X chromosome inactivation in females with multiple sclerosis

The aetiology of multiple sclerosis (MS) is unknown. Autoimmune mechanisms are most probably involved. Loss of immunological tolerance to self-antigens is a common feature of autoimmune disorders. Response to X-linked self-antigens could be influenced by X-chromosome inactivation, and contribute to the gender bias observed in autoimmune disorders. Previous studies have indicated an association between skewed X inactivation and autoimmune thyroid disease and scleroderma. To investigate a potential role of X inactivation in MS, we compared the X-inactivation pattern in 568 female MS patients with controls. We found no difference in degree of skewing between patients (median 64%) and controls (median 65%) ( P  = 0.474). The X-inactivation pattern did thus not explain the female predominance of MS patients in general. As the aetiology of different subgroups of MS may differ, patients were grouped according to disease course: relapsing–remitting (RR-MS), secondary progressive (SP-MS) and primary progressive (PP-MS). A comparison of the X-inactivation pattern between subgroups indicated a possible difference in degree of skewing between patients with a progressive versus a relapsing course ( P  = 0.05).     

Patterns of X chromosome inactivation in the Rett syndrome

The Rett syndrome (RS) is a degenerative neurological disorder occurring exclusively in young females. The disorder is sporadic in the majority of the cases, however a few familial cases with inheritance through maternal lines have been identified. Based on these observations the condition could be due to an X chromosome mutation which is lethal in males. To explain the familial cases, a hypothesis of possible non-random X inactivation is proposed. To investigate the possibility of non-random X chromosome inactivation in RS, we carried out analysis using restriction fragment length polymorphisms (RFLPs) and methylation sensitive enzymes at the PGK and HPRT loci. The results show that there is increased incidence of non-random X chromosome inactivation in peripheral blood leukocytes in sporadic RS patient (36%), as compared to healthy controls (8%). Using brain tissue from three patients, only a random pattern was detected, although varying degrees of skewing were detected in the peripheral tissues of these patients. Analysis of leukocyte DNA from a mother of two affected half-sisters revealed non-random X chromosome inactivation suggesting a possible selection against RS allele. Additional familial cases of RS should be evaluated to determine if this observation is common to all female carriers. If non-random X chromosome inactivation occurs in all the putative "carriers," this would be the first evidence to support the hypothesis of an X linked mutation which is lethal in males.     

Psychiatric disability in female carriers of the fragile X chromosome

Fragile X syndrome, an X-linked genetic condition, is an important genetic cause of mental retardation in males. In addition to mental retardation, hemizygous males with fragile X syndrome appear to have a greater likelihood of displaying behaviors classified under the diagnostic category of pervasive developmental disorder than would be expected on the basis of mental retardation alone. Although the majority of female heterozygotes with the fragile X genetic defect are of normal intelligence, our clinical work with this population and a recent case report have suggested that females with fragile X syndrome have an increased rate of schizophrenia spectrum and affective disorders. In this study, the relationship of the fragile X genetic defect to psychopathology in female heterozygotes is investigated by psychiatric evaluation of 35 obligate female carriers of the fragile X chromosome and a comparison group of 24 fragile X-negative controls. Female fragile X carriers were found to have a greater frequency of psychopathology associated with schizophrenia spectrum diagnoses, particularly schizotypal features. A weaker association between the fragile X genetic defect and chronic affective disorders was detected. The specificity of the neuropsychiatric phenotype occurring in particular genetic conditions such as the fragile X syndrome adds a potentially valuable tool to the study of psychopathology in the general population.     

Molecular-cytogenetic investigation of skewed chromosome X inactivation in Rett syndrome

We have developed an approach to differentiate homologous X chromosomes in metaphase chromosomes and interphase nuclei by a fluorescence in situ hybridization (FISH) technique with chromosome X-specific alpha-satellite DNA probe. FISH analysis of metaphase chromosomes in a cohort of 33 girls with Rett syndrome (RTT) allowed us to detect eight girls with structurally different X chromosomes, one X chromosome with a large and another one with a small centromeric heterochromatin (so-called chromosomal heteromorphism). Step-wise application of differential replication staining and the FISH technique to identify the inactivation status of paternal and maternal chromosome X in RTT girls was applied. Skewed X inactivation in seven RTT girls with preferential inactivation of one X chromosome over the other X chromosome was detected in 62–93% of cells. Therefore, non-random or skewed X inactivation with variable penetrance in blood cells could take place in RTT.     

