X-chromosomal Inactivation Research Articles

Skewed X-inactivation is associated with retinal dystrophy in female carriers of RPGR mutations.

Progressive degeneration of rod and cone photoreceptors frequently is caused by mutations in the X-chromosomal gene Retinitis Pigmentosa GTPase Regulator (RPGR). Males hemizygous for a RPGR mutation often are affected by Retinitis Pigmentosa (RP), whereas female mutation carriers only occasionally present with severe RP phenotypes. The underlying pathomechanism leading to RP in female carriers is not well understood. Here, we analyzed a three-generation family in which two of three female carriers of a nonsense RPGR mutation presented with RP. Among two cell lines derived from the same female family members, differences were detected in RPGR transcript expression, in localization of RPGR along cilia, as well as in primary cilium length. Significantly, these differences correlated with alterations in X-chromosomal inactivation patterns found in the patient-derived cell lines from females. In summary, our data suggest that skewed X-chromosomal inactivation is an important factor that determines the disease manifestation of RP among female carriers of pathogenic sequence alterations in the RPGR gene.

Identification of a novel CACNA1F mutation in a Chinese family with CORDX3.

X-linked cone-rod dystrophy (CORDX) is one form of inherited retinal disorders (IRDs) characterized by progressive dysfunction of photoreceptor. Three types of CORDX were reported and CACNA1F gene defect can cause CORDX3. The aim of this study was to investigate the pathogenic variant in a Chinese family with IRD. The two affected subjects including the proband and his elder sister underwent ophthalmic examinations. Whole exome sequencing (WES) was performed in the proband at first, then co-segregation analysis was performed in the family by Sanger sequencing. Minigene approach was used to verify the effect of the mutation on the splicing of CACNA1F. X-chromosomal inactivation assay was performed to evaluate the inactivation patterns of the female carriers. The ophthalmic examination results of the proband fit the clinical description of CORDX3, and the female patient presented with only mild symptoms due to mildly skewed X-chromosomal inactivation (ratio 67: 33). Molecular genetic testing identified a novel splice-site mutation c.3847-2A > G in CACNA1F (NM_005183.4) gene in the patients, which inherited from their asymptomatic mother. Minigene approach confirmed that c.3847-2A > G could affect the splicing of CACNA1F. Our study identified a novel splice-site mutation in the CACNA1F gene, which expanded the mutational spectrum of CACNA1F-releated diseases and demonstrated the importance of combining clinical and genetic testing in the diagnosis of IRDs.

X-chromosomal inactivation patterns in women with Fabrydisease.

BackgroundAlthough Fabry disease (FD) is an X‐linked lysosomal storage disorder caused by mutations in the α‐galactosidase A gene (GLA), women may develop severe symptoms. We investigated X‐chromosomal inactivation patterns (XCI) as a potential determinant of symptom severity in FD women.Patients and MethodsWe included 95 women with mutations in GLA (n = 18 with variants of unknown pathogenicity) and 50 related men, and collected mouth epithelial cells, venous blood, and skin fibroblasts for XCI analysis using the methylation status of the androgen receptor gene. The mutated X‐chromosome was identified by comparison of samples from relatives. Patients underwent genotype categorization and deep clinical phenotyping of symptom severity.Results43/95 (45%) women carried mutations categorized as classic. The XCI pattern was skewed (i.e., ≥75:25% distribution) in 6/87 (7%) mouth epithelial cell samples, 31/88 (35%) blood samples, and 9/27 (33%) skin fibroblast samples. Clinical phenotype, α‐galactosidase A (GAL) activity, and lyso‐Gb3 levels did not show intergroup differences when stratified for X‐chromosomal skewing and activity status of the mutated X‐chromosome.ConclusionsX‐inactivation patterns alone do not reliably reflect the clinical phenotype of women with FD when investigated in biomaterial not directly affected by FD. However, while XCI patterns may vary between tissues, blood frequently shows skewing of XCI patterns.

Identification of a novel mutation in thyroxine-binding globulin (TBG) gene associated with TBG-deficiency and its effect on the thyroid function.

