell. Each of these 11 sites was also found in at least one among 12 individuals further examined. Some of these individuals showed six of these 11 sites. The fragile site 3p14 was detected in all individuals examined. The homologous sites 3p14 of one individual differed from each other in their frequency of lesions induced by methotrexate as well as fluorodeoxyuridine. This observation suggests that folic acid sensitivity is a property inherent in the chromatin of an individual chromosome at the site involved in fragility. This property seems to be responsible for the nonrandom fragility at that site and also for the individual sensitivity of each chromosomal site.

Genotype mosaicism in fragile X fetal tissues

SummaryThe fragile X syndrome is one of the most common familial causes of mental retardation. It is associated with the expression of a fragile site at Xq27.3, although not all individuals carrying the mutation are fragile-X-positive. Recently, the mutation causing this disease has been identified as the amplification of, or insertion into, a CGG repeat sequence at the fragile site. The mutated chromosome can be recognised by the decrease in mobility of the EcoRI fragment that covers the mutated region. Analysis of lymphocytes of affected males often gives a number of different sized fragments indicating somatic heterogeneity. We have investigated this mosaicism in various tissues of an affected fetus in order to determine the extent of the variation between tissues, and to ascertain how to interpret the results in lymphocytes. Our results suggest that the heterogeneity occurs in all fetal tissues, but that the pattern of fragments observed varies between tissues. Methylation across the region also varies. These differences may be reflected in the cellular phenotypes and may influence the ultimate expression of the clinical phenotype.

Quantification of brain atrophy in patients with myotonic dystrophy and proximal myotonic myopathy: a controlled 3-dimensional magnetic resonance imaging study

Myotonic dystrophy (DM1) and proximal myotonic myopathy (PROMM or DM2) are two distinct muscular disorders with multisystemic involvement. Both have previously been reported to be associated with cognitive impairment and white matter lesions detected by cerebral magnetic resonance imaging (MRI). In this study, the extent of brain atrophy was investigated in vivo in ten DM1 and nine PROMM patients in comparison to age-matched healthy controls for each group. The diagnosis was confirmed by DNA analysis of all patients. As a quantitative marker, the ratio of brain parenchymal to intracranial volume, called brain parenchymal fraction (BPF), was calculated from 3-dimensional MRI data using an automated analysis technique. Compared to age-matched healthy controls (mean BPF 0.852 +/- 0.032), the BPF in DM1 patients (0.713 +/- 0.031) was highly significantly decreased (P < 0.001). In contrast, the PROMM patients (mean BPF 0.792 +/- 0.029) showed only slightly decreased BPF values (P < 0.05). BPF was not significantly correlated to any of the clinical or genetic parameters in both diseases (disease duration, motor score, educational level, and number of CTG repeats in the expanded allele). In summary, global brain atrophy was demonstrated to occur in both diseases, but was more severely manifestated in DM1 patients.

Basal ganglia involvement of a patient with SCA 17--a new form of autosomal dominant spinocerebellar ataxia.

Sirs: We describe a patient with spinocerebellar ataxia 17 (SCA17), a condition that has recently been linked to an abnormal CAG expansion in the TATA-binding protein, a general transcription factor [4–7]. A 35-year-old male was referred to our hospital with a five year history of slowly progressive speech disorder and a one year history of dysphagia. The patient recalled “movement problems” in his father’s mother who died with dementia of unknown aetiology at age 57 years. A son of his father’s brother started to experience cognitive deficits, speech problems and loss of fine motor skills at the age of 31 years. The father, however, has never experienced any neurological problems. Neurological examination revealed marked cerebellar dysarthria, a diminished gag reflex, but no other neurological abnormalities. Mild cognitive impairment was suggested with a Mini Mental State (MMS) of 26 (maximum 30) and SISCO (SIDAM) score of 40 (maximum 55) points. Laboratory findings were normal. The cerebrospinal fluid (CSF) analysis showed a total protein concentration of 675 mg/l, other results were normal. Brain stem auditory evoked potentials (BEAP) detected a mesencephalic lesion bilaterally. The latencies of the masseter and blink reflex responses were within normal limits. Electrooculography revealed hypometric saccades. Magnetic resonance imaging demonstrated cortical cerebellar atrophy. Striatal dopamine transporter (DAT) availability and striatal dopamine D2 receptor (D2R) were measured with [123I]FP-CIT, and [123I]IBZM (Amersham Health), respectively, applying a brain dedicated single-photon emission computed tomography (SPECT) system (Ceraspect, DSI) and an MRI-based region of interest (ROI) technique to calculate specific-to-non-specific binding ratios. DAT availability was 23–25 % lower than our agematched controls at baseline [right striatum: 4.43 vs. 5.74 ± 1.40 [mean ± SD], 95 %-confidence interval: upper limit 6.80, lower limit 5.09, left striatum: 4.25 vs. 5.67 ± 1.23 (6.74, 5.07), reference region: occipital cortex], which was clearly beyond our test-retest variability [3]. This reduction declined over a twoyear follow-up period (ratios: 3.10, right striatum, 3.24, left striatum, Fig. 1). Positron emission tomography (PET) with [18F]Fluorodeoxyglucose revealed a reduced striatal glucose metabolic rate (32.7 μmol/ min/100 mL brain tissue), which exceeds an only slightly diminished D2R binding capacity [right striatum: 2.18 vs. 2.34 ± 0.20 (2.46, 2.21), left striatum: 2.13 vs. 2.32 ± 0.15 (2.43, 2.18), reference region: frontal cortex, Fig. 2]. Neither abnormalities in the cerebral cortical and cerebellar glucose metabolic rate (36.6 μmol/min/100 mL, and 35.3 μmol/min/100 mL, respectively) nor significant follow-up changes of glucose metabolism and D2R binding capacity were found. An oesophagogram and videocinematography revealed dysphagia and a prolonged pharyngeal phase. In conclusion, an autosomal dominant degenerative disorder involving cerebellum and basal ganglia was diagnosed. Genotyping revealed an abnormal expansion in the TATA-binding protein (TBP) gene demonstrating one allele with one pathological expanded fragment of TBP gene with 50 CAG repeats and one normal allele with 37 CAG repeats (normal range: 27 to 44 CAG repeats) confirming spinocerebellar ataxia SCA 17. LETTER TO THE EDITORS