Influence of mutation type and X chromosome inactivation on Rett syndrome phenotypes

We screened 71 sporadic and 7 familial Rett syndrome (RTT) patients for MECP2 mutations by direct sequencing and determined the pattern of X chromosome inactivation (XCI) in 39 RTT patients. We identified 23 different disease-causing MECP2 mutations in 54 of 71 (76%) sporadic patients and in 2 of 7 (29%) familial cases. We compared electrophysiological findings, cerebrospinal fluid neurochemistry, and 13 clinical characteristics between patients carrying missense mutations and those carrying truncating mutations. Thirty-one of 34 patients (91%) with classic RTT had random XCI. Nonrandom XCI was associated with milder phenotypes, including a mitigated classic RTT caused by a rare early truncating mutation. Patients with truncating mutations have a higher incidence of awake respiratory dysfunction and lower levels of cerebrospinal fluid homovanillic acid. Scoliosis is more common in patients with missense mutations. These data indicate that different MECP2 mutations have similar phenotypic consequences, and random XCI plays an important role in producing the full phenotypic spectrum of classic RTT. The association of early truncating mutations with nonrandom XCI, along with the fact that chimeric mice lacking methyl-CpG-binding protein 2 (MeCP2) function die during embryogenesis, supports the notion that RTT is caused by partial loss of MeCP2 function.     

Absence of correlation between X chromosome inactivation pattern and plasma concentration of factor VIII and factor IX in carriers of haemophilia A and B

Haemophilia A and B are X-linked disorders which are due to a reduced activity of coagulation factor VIII or IX, respectively. Female carriers have a wide range of plasma concentration of factor VIII or factor IX, and may in rare cases have an affected phenotype. In order to investigate if this variation is related to X chromosome inactivation, we determined the X inactivation pattern in 31 haemophilia A and 15 haemophilia B carriers, using a PCR in the androgen receptor locus in blood DNA. Seven of the haemophilia A carriers and none of the haemophilia B carriers had a skewed pattern (> or =80:20). One of the skewed haemophilia A carriers had a low plasma concentration of factor VIII (0.15 U/ml), but the remaining 6 carriers did not differ in factor VIII concentration from that of carriers with a random X inactivation pattern. One carrier with a high factor VIII concentration (2.0 U/ml) did not have a skewed pattern. Similarly, for the haemophilia B carriers, there was no tendency to a more skewed X inactivation pattern in the carriers with low or high factor IX concentrations. In addition, we analysed a female with haemophilia B who was heterozygous for the mutation R180W in the factor IX gene. She had a random X chromosome inactivation pattern. We conclude that the wide range in plasma concentration of factor VIII and factor IX in haemophilia A and B carriers cannot in general be explained by the X chromosome inactivation pattern in peripheral blood cells.     

X chromosome inactivation in Rett Syndrome and its correlations with MECP2 mutations and phenotype

Rett syndrome (RTT) is an X-linked dominant neurodevelopment disorder, which is mainly caused by gene mutation of methyl-CpG-binding protein 2 (MECP2). The correlations between genotype, X chromosome inactivation (XCI), and phenotype have been studied, but the results are conflicting. In the present study, XCI patterns in patients and their mothers, parental origin of skewed X chromosome in patients, and the correlations between XCI, genotype, and phenotype were analyzed in 52 cases of RTT with MECP2 mutations, 50 RTT mothers, and 48 normal female controls. The results showed XCI and genotype had limitations in explaining all the phenotypic manifestations of RTT. Other genomic factors have to be considered to explain the phenotypic differences.     