This study presents a case of familial transmission of thyroxine-binding globulin (TBG) deficiency. The SERPINA7-gene which codes for TBG is located on the X-chromosome (Xq21-22). More than 45 mutations have been reported to cause TBG- deficiency from various countries, but none from India so far. Genetic analysis of SERPINA7 gene was carried out to determine the cause of low TBG levels in one family. DNA samples of the propositus and the family members were subjected to Polymerase Chain Reaction (PCR) followed by direct sequencing. Allele-specific PCR and Next-gen sequencing (NGS) were employed to confirm the site of the mutation. Thyroid function tests were estimated by Radioimmunoassay (RIA) and Immunoradiometric assay (IRMA) kits. X-chromosomal inactivation status was analyzed in the female members harboring the mutation. A mutational screening in this family revealed a novel frame-shift mutation S353Q, 354fs3X in the exon 4 ofthe SERPINA7gene which will be referred to as TBG-complete deficiency-India (TBG-CD-Ind). One out of four female family members harboring the mutation showed selective X-chromosomal inactivation. The affected family members were clinically euthyroid initially, showed changes in the thyroid function when tested after a long time span. However, the changes in the thyroid function in the affected family members had an autoimmune etiology. This study presents the first report of TBG-CD from India wherein a novel frameshift mutation referred to as TBG-CD-Ind (S353Q, 354fs3X)in the SERPINA7 gene was detected. No apparent association was identified between thyroid function and the TBG-mutation in the affected subjects. A detailed biochemical and genomic testing to determine the exact cause of discordant TFT in the patients would certainly aid in the unequivocal diagnosis of the thyroid function and for the precise individualized treatment.

The treatment for Fabry disease: focus on agalsidase alpha and beta

Fabry disease (FD) is an X-linked lysosomal storage disorder resulting from the deficiency of the hydrolytic enzyme α-galactosidase A (α-Gal A), with consequent accumulation of globotrioasoylceramide in cells and tissues of the body, resulting in a multi-system pathology. Classically affected hemizygous males may display all the characteristic neurological (pain), cutaneous (angiokeratoma), renal (proteinuria, kidney failure), cardiovascular (cardiomyopathy, arrhythmia), and cerebrovascular (transient ischemic attacks, strokes) signs of the disease, while heterozygous females have symptoms ranging from very mild to severe. End-stage renal disease and cardiovascular or cerebrovascular complications limit life-expectancy of untreated patients. Demonstration of α-Gal A deficiency is the definitive method for the diagnosis of hemizygous males, while it's often inconclusive due to random X-chromosomal inactivation so that molecular testing (genotyping) of females is mandatory. The treatment options for FD are enzyme replacement therapy (ERT), and the oral pharmacological chaperone migalastat. Two different products, agalsidase alfa and agalsidase beta, have been commercially available in Europe for 20 years and they are both indicated for long-term ERT. In fact, clinical trials, observational studies and registry data have provided abundant evidence for the safety and efficacy of ERT in improving symptoms and disease progression. Agalsidase alpha and beta are two almost identical recombinant proteins although they are used clinically with a different dosage regimen. In this chapter we aim to clarify the differences between the two ERTs and how these can affect the pharmacokinetic/pharmacodynamic (PK/PD) characteristics and ultimately the risk/benefit profile. The chaperone migalastat, available in Europe since 2016, is the only oral treatment for FD, and acts stabilizing specific mutant forms of α-Gal, defined "amenable" to migalastat. A multitude of therapies are now under investigation in various phases of clinical trials. These include pegylated form of α-Gal (pegunigalsidase alpha), gene therapy (both in-vivo and ex-vivo methods), mRNA therapy (inducing production of α-Gal) and substrate reduction therapy (inhibitors of glucosylceramide synthase leading to reduction of Gb-3).

The Effect of Genetic Variation on the Placental Transcriptome in Humans.

