Skewed X-inactivation Research Articles

Variable clinical consequences of mosaicism for c.1167dupC in IKBKG in male and female patients with incontinentia pigmenti and related phenotypes.

In the present study, we report a duplication variant found in two families with incontinentia pigmenti, which exhibited a variety of phenotypes in both male and female carriers. Although natural mosaicism through Lyonization, with variably skewed X-inactivation, accounts for the phenotype in females, our study also demonstrates that mosaicism, albeit through a different mechanism, should also be remembered in understanding the phenotypic expression of IKBKG-related disorders in some males.

PB0218 Trisomy X Conferring Moderate Hemophilia A by Extremely Skewed X-Chromosome Inactivation

PB0218 Trisomy X Conferring Moderate Hemophilia A by Extremely Skewed X-Chromosome Inactivation

Whole-Genome Sequencing Identified New Structural Variations in the DMD Gene That Cause Duchenne Muscular Dystrophy in Two Girls.

Dystrophinopathies are the most common muscle diseases, especially in men. In women, on the other hand, a manifestation of Duchenne muscular dystrophy is rare due to X-chromosomal inheritance. We present two young girls with severe muscle weakness, muscular dystrophies, and creatine kinase (CK) levels exceeding 10,000 U/L. In the skeletal muscle tissues, dystrophin staining reaction showed mosaicism. The almost entirely skewed X-inactivation in both cases supported the possibility of a dystrophinopathy. Despite standard molecular diagnostics (including multiplex ligation-dependent probe amplification (MLPA) and next generation sequencing (NGS) gene panel sequencing), the genetic cause of the girls' conditions remained unknown. However, whole-genome sequencing revealed two reciprocal translocations between their X chromosomes and chromosome 5 and chromosome 19, respectively. In both cases, the breakpoints on the X chromosomes were located directly within the DMD gene (in introns 54 and 7, respectively) and were responsible for the patients' phenotypes. Additional techniques such as Sanger sequencing, conventional karyotyping and fluorescence in situ hybridization (FISH) confirmed the disruption of DMD gene in both patients through translocations. These findings underscore the importance of accurate clinical data combined with histopathological analysis in pinpointing the suspected underlying genetic disorder. Moreover, our study illustrates the viability of whole-genome sequencing as a time-saving and highly effective method for identifying genetic factors responsible for complex genetic constellations in Duchenne muscular dystrophy (DMD).

Recapitulation of Skewed X-Inactivation in Female Ornithine Transcarbamylase-Deficient Primary Human Hepatocytes in the FRG Mouse: A Novel System for Developing Epigenetic Therapies.

Realization of the immense therapeutic potential of epigenetic editing requires development of clinically predictive model systems that faithfully recapitulate relevant aspects of the target disease pathophysiology. In female patients with ornithine transcarbamylase (OTC) deficiency, an X-linked condition, skewed inactivation of the X chromosome carrying the wild-type OTC allele is associated with increased disease severity. The majority of affected female patients can be managed medically, but a proportion require liver transplantation. With rapid development of epigenetic editing technology, reactivation of silenced wild-type OTC alleles is becoming an increasingly plausible therapeutic approach. Toward this end, privileged access to explanted diseased livers from two affected female infants provided the opportunity to explore whether engraftment and expansion of dissociated patient-derived hepatocytes in the FRG mouse might produce a relevant model for evaluation of epigenetic interventions. Hepatocytes from both infants were successfully used to generate chimeric mouse-human livers, in which clusters of primary human hepatocytes were either OTC positive or negative by immunohistochemistry (IHC), consistent with clonal expansion from individual hepatocytes in which the mutant or wild-type OTC allele was inactivated, respectively. Enumeration of the proportion of OTC-positive or -negative human hepatocyte clusters was consistent with dramatic skewing in one infant and minimal to modest skewing in the other. Importantly, IHC and fluorescence-activated cell sorting analysis of intact and dissociated liver samples from both infants showed qualitatively similar patterns, confirming that the chimeric mouse-human liver model recapitulated the native state in each infant. Also of importance was the induction of a treatable metabolic phenotype, orotic aciduria, in mice, which correlated with the presence of clonally expanded OTC-negative primary human hepatocytes. We are currently using this unique model to explore CRISPR-dCas9-based epigenetic targeting strategies in combination with efficient adeno-associated virus (AAV) gene delivery to reactivate the silenced functional OTC gene on the inactive X chromosome.