Random X chromosome methylation patterns in the carriers with clinical phenotypic expressions of X-linked recessive bulbospinal neuronopathy

OBJECTIVES: We report the unusual phenotypic expression in 2 female carriers of a family with X-linked recessive bulbospinal neuronopathy (X-BSN). We analyze the methylation pattern of the androgen receptor (AR) gene to inspect the possibility of non-random X chromosome inactivation to be the underlying mechanism. MATERIAL AND METHODS: Twenty-three members in 3 generations of a Taiwanese family were examined and studied for genomic DNA analysis. We analyzed the sequence of the first exon of the AR gene to identify the numbers of CAG repeats, and to determine the methylation pattern by using the restriction enzymes HpaII and HhaI. RESULTS: There were 3 probands and 5 carriers and 2 of the carriers manifested clinical symptoms. Sequence analysis revealed that the numbers of trinucleotide repeats ranged from 42 to 45 in one allele of the X-chromosome in the 5 female carriers. The restriction pattern of the HpaII and HhaI recognizable sites of the X-chromosome indicated a random methylation. CONCLUSION: Our data suggest that molecular genetic studies are important in confirming the diagnosis of X-BSN and early detection of female carriers, and the random or non-random methylation pattern of the X-chromosome is not a determining factor for partial expression of the abnormal AR gene in some carriers.     

Chromosome fragility and psychopathology in obligate female carriers of the fragile X chromosome.

The relationship between fragility (the percentage of cells exhibiting the fragile X chromosome abnormality) and psychopathological conditions was investigated in a sample of 40 obligate female carriers of the fragile X chromosome. Subjects were categorized by those with positive fragility greater than 0% (n = 19) and those with 0% fragility (n = 21). Compared with women with 0% fragility, it was expected that women with positive fragility would have a higher likelihood of manifesting a spectrum of social and psychological disability previously shown to be associated with fragile X syndrome in women. It was also expected that within the group with positive fragility, degree of fragility would be related to severity of symptoms. Results partially supported the hypotheses: women with fragility over 0% were more likely to be assigned a diagnosis of schizotypal features, were rated higher on symptoms associated with the schizophrenia spectrum, and scored lower on IQ, level of healthiest functioning, education, and socioeconomic status than women with 0% fragility. Subsequent comparisons with a control group indicated that the group with 0% fragility and normal controls did not differ on these variables. Within the group with positive fragility, increasing fragility was related to greater severity of symptoms and lower IQ, education, socioeconomic status, and levels of adaptive functioning, as predicted. Contrary to expectations, positive fragility was not associated with proportion of affective disorder diagnoses or ratings on affective disorder symptoms. The results of the study provide evidence that degree of fragility is a potentially important predictor of psychopathology among women with normal IQ who are carriers of the fragile X chromosome abnormality.     

Possible manifestation of the dystrophic X chromosome in muscle cultures from carriers of Duchenne muscular dystrophy

Multilayer cell clusters have been observed before confluence and before myotube formation in muscle cell cultures derived from open biopsies of 7 of 14 (50%) female carriers of Duchenne muscular dystrophy, and in a high percentage of other dystrophic cultures. By contrast, this abnormality was seen in only 12 of 204 (6%) muscle biopsies from patients with other neuromuscular disorders. It appears that cluster formation is independent of the amount of connective tissue present in vivo, because histopathological analysis of the carrier biopsies showed increased endomysial connective tissue in only two cases. These results suggest that cluster formation is an expression of a myogenic defect and that it may be a manifestation of the genetic abnormality in X-linked muscular dystrophy.     

X chromosome inactivation patterns in brain in Rett syndrome: implications for the disease phenotype

Skewed X chromosome inactivation (XCI) has been implicated in modulating the severity of Rett syndrome (RTT), although studies by different groups have yielded conflicting results. In this study we have characterised the XCI pattern in various neuroanatomical regions of nine RTT brains and non-neural tissue in two of these patients to determine whether or not variable XCI patterns occur in different brain regions or somatic tissues of the same patient. The mean XCI patterns for frontal and occipital cortex were compared between RTT and control subjects, and showed no significant differences when comparing RTT frontal to control frontal cortex or RTT occipital to control occipital cortex. However, one RTT subject displayed variability across the different neuroanatomical regions of the brain and skewing in some non-neural tissues. This observation adds another dimension to the epigenetic factors that may contribute to the phenotype in RTT. It also mandates that caution should be exercised in factoring XCI, including assumptions based on the blood XCI pattern, into the development of phenotype-genotype correlations.     