The knowledge of genetic variants shaping human placental transcriptome is limited and they are not cataloged in the Genotype-Tissue Expression project. So far, only one whole genome analysis of placental expression quantitative trait loci (eQTLs) has been published by Peng et al. (2017) with no external independent validation. We report the second study on the landscape of placental eQTLs. The study aimed to generate a high-confidence list of placental cis-eQTLs and to investigate their potential functional implications. Analysis of cis-eQTLs (±100 kbp from the gene) utilized 40 placental RNA sequencing and respective whole genome genotyping datasets. The identified 199 placental cis-eSNPs represented 88 independent eQTL signals (FDR < 5%). The most significant placental eQTLs (FDR < 10-5) modulated the expression of ribosomal protein RPL9, transcription factor ZSCAN9 and aminopeptidase ERAP2. The analysis confirmed 50 eSNP-eGenes pairs reported by Peng et al. (2017) and thus, can be claimed as robust placental eQTL signals. The study identified also 13 novel placental eGenes. Among these, ZSCAN9 is modulated by several eSNPs (experimentally validated: rs1150707) that have been also shown to affect the methylation level of genes variably escaping X-chromosomal inactivation. The identified 63 placental eGenes exhibited mostly mixed or ubiquitous expression. Functional enrichment analysis highlighted 35 Gene Ontology categories with the top ranking pathways “ruffle membrane” (FDR = 1.81 × 10-2) contributing to the formation of motile cell surface and “ATPase activity, coupled” (FDR = 2.88 × 10-2), critical for the membrane transport. Placental eGenes were also significantly enriched in pathways implicated in development, signaling and immune function. However, this study was not able to confirm a significant overrepresentation of genome-wide association studies top hits among the placental eSNP and eGenes, reported by Peng et al. (2017). The identified eSNPs were further analyzed in association with newborn and pregnancy traits. In the discovery step, a suggestive association was detected between the eQTL of ALPG (rs11678251) and reduced placental, newborn’s and infant’s weight. Meta-analysis across REPROMETA, HAPPY PREGNANCY, ALSPAC cohorts (n = 6830) did not replicate these findings. In summary, the study emphasizes the role of genetic variation in driving the transcriptome profile of the human placenta and the importance to explore further its functional implications.

Distinctions in gene-expression in PBMC from male and female SLE patients

Abstract The female predisposition for multiple autoimmune diseases is well known. The mechanisms might be related to the female reproductive system, sex hormones, incomplete X-chromosomal inactivation etc. We investigated gene expression in PBMC of quiescent SLE patients and corresponding healthy donors of both genders (10 matching samples per group) using the RNA-seq approach. Principal component analysis differentiated the 4 donor groups, with PC1 reflecting disease and PC2 reflecting gender. Male SLE patients received the highest score on PC1 and were placed closer to females than healthy males on PC2. We identified 796 and 99 differentially expressed genes in male and female SLE patients compared to HD (p&amp;lt;0.05, logFC=1.5). Fewer genes (20 vs. 27) differed between genders in SLE vs. HD (p&amp;lt;0.05, logFC=1.5). Weighted Gene Co-expression Network Analysis (WGCNA) identified 24 clusters of co-expressed genes. The most intriguing were clusters that differed in male and female HD and were upregulated in SLE of both genders. Those clusters include genes related to EGF receptor ligands (AREG, EREG, HBEGF), transcription factors ATF3, NR4A1, NR4A2, NR4A3, cytokines and chemokines (CCL3, CXCL2, CXCL8, CCL3L3, IL1A, IL1B). Despite being clinically similar, male SLE patients showed a higher level of affected genes and had a lower divergence from females based on gender-related genes. Male HD had the lowest basal level of pro-inflammatory genes and EGF receptor ligands. EGFR is expressed in pituitary and involved in the reproductive hypothalamic-pituitary axis, possibly providing relative protection of males. We propose that gender-specific differences in gene expression reveal immunopathogenic mechanisms in autoimmune disease.

Sex-specific differences in the intracellular proteome of human endothelial cells from dizygotic twins