Clinical, pathological, and genetic characterization in a large Chinese cohort with female dystrophinopathy

We aimed to investigate the clinical, pathological, and genetic characteristics of Chinese female dystrophinopathy and to identify possible correlations among them. One hundred forty genetically and/or pathologically confirmed female DMD variant carriers were enrolled, including 104 asymptomatic carriers and 36 symptomatic carriers. Twenty of 36 symptomatic and 16 of 104 asymptomatic carriers were sporadic with no family history. Muscle pathological analysis was performed in 53 carriers and X chromosome inactivation (XCI) analysis in 19 carriers. In asymptomatic carriers, the median age was 35.0 (range 2.0–58.0) years, and the serum creatine kinase (CK) level was 131 (range 60–15,745) IU/L. The median age, age of onset, and CK level of symptomatic carriers were 15.5 (range 1.8–62.0) years, 6.3 (range 1.0–54.0) years, and 6,659 (range 337–58,340) IU/L, respectively. Four female carriers with X-autosome translocation presented with a Duchenne muscular dystrophy (DMD) phenotype. Skewed XCI was present in 70.0% of symptomatic carriers. Compared to Becker muscular dystrophy (BMD)-like carriers, DMD-like carriers were more likely to have an early onset age, rapidly progressive muscle weakness, delayed walking, elevated CK levels, severe reduction of dystrophin, and skewed XCI. Our study reports the largest series of symptomatic female DMD carriers and suggests that delayed walking, elevated CK levels, severe reduction of dystrophin, X-autosome translocation, and skewed XCI pattern are associated with a severe phenotype in female dystrophinopathy.

Molecular Challenges in the Diagnosis of X-Linked Chronic Granulomatous Disease: CNVs, Intronic Variants, Skewed X-Chromosome Inactivation, and Gonosomal Mosaicism.

Chronic granulomatous disease (CGD) is a prototypical inborn error of immunity affecting phagocytes, in which these cells are unable to produce reactive oxygen species. CGD is caused by defects in genes encoding subunits of the NADPH oxidase enzyme complex (CYBA, CYBB, CYBC1, NCF1, NCF2, NCF4); inflammatory responses are dysregulated, and patients are highly susceptible to recurrent severe bacterial and fungal infections. X-linked CGD (XL-CGD), caused by mutations in the CYBB gene, is the most common and severe form of CGD. In this study, we describe the analytical processes undertaken in 3 families affected with XL-CGD to illustrate several molecular challenges in the genetic diagnosis of this condition: in family 1, a girl with a heterozygous deletion of CYBB exon 13 and skewed X-chromosome inactivation (XCI); in family 2, a boy with a hemizygous deletion of CYBB exon 7, defining its consequences at the mRNA level; and in family 3, 2 boys with the same novel intronic variant in CYBB (c.1151 + 6T > A). The variant affected the splicing process, although a small fraction of wild-type mRNA was produced. Their mother was a heterozygous carrier, while their maternal grandmother was a carrier in form of gonosomal mosaicism. In summary, using a variety of techniques, including an NGS-based targeted gene panel and deep amplicon sequencing, copy number variation calling strategies, microarray-based comparative genomic hybridization, and cDNA analysis to define splicing defects and skewed XCI, we show how to face and solve some uncommon genetic mechanisms in the diagnosis of XL-CGD.

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.