X inactivation in females with X-linked Charcot-Marie-Tooth disease

X-linked Charcot-Marie-Tooth disease (CMT1X) is the second most common inherited neuropathy, caused by mutations in gap junction beta-1 (GJB1). Males have a uniformly moderately severe phenotype while females have a variable phenotype, suggested to be due to X inactivation. We aimed to assess X inactivation pattern in females with CMT1X and correlate this with phenotype using the CMT examination score to determine whether the X inactivation pattern accounted for the variable phenotype in females with CMT1X. We determined X inactivation pattern in 67 females with CMT1X and 24 controls using the androgen receptor assay. We were able to determine which X chromosome carried the GJB1 mutation in 30 females. There was no difference in X inactivation pattern between patients and controls. In addition, there was no correlation between X inactivation pattern in blood and phenotype. A possible explanation for these findings is that the X inactivation pattern in Schwann cells rather than in blood may explain the variable phenotype in females with CMT1X.     

Dystrophin gene analysis on 76 families with dystrophinopathy

We report for the last 1 year and 9 months results of the molecular diagnosis of Duchenne (DMD) and Becker (BMD) muscular dystrophy at Kobe University. Analysis was done on 87 patients belonging to 76 families (66 DMD cases, 10 BMD cases). We first determined the presence of deletions or duplications using Southern blot analysis. Then analyzed small mutations (point mutations or small deletions/insertions) by mRNA analysis using RT-PCR and direct sequencing. 69 mutations (89.5%) were found, 46 deletions (60.5%), 5 duplications (6.6%) and 17 point mutations (22.4%) including 15 nonsense mutation. mRNA analysis from lymphocytes or muscle was useful for screening patients without a mutation identifiable by Southern blot analysis.     

Symptomatic generalized epilepsy associated with an inverted duplication of chromosome 15.

An inverted duplication of chromosome 15 (inv dup[15] chromosome) is the most common supernumerary marker chromosome in humans. Inv dup(15) chromosomes are commonly associated with mental retardation, epilepsy, behavioral problems and structural malformations. Though epilepsies associated with inv dup(15) chromosomes are often intractable, there have been very few reports regarding the seizure manifestations or types. We report a patient with severe mental retardation and intractable epilepsy, associated with an inv dup(15) chromosome. The seizures recorded with EEG-VTR monitoring were axial and generalized tonic seizures, and our case was diagnosed as symptomatic generalized epilepsy. Molecular and cytogenetic analysis showed an inv dup(15) chromosome containing the Prader-Willi syndrome/Angelman syndrome region mapped within bands 15q 11-q13.     

Ataluren treatment of patients with nonsense mutation dystrophinopathy

Introduction: Dystrophinopathy is a rare, severe muscle disorder, and nonsense mutations are found in 13% of cases. Ataluren was developed to enable ribosomal readthrough of premature stop codons in nonsense mutation (nm) genetic disorders. Methods: Randomized, double‐blind, placebo‐controlled study; males ≥5 years with nm‐dystrophinopathy received study drug orally 3 times daily, ataluren 10, 10, 20 mg/kg (N = 57); ataluren 20, 20, 40 mg/kg (N = 60); or placebo (N = 57) for 48 weeks. The primary endpoint was change in 6‐Minute Walk Distance (6MWD) at Week 48. Results: Ataluren was generally well tolerated. The primary endpoint favored ataluren 10, 10, 20 mg/kg versus placebo; the week 48 6MWD Δ = 31.3 meters, post hoc P = 0.056. Secondary endpoints (timed function tests) showed meaningful differences between ataluren 10, 10, 20 mg/kg, and placebo. Conclusions: As the first investigational new drug targeting the underlying cause of nm‐dystrophinopathy, ataluren offers promise as a treatment for this orphan genetic disorder with high unmet medical need. Muscle Nerve 50 : 477–487, 2014