Increase of FMRP expression, raised levels of FMR1 mRNA, and clonal selection in proliferating cells with unmethylated fragile X repeat expansions: a clue to the sex bias in the transmission of full mutations?

Fragile X syndrome is a triplet repeat disorder caused by expansions of a CGG repeat in the fragile X mental retardation gene (FMR1) to more than 220 triplets (full mutation) that usually coincide with hypermethylation and transcriptional silencing. The disease phenotype results from deficiency or loss of FMR1 protein (FMRP) and occurs in both sexes. The underlying full mutations arise exclusively on transmission from a mother who carries a premutation allele (60-200 CGGs). While the absolute requirement of female transmission could result from different mechanisms, current evidence favours selection or contraction processes acting at gametogenesis of pre- and full mutation males. To address these questions experimentally, we used a model system of cultured fibroblasts from a male who presented heterogeneous unmethylated expansions in the pre- and full mutation size range. On continual cell proliferation to 30 doublings we re-examined the behaviour of the expanded repeats on Southern blots and also determined the expression of theFMR1 gene by FMRP immunocytochemistry, western analysis, and RT-PCR. With increasing population doublings, expansion patterns changed and showed accumulation of shorter alleles. The FMRP levels were below normal but increased continuously while the cells that were immunoreactive for FMRP accumulated. The level ofFMR1 mRNA was raised with even higher levels of mRNA measured at higher passages. Current results support the theory of a selection advantage of FMRP positive over FMRP deficient cells. During extensive proliferation of spermatogonia in fragile X males, this selection mechanism would eventually replace all full mutations by shorter alleles allowing more efficient FMRP translation. At the proliferation of oogonia of carrier females, the same mechanism would, in theory, favour transmission of any expandedFMR1 allele on inactive X chromosomes.

Detection of long-term progression of myocardial fibrosis in Duchenne muscular dystrophy in an affected family: a cardiovascular magnetic resonance study.

BACKGROUND Detection of myocardial fibrosis and left ventricular dysfunction in Duchenne muscular dystrophy (DMD) is the corner stone for further therapeutic studies. Little is known about the ability of cardiac magnetic resonance imaging (CMR) to evaluate progression of myocardial fibrosis. Aim of our study was to provide CMR data in a previously genotyped DMD family and to evaluate whether progression of myocardial fibrosis could be visualized. METHODS AND RESULTS DMD genotypes were available in 14 family members. CMR was performed in 4/5 carrier females, in 2/2 affected males and in one healthy family member with normal genotype. Functional images and late gadolinium enhanced (LGE) images in contiguous short-axis orientation were acquired at baseline and follow-up of 1231 days CMR examination could be repeated in three carrier females, in one affected male and in the healthy subject previously scanned. Mean decrease of left ventricular ejection fraction during the follow-up period was 10.5±11.0%, mean progression of LGE volume 11.7±9.5%. CONCLUSIONS Myocardial fibrosis seems to occur prior to global left ventricular dysfunction in DMD diseased males and carrier females. CMR could be used to evaluate progression of myocardial fibrosis and left ventricular function and may thus serve as an important diagnostic tool in the evaluation of therapeutical options in DMD.