Four Decades of Carrier Detection and Prenatal Diagnosis in Hemophilia A: Historical Overview, State of the Art and Future Directions.

Hemophilia A (HA), a rare recessive X-linked bleeding disorder, is caused by either deficiency or dysfunction of coagulation factor VIII (FVIII) resulting from deleterious mutations in the F8 gene encoding FVIII. Over the last 4 decades, the methods aimed at determining the HA carrier status in female relatives of HA patients have evolved from phenotypic studies based on coagulation tests providing merely probabilistic results, via genetic linkage studies based on polymorphic markers providing more accurate results, to next generation sequencing studies enabling highly precise identification of the causative F8 mutation. In parallel, the options for prenatal diagnosis of HA have progressed from examination of FVIII levels in fetal blood samples at weeks 20-22 of pregnancy to genetic analysis of fetal DNA extracted from chorionic villus tissue at weeks 11-14 of pregnancy. In some countries, in vitro fertilization (IVF) combined with preimplantation genetic diagnosis (PGD) has gradually become the procedure of choice for HA carriers who wish to prevent further transmission of HA without the need to undergo termination of pregnancies diagnosed with affected fetuses. In rare cases, genetic analysis of a HA carrier might be complicated by skewed X chromosome inactivation (XCI) of her non-hemophilic X chromosome, thus leading to the phenotypic manifestation of moderate to severe HA. Such skewed XCI may be associated with deleterious mutations in X-linked genes located on the non-hemophilic X chromosome, which should be considered in the process of genetic counseling and PGD planning for the symptomatic HA carrier. Therefore, whole exome sequencing, combined with X-chromosome targeted bioinformatic analysis, is highly recommended for symptomatic HA carriers diagnosed with skewed XCI in order to identify additional deleterious mutations potentially involved in XCI skewing. Identification of such mutations, which may profoundly impact the reproductive choices of HA carriers with skewed XCI, is extremely important.

X chromosome inactivation skewing is common in advanced carotid atherosclerotic lesions in females and predicts secondary peripheral artery events

Background and aimSex differences in atherosclerosis have been described with female plaques being mostly perceived as stable and fibrous. Sex-specific mechanisms such as mosaic loss of the Y chromosome in men have been linked to cardiovascular health. In women, X-linked mechanisms such as X chromosome inactivation (XCI) skewing is common in several tissues. Yet, information on the role of XCI in female atherosclerotic plaques is lacking. Here, we investigated the presence of XCI skewing in advanced atherosclerotic lesions and its association with cardiovascular risk factors, histological plaque data, and clinical data.MethodsXCI skewing was quantified in 154 atherosclerotic plaque and 55 blood DNA samples of women included in the Athero-Express study. The skewing status was determined performing the HUMARA assay. Then, we studied the relationship of XCI skewing in female plaque and cardiovascular risk factors using regression models. In addition, we studied if plaque XCI predicted plaque composition, and adverse events during 3-years follow-up using Cox proportional hazard models.ResultsXCI skewing was detected in 76 of 154 (49.4%) plaques and in 27 of 55 (67%) blood samples. None of the clinical risk factors were associated with plaque skewing. Plaque skewing was more often detected in plaques with a plaque hemorrhage (OR [95% CI]: 1.44 [1.06–1.98], P = 0.02). Moreover, skewed plaques were not associated with a higher incidence of composite and major events but were specifically associated with peripheral artery events during a 3-year follow-up period in a multivariate model (HR [95%CI]: 1.46 [1.09–1.97]; P = 0.007).ConclusionsXCI skewing is common in carotid plaques of females and is predictive for the occurrence of peripheral artery events within 3 years after carotid endarterectomy.