Differences between men and women are being continuously identified in many human diseases. The underlying reasons are not yet fully understood. Beside the influence of endogenous hormones and life style, intrinsic sex-specific dimorphisms at the cellular level may also play a role. HUVECs from twin pairs of opposite sex provide an excellent tool to address the question of sex-specific differences at the molecular level. We compared for the first time protein levels of male and female HUVECs from dizygotic twins using a proteomic approach. To investigate differences under basal and stress conditions, cells were either left untreated or wounded and serum starved for different time points. Approximately 10% of all proteins monitored showed significant sexual dimorphisms in their level under the different conditions tested. The majority of the proteins displayed a higher abundance in female cells. The magnitude of the difference in protein levels between male and female cells was rather small. The most prominent differences throughout all conditions were observed for several X-chromosome encoded proteins with higher levels in female (UBA1, HDHD1) or in male cells (G6PD). Proteins involved in basic cellular processes, such as gene expression and translation (e.g. HMGN1, SRP54) displayed sex-specific levels in particular conditions only. SignificanceThis study provides novel insights into sexual dimorphic protein levels in HUVECs from twin pairs of the opposite sex. The findings identify proteins with sex-specific differences in their levels under different cell culture conditions. The study also highlights the presence of X-chromosome encoded proteins escaping X-chromosomal inactivation. The results emphasize the need to consider the cellular sex of male and female HUVECs in in vitro experiments.

Generation of Isogenic Controls for In Vitro Disease Modelling of X-Chromosomal Disorders

Generation of proper controls is crucial in induced pluripotent stem cell (iPSC) studies. X-chromosomal disorders offer the potential to develop isogenic controls due to random X-chromosomal inactivation (XCI). However, the generation of such lines is currently hampered by skewed X-inactivation in fibroblast lines and X-chromosomal reactivation (XCR) after reprogramming. Here we describe a method to generate a pure iPSC population with respect to the specific inactivated X-chromosome (Xi). We used fibroblasts from Rett patients, who all have a causal mutation in the X-linked MeCP2 gene. Pre-sorting these fibroblasts followed by episomal reprogramming, allowed us to overcome skewness in fibroblast lines and to retain the X-chromosomal state, which was unpredictable with lentiviral reprogramming. This means that fibroblast pre-sorting followed by episomal reprogramming can be used to reliably generate iPSC lines with specified X-chromosomal phenotype such as Rett syndrome.

P191 – 2505: Two siblings with a CDKL5 mutation: Genotype and phenotype evaluation

Objective This is the second report of a family with a recurrence of a CDKL5 mutation (c. 283-3_290del) in two sisters. Their phenotype is different despite an identical genotype Clinically CDKL5 patients resemble those with Rett syndrome, caused by a MECP2 mutation, who experience a regression, after an initial normal development. However, CDKL5 patients experience a developmental delay from from birth onwards and seizures starts the first three months of life. Methods Advanced genetic evaluations of the sisters and parents, the electropherogram, clinical and electroencephalographic (EEG) features of both sisters are discussed. Results In the youngest patient, who was known to us prior to appearance of the clinical symptoms, neurological and EEG evaluations at the age of 6 weeks were normal. Infantile spasms evolved at the age of 10 weeks. Simultaneously, she showed a psychomotor regression and deterioration of the EEG into a severe epileptic pattern. Her older sister, was from the start severely hypotonic, had a developmental delay (without regression) and feeding problems and experiences seizures from the age of 8 weeks. In both seizures are refractory. The phenotype of the youngest sister is worse than that of her older sister, despite an identical genotype. This may be explained by differences in X-chromosomal inactivation and other unknown epigenetic and environmental factors. Unfortunately, X inactivation pattern could not be determined in the youngest sister. Both parents tested negative for the mutation in all tissues, but germline mosaicism is likely. Conclusion This report of familial recurrence with suspected germline mosaicism in a healthy parent, has important consequences for genetic counselling. Although it is not possible to predict an exact recurrence risk, it is likely to be increased.

X-chromosomal inactivation directly influences the phenotypic manifestation of X-linked protoporphyria.

X-linked protoporphyria (XLP), a rare erythropoietic porphyria, results from terminal exon gain-of-function mutations in the ALAS2 gene causing increased ALAS2 activity and markedly increased erythrocyte protoporphyrin levels. Patients present with severe cutaneous photosensitivity and may develop liver dysfunction. XLP was originally reported as X-linked dominant with 100% penetrance in males and females. We characterized 11 heterozygous females from six unrelated XLP families and show markedly varying phenotypic and biochemical heterogeneity, reflecting the degree of X-chromosomal inactivation of the mutant gene. ALAS2 sequencing identified the specific mutation and confirmed heterozygosity among the females. Clinical history, plasma and erythrocyte protoporphyrin levels were determined. Methylation assays of the androgen receptor and zinc-finger MYM type 3 short tandem repeat polymorphisms estimated each heterozygotes X-chromosomal inactivation pattern. Heterozygotes with equal or increased skewing, favoring expression of the wild-type allele had no clinical symptoms and only slightly increased erythrocyte protoporphyrin concentrations and/or frequency of protoporphyrin-containing peripheral blood fluorocytes. When the wild-type allele was preferentially inactivated, heterozygous females manifested the disease phenotype and had both higher erythrocyte protoporphyrin levels and circulating fluorocytes. These findings confirm that the previous dominant classification of XLP is inappropriate and genetically misleading, as the disorder is more appropriately designated XLP.