#3067 ALPORT SYNDROME NATURAL HISTORY FROM THE RADAR REGISTRY: ASSOCIATIONS WITH GENE, VARIANT TYPE AND SEX

Abstract Background and Aims Alport Syndrome (AS) is caused by pathogenic variants in COL4A3, COL4A4 & COL4A5 genes. The clinical course of AS can be highly variable, depending on gene affected, mutation type and Male (M) or Female (F) sex. Proteinuria is associated with worse prognosis. Previous genotype-phenotype correlation studies have shown protein length altering variants are associated with a more severe phenotype in males with COL4A5 variants, however studies in females have shown contradicting results. This study aims to describe demographics and investigate renal outcomes associated with pathogenic variant type in M vs F AS patients, using longitudinal data from the National Registry of Rare Kidney Diseases (RaDaR) which recruits patients at 108 UK renal clinics. Method RaDaR is linked to Regional Genetics hubs for clinical genetic reports, renal IT systems for routine test results and the UK Renal Registry for Renal Replacement Therapy (RRT) initiation data. eGFR was calculated using CKD-EPI Cr equation (2021) or Schwartz equation for those ≤ 16 yrs. eGFR slope was calculated over the last 3 years (or 3 years prior to End Stage Kidney Disease (ESKD)/death for patients who reached those outcomes), with patients required to have a minimum of 4 values over 2 years. For genotype data, variants classified in clinically issued reports as “Pathogenic” or “Likely Pathogenic” were included and classed as: 1. Protein Length Altering and 2. Missense variants. Where both protein length altering and missense variants in the same gene were identified in an individual, the missense variant was used. Data are presented as percentages for categorical variables and mean ± SD for continuous variables. Kaplan Meier analysis and the log rank statistic were used to compare age at RRT start, stratified by gender and variant types. Lower quartile (LQ) (25%) estimates are presented where too few events have occurred to calculate a median (50%) estimate. Results Between Jan 2013–Jul 2022, 920 AS patients were recruited; 53% M vs. 47% F. Genetic test reports were available for 343/920 (37%) patients. 294/343 (86%) reported a pathogenic or likely pathogenic variant, 140 protein length altering and 154 missense. Individuals with 2x COL4A3/COL4A4 variants were youngest at RRT start, whilst females with COL4A5 variants were oldest (LQ 20.1 vs 60.9 yrs, log-rank p<0.0001) (Figure 1a). COL4A5 variants Males with protein length altering COL4A5 variants were younger at ESKD compared with missense variants (median age 30.2 vs 52.1 yrs, p = 0.01). Conversely 0/36 females with protein length altering COL4A5 variants had started RRT, compared with 6/37 with missense variants (p = 0.01) (Figure 1bi-ii). COL4A3 or COL4A4 variants Whilst females with heterozygous protein length altering COL4A3/4 variants were younger at RRT start vs. those with missense variants (LQ 47.1 vs 64.9 yrs, p = 0.05), age at RRT start did not differ by mutation type for males with COL4A3/4 heterozygous variants (Figure 1b iii-iv). However, males with homozygous or 2x protein length altering COL4A3/4 variants were younger at RRT start than those with missense variants (median age 20.1 vs 24.3 yrs, P = 0.05), whilst no difference was observed by variant type for females with 2x COL4A3/4 variants (Figure 1b v-vi). Proteinuria and eGFR slope were generally correlated with age at ESKD. Conclusion The observed effect of pathogenic variant type on renal outcomes varied by gene affected, number of mutations and sex. The relatively reduced severity among females harbouring a protein length altering COL4A5 variant may represent an effect of skewed X-inactivation or a missense gain-of-function mechanism. RaDaR recruits patients with a clinical diagnosis of Alport syndrome or Thin Basement Membrane Nephropathy; ascertainment of individuals with a single COL4A3 or COL4A4 variant is therefore likely to favour those with more severe kidney disease. Linkage of the RaDaR AS cohort with genetic report data is ongoing; further correlations may be observed with larger numbers.