Development of a Highly Sensitive Immuno-PCR Assay for the Measurement of α-Galactosidase A Protein Levels in Serum and Plasma

Fabry disease is an X-linked genetic disorder caused by defects in the α-galactosidase A (GLA) gene, and heterogeneous mutations lead to quantitative and/or qualitative defects in GLA protein in male patients with Fabry disease. Random X-chromosomal inactivation modifies the clinical and biochemical features of female patients with Fabry disease. Functional polymorphisms have been frequently reported in recent times, and these increase the difficulty of understanding the pathogenetic basis of the disease. To date, GLA protein level has been measured using an enzyme-linked immunosorbent assay (ELISA). However, ELISA is not highly sensitive due to the high background noise. In this paper, we introduce a novel application of the immuno-polymerase chain reaction (PCR) method (termed Multiple Simultaneous Tag [MUSTag]) for measurement of the GLA protein level in blood samples. We compared the sensitivities of the MUSTag method with plates or magnetic beads with those of ELISA for recombinant human GLA and found that the apparent maximal sensitivity was higher for the former than for the latter. We then measured the GLA concentrations in serum and plasma from male patients with classic Fabry disease (Male Fabry), females with Fabry disease (Female Fabry), male subjects harboring the functional polymorphism p.E66Q (E66Q), and control (Control) subjects. Our results revealed that compared to the MUSTag plate and ELISA, the MUSTag beads assay afforded a clearer estimation of the GLA protein levels in the serum and plasma with minimal or no background noise, although all the methods could differentiate between the Male Fabry, E66Q, and Control groups. The Female Fabry group showed characteristic heterogeneity, which was consistent with the X-linked inheritance. This novel method is expected to be useful for the sensitive determination of GLA level in blood and elucidation of the pathogenetic basis of Fabry disease.

Mosaic synaptopathy and functional defects in Cav1.4 heterozygous mice and human carriers of CSNB2

Mutations in CACNA1F encoding the α1-subunit of the retinal Cav1.4 L-type calcium channel have been linked to Cav1.4 channelopathies including incomplete congenital stationary night blindness type 2A (CSNB2), Åland Island eye disease (AIED) and cone-rod dystrophy type 3 (CORDX3). Since CACNA1F is located on the X chromosome, Cav1.4 channelopathies are typically affecting male patients via X-chromosomal recessive inheritance. Occasionally, clinical symptoms have been observed in female carriers, too. It is currently unknown how these mutations lead to symptoms in carriers and how the retinal network in these females is affected. To investigate these clinically important issues, we compared retinal phenotypes in Cav1.4-deficient and Cav1.4 heterozygous mice and in human female carrier patients. Heterozygous Cacna1f carrier mice have a retinal mosaic consistent with differential X-chromosomal inactivation, characterized by adjacent vertical columns of affected and non-affected wild-type-like retinal network. Vertical columns in heterozygous mice are well comparable to either the wild-type retinal network of normal mice or to the retina of homozygous mice. Affected retinal columns display pronounced rod and cone photoreceptor synaptopathy and cone degeneration. These changes lead to vastly impaired vision-guided navigation under dark and normal light conditions and reduced retinal electroretinography (ERG) responses in Cacna1f carrier mice. Similar abnormal ERG responses were found in five human CACNA1F carriers, four of which had novel mutations. In conclusion, our data on Cav1.4 deficient mice and human female carriers of mutations in CACNA1F are consistent with a phenotype of mosaic CSNB2.