Gustavson syndrome is caused by an in-frame deletion in RBMX associated with potentially disturbed SH3 domain interactions

RNA binding motif protein X‐linked (RBMX) encodes the heterogeneous nuclear ribonucleoprotein G (hnRNP G) that regulates splicing, sister chromatid cohesion and genome stability. RBMX knock down experiments in various model organisms highlight the gene’s importance for brain development. Deletion of the RGG/RG motif in hnRNP G has previously been associated with Shashi syndrome, however involvement of other hnRNP G domains in intellectual disability remain unknown. In the current study, we present the underlying genetic and molecular cause of Gustavson syndrome. Gustavson syndrome was first reported in 1993 in a large Swedish five-generation family presented with profound X-linked intellectual disability and an early death. Extensive genomic analyses of the family revealed hemizygosity for a novel in-frame deletion in RBMX in affected individuals (NM_002139.4; c.484_486del, p.(Pro162del)). Carrier females were asymptomatic and presented with skewed X-chromosome inactivation, indicating silencing of the pathogenic allele. Affected individuals presented minor phenotypic overlap with Shashi syndrome, indicating a different disease-causing mechanism. Investigation of the variant effect in a neuronal cell line (SH-SY5Y) revealed differentially expressed genes enriched for transcription factors involved in RNA polymerase II transcription. Prediction tools and a fluorescence polarization assay imply a novel SH3-binding motif of hnRNP G, and potentially a reduced affinity to SH3 domains caused by the deletion. In conclusion, we present a novel in-frame deletion in RBMX segregating with Gustavson syndrome, leading to disturbed RNA polymerase II transcription, and potentially reduced SH3 binding. The results indicate that disruption of different protein domains affects the severity of RBMX-associated intellectual disabilities.

Genetic features and kidney morphological changes in women with X-linked Alport syndrome

BackgroundX-linked Alport syndrome (XLAS) caused by COL4A5 pathogenic variants usually has heterogeneous phenotypes in female patients. The genetic characteristics and glomerular basement membrane (GBM) morphological changes in women with XLAS...

Germline C1GALT1C1 mutation causes a multisystem chaperonopathy

Mutations in genes encoding molecular chaperones can lead to chaperonopathies, but none have so far been identified causing congenital disorders of glycosylation. Here we identified two maternal half-brothers with a novel chaperonopathy, causing impaired protein O-glycosylation. The patients have a decreased activity of T-synthase (C1GALT1), an enzyme that exclusively synthesizes the T-antigen, a ubiquitous O-glycan core structure and precursor for all extended O-glycans. The T-synthase function is dependent on its specific molecular chaperone Cosmc, which is encoded by X-chromosomal C1GALT1C1. Both patients carry the hemizygous variant c.59C>A (p.Ala20Asp; A20D-Cosmc) in C1GALT1C1. They exhibit developmental delay, immunodeficiency, short stature, thrombocytopenia, and acute kidney injury (AKI) resembling atypical hemolytic uremic syndrome. Their heterozygous mother and maternal grandmother show an attenuated phenotype with skewed X-inactivation in blood. AKI in the male patients proved fully responsive to treatment with the complement inhibitor Eculizumab. This germline variant occurs within the transmembrane domain of Cosmc, resulting in dramatically reduced expression of the Cosmc protein. Although A20D-Cosmc is functional, its decreased expression, though in a cell or tissue-specific manner, causes a large reduction of T-synthase protein and activity, which accordingly leads to expression of varied amounts of pathological Tn-antigen (GalNAcα1-O-Ser/Thr/Tyr) on multiple glycoproteins. Transient transfection of patient lymphoblastoid cells with wild-type C1GALT1C1 partially rescued the T-synthase and glycosylation defect. Interestingly, all four affected individuals have high levels of galactose-deficient IgA1 in sera. These results demonstrate that the A20D-Cosmc mutation defines a novel O-glycan chaperonopathy and causes the altered O-glycosylation status in these patients.