Identification of genuine primary pulmonary NK cell lymphoma via clinicopathologic observation and clonality assay

Extranodal natural killer (NK)/T-cell lymphoma, nasal type, is an uncommon lymphoma associated with the Epstein-Barr virus (EBV). It most commonly involves the nasal cavity and upper respiratory tract. Primary pulmonary NK/T cell lymphoma is extremely rare. If a patient with a NK or T-cell tumor has an unusual reaction to treatment or an unusual prognosis, it is wise to differentiate NK from T-cell tumors. The clinicopathologic characteristics, immunophenotype, EBV in situ hybridization, and T cell receptor (TCR) gene rearrangement of primary pulmonary NK cell lymphoma from a 73-year-old Chinese woman were investigated and the clonal status was determined using female X-chromosomal inactivation mosaicism and polymorphisms at the phosphoglycerate kinase (PGK) gene. The lesion showed the typical histopathologic characteristics and immunohistochemical features of NK/T cell lymphoma. However, the sample was negative for TCR gene rearrangement. A clonality assay demonstrated that the lesion was monoclonal. It is concluded that this is the first recorded case of genuine primary pulmonary NK cell lymphoma. The purpose of the present work is to recommend that pathologists carefully investigate the whole lesion to reduce the likelihood that primary pulmonary NK cell lymphoma will be misdiagnosed as an infectious lesion. In addition, TCR gene rearrangement and clonal analysis, which is based on female X-chromosomal inactivation mosaicism and polymorphisms at PGK and androgen receptor (AR) loci, were found to play important roles in differentiating NK cell lymphoma from T cell lymphoma.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5205300349457729

Visual Function in CaV1.4-Deficient Mice

CACNA1F encodes the alpha1 subunit of retina-specific Cav1.4 voltage-gated L type calcium channels. Mutations in this gene cause congenital stationary night blindness type 2A (CSNB2), Aland Island eye disease (AIED) and cone-rod dystrophy type 3 (CORDX3). The clinical phenotypes of these eye diseases vary substantially regarding the ratio of rod to cone functional impairment. The reasons for this variability are not known. To gain more insights into the pathophysiology caused by loss of Cav1.4 function we analyzed the visual phenotype of Cav1.4-deficient mice. To this end, we combined immunohistochemistry, electroretinography (ERG) and vision-dependent behavioral testing. Immunohistochemical analysis using synaptic and postsynaptic markers revealed severe synaptic defects in Cav1.4-deficient mice. Heterozygous Cav1.4 mice showed mosaic synaptic defects most probably caused by random X-chromosomal inactivation of the healthy allele. Electroretinography revealed a loss of scotopic and photopic photoreceptor function. This loss of retinal network function resulted in impaired perfomance of Cav1.4 knockout mice in a water maze-based behavioral test of rod and cone function. In conclusion, loss of Cav1.4 channels strongly impairs rod and cone retinal function and vision in mice.

Fabry Disease: from Diagnosis to Therapy

Fabry disease (FD) is an accelerating, X-linked hereditary disorder of glycosphingolipid metabolism which occurs due to improper lysosomal α-galactosidase A activity. Its pan-ethnic and the annually reported occurrence may underestimate the true frequency of the disease. Classically affected hemizygous males, with no residual α-galactosidase A activity may display all the characteristics like cardiovascular (cardiomyopathy), cutaneous (angiokeratoma), renal (kidney failure), neurological (pain), cerebrovascular and cochleo-vestibular signs of the disease while heterozygous females have similar symptoms ranging from very mild to severe. Incomplete action of lysosomal α-galactosidase A results in gradual increase of globotriaosylceramide within lysosomes, supposed to set off a flow of cellular events. Enzyme analysis may occasionally help to detect heterozygote but in females results are often questionable because of random X-chromosomal inactivation that obligates the molecular testing (genotyping). Due to ethical reasons determination of enzyme activity through prenatal diagnosis is carried out only in male's fetus. The subsistence of atypical variants and the unavailability of a specific therapy complicate the genetic counseling. Enzyme replacement therapy (ERT) is the only specific therapeutic option that introduced recombinant human α-galactosidase A. End stage renal disease and critical cardiovascular or cerebrovascular complications limit the life-expectancy of untreated males and females. Oral therapy drives the research forward into active site specific chaperones. Adjunctive therapy of ERT with