Immunodeficiency in a female patient with Incontinentia Pigmenti

Incontinentia pigmenti (IP) is a rare X-linked dominant neuroectodermal disorder characterized by ocular, dermatologic, and neurological abnormalities. It is caused by heterozygous mutations in IKBKG (NEMO), which encodes a regulatory factor involved in NF-kB activation. Random and skewed X-inactivation allows for female survival and determines extent of disease. Although immune function is typically normal in IP patients, hypomorphic IKBKG mutations are known to cause immunodeficiency, as occurs in X-linked NEMO deficiency. A female newborn presented with bullous vesicles, peeling skin, alopecia, hearing loss, and tonic-clonic seizures with MRI demonstrating evidence of in utero stroke. She developed intermittent fevers and cellulitis and was diagnosed with IP based on clinical findings. Following discharge, she was readmitted at 1 month of age due to bronchiolitis complicated by pulmonary abscess. Labs demonstrated hyperleukocytosis with hypereosinophilia, normal T, B, and NK lymphocyte enumeration, abnormal lymphocyte proliferation to phytohemagglutinin (PHA) and anti-CD3, and abnormal toll-like receptor (TLR) function. Genetic analysis revealed a heterozygous deletion of exons 4–10 in IKBKG. Labs at 1 year follow up showed normal lymphocyte (T, B, and NK) enumeration and mitogen responses, normal immunoglobulin levels, and normal responses to pneumococcal and tetanus vaccines. While about 70% of IP patients harbor a deletion of exons 4–10 in IKBKG. unlike other hypomorphic NEMO mutations, this mutation is not typically associated with altered immune function. This case highlights that immunodeficiency may be an early clinical manifestation in female patients with IP.

X-Linked Chronic Granulomatous Disease in a Female Carrier: A Management Challenge

Abstract Chronic granulomatous disease (CGD) is characterized by recurrent bacterial and fungal infections due to reduced nicotinamide adenine dinucleotide phosphate oxidase resulting in impaired production of reactive oxidant species, and aberrant inflammation. X-linked CGD (XL-CGD) is associated with a mutation in the CYBB gene. XL-CGD affects more males, although some females are affected affected due to skewed X-inactivation. Though management guidelines for CGD patients exist, they remain lacking for clinically affected XL-CGD female carriers. A 3 year old female with history of hematochezia, oral ulcers, and recurrent skin infections who presented with fevers and skin pustules. Pustule cultures were positive for B. cepacian, and colonoscopy was non-acute. Immunology had concern for XL-CGD with recent atypical infections and dihydrorhodamine (DHR) flow cytometry was abnormal, 17.7% (normal greater than 95%). DHR 2 months later showed 13% activation. Genetic testing showed a CYBB, c.45+2dup (intronic), heterozygous, VUS, gene mutation affecting the splice site, associated with XL-CGD, and a NOD2, c.3019dup, heterozygous, with a 2–4 fold increased risk for Crohn’s disease. She was started on bacterial prophylaxis only. She is awaiting gp91phox expression for gene sequencing. XL-CGD carriers can be at higher risk for infections, and risk is inversely correlated with %DHR neutrophil counts, but only clinically apparent with %DHR less than 20%, and more as levels are less than 10%. Though our patient was started on Bactrim prophylaxis with her %DHR less than 20%, there remains the question of when and if she should be started on anti-fungal prophylaxis. There remain no clear guidelines on the management of XL-CGD carriers.

Hypomorphic splice site IL2RG variants associated with Cryptosporidium liver disease