Diagnostic dilemma: A young woman with Fabry disease symptoms, no family history, and a “sequencing cryptic” α-galactosidase a large deletion

Fabry disease, an X-linked lysosomal storage disorder, results from the deficient activity of α-galactosidase A (α-Gal A). In affected males, the clinical diagnosis is confirmed by the markedly decreased α-Gal A activity. However, in female heterozygotes, the α-Gal A activity can range from low to normal due to random X-chromosomal inactivation, and diagnostic confirmation requires identification of the family's α-Gal A gene mutation. In a young female who had occasional acroparesthesias, corneal opacities, and 15 to 50% of the lower limit of normal leukocyte α-Gal A activity, α-Gal A sequencing in two expert laboratories did not identify a confirmatory mutation, presenting a diagnostic dilemma. A renal biopsy proved diagnostic and renewed efforts to detect an α-Gal A mutation. Subsequent gene dosage analyses identified a large α-Gal A deletion confirming her heterozygosity, and she was started on enzyme replacement therapy. Thus, gene dosage analyses can detect large deletions (>50bp) in suspect heterozygotes for X-linked and autosomal dominant diseases that are “sequencing cryptic,” resolving molecular diagnostic dilemmas.

Skewed X-chromosomal inactivation impacts T regulatory cell function in systemic sclerosis

OBJECTIVES: > To investigate the role of X chromosomal inactivation (XCI) in systemic sclerosis (SSc) and its effects on forkhead box P3 (Foxp3) expression in T regulatory cells (Tregs). METHODS: 217 women with SSc and 107 healthy women (controls) were included in the study. From these subjects, DNA was isolated from total peripheral blood mononuclear cells, plasmacytoid dendritic cells, T cells, B cells, myeloid dendritic cells and monocytes after magnetic bead separation. All samples were assessed for skewed XCI patterns with the Human Androgen Receptor Assay. The outcome was assessed by linear regression. CD4+ CD25+ cells were then isolated and intracellular Foxp3 expression was assessed by flow cytometry. RESULTS: Skewing was not associated with increased age in patients with SSc, in contrast to the control population (r = 0.45, p < 0.0001). Taking this into account, a significantly higher frequency of skewed XCI was found in patients with SSc compared with controls (p = 0.001). No difference in skewing was observed between the immune cell subsets. In addition, a higher concentration of Foxp3+ cells exhibiting a lower Foxp3 mean fluorescence intensity was found in the patients with SSc, with profound XCI skewing (both p < 0.001) associated with less efficient suppressive activity (p=0.012). CONCLUSIONS: Skewed XCI plays a role in susceptibility to SSc, is not restricted and influences Foxp3 expression and the suppressive capacity of Tregs.

Skewed X chromosomal inactivation impacts T regulatory cell function in systemic sclerosis

ObjectivesTo investigate the role of X chromosomal inactivation (XCI) in systemic sclerosis (SSc) and its effects on forkhead box P3 (Foxp3) expression in T regulatory cells (Tregs).Methods217 women with SSc...

Clonal Status and Clinicopathological Features of Langerhans Cell Histiocytosis

Some studies have demonstrated that Langerhans cell histiocytosis (LCH) is a neoplastic hyperplasia of Langerhans cells, however some researchers consider that clonality should be assessed in more patients with LCH, both at disease presentation and during the disease course. Monoclonality is a major characteristic of most tumours, whereas normal tissue and reactive hyperplasia are polyclonal. To elucidate the nature of Langerhans cells further, the present study investigated the clinicopathological features and clonality of three cases of LCH in female patients using laser microdissection and a clonality assay, based on X-chromosomal inactivation mosaicism in somatic tissues and polymorphism of the androgen receptor gene. The results indicated that LCH was composed of Langerhans cells with a characteristic morphological appearance, eosinophils, giant cells, neutrophils and foamy cells. Immunohistochemically, the Langerhans cells were positive for CD1a, S-100 protein and vimentin. The clonality assay demonstrated that the Langerhans cells formed a monoclonal population, showing that LCH is neoplastic. We conclude that LCH is characterized by clonal proliferation, although additional studies with larger sample sizes are required to prove this conclusively.