BackgroundX-linked severe combined immunodeficiency (X-SCID) is caused by mutations in IL2RG encoding for the common gamma chain (γc). This disease is typically fatal within the first year without immune reconstitution. We present two patients with IL2RG variants leading to a phenotype of Cryptosporidium-related sclerosing cholangitis. MethodsMedical history and laboratories were reviewed for two patients with IL2RG variants at c.270G. For one patient, we examined cDNA, γc expression, TCR repertoire, cytokine signaling, in vitro B-cell plasmablast differentiation, and in vitro T-cell differentiation of peripheral blood CD34+ cells. ResultsTwo unrelated male patients (17 and 21 years old) were identified with liver disease, recurrent childhood pneumonia and resultant bronchiectasis. P1 has persistent warts. Elevated transaminases in P1 led to liver imaging and biopsy consistent with sclerosing cholangitis. Stool PCR was positive for Cryptosporidium. P2 was diagnosed at age 6 with cryptogenic cirrhosis. He underwent orthotopic liver transplant at age 19; diarrhea and cholestatic liver disease developed 4 months post-transplant, and cryptosporidium was found in stool and liver. Cryptococcus meningitis also developed on immunosuppression. Both patients were hemizygous for an IL2RG variant resulting in p.W90C (P1:c.270G>T; P2:c.270G>C). P1 had mildly reduced CD4+ lymphocytes, CD4+ and CD8+ central/ effector memory T-lymphocytes, memory B-lymphocytes, and normal NK counts. P1 cDNA revealed a leaky splicing defect with a mix of exons 2–4 skipping along with full-length transcript encoding p.W90C. Cells from heterozygous mother expressed wild-type sequence suggesting skewed X-inactivation. Surface protein expression and distribution of TCR V-Beta repertoire was largely normal. IL-2-mediated STAT5 phosphorylation was impaired in T- and NK-cells. P1’s B-lymphocytes proliferated in response to in vitro IL-21/CD40L co-stimulation but failed to differentiate into CD27+CD38+ plasmablasts (< 1%). In vitro T-cell differentiation showed impaired survival of developing thymocytes, despite capability of maturing intoTCRaB+ CD3+ T cells. ConclusionsWe present two patients hemizygous for IL2RG variants leading to milder clinical phenotype compared to classic X-SCID. Consistent with other diseases of impaired IL-21 signaling, there was significant Cryptosporidium-related liver disease in addition to clinical manifestations of hypomorphic IL-2 signaling defect. Further work is necessary to address restoring IL-21 signaling to improve liver disease.

CDKL5 Deficiency Disorder Without Epilepsy

Cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder (CDD) has epilepsy as a cardinal feature. Here we report two new female patients and review six previously published patients, one male and five females, with features of CDD but who never developed epilepsy. In contrast with the classical and severe CDD phenotype, they presented with milder gross motor delays, autism spectrum disorder, and no visual cortical impairment. Prolonged video electroencephalography was normal in adult cases but showed interictal frontal-temporal bilateral spikes and sharp waves in sleep in the three-year-old girl. Causative CDKL5 variants included two likely gene damaging (nonsense and frameshift) and six missense variants, being de novo or maternally inherited from asymptomatic females with skewed X-chromosome inactivation (two missense variants). Our data indicate that a milder form of CDD without epilepsy can occur in some cases without clear correlation with specific variants in the CDKL5 gene.

Skewed X-chromosome Inactivation in Women with Idiopathic Intellectual Disability is Indicative of Pathogenic Variants.

Intellectual disability (ID) is an early onset impairment in cognitive functioning and adaptive behavior, affecting approximately 1% of the population worldwide. Extreme skewing of X-chromosome inactivation (XCI) can be associated with ID phenotypes caused by pathogenic variants in the Xchromosome. We analyzed the XCI pattern in blood samples of 194 women with idiopathic ID, using the androgen receptor gene (AR) methylation assay. Among the 136 patients who were informative, 11 (8%) presented with extreme or total XCI skewing (≥ 90%), which was significantly higher than expected by chance. Whole-exome data obtained from these 11 patients revealed the presence of dominant pathogenic variants in eight of them, all sporadic cases, resulting in a molecular diagnostic rate of 73% (8/11 patients). All variants were mapped to ID-related genes with dominant phenotypes: four variants in the X-linked genes DDX3X (an XCI escape gene; two cases), WDR45, and PDHA1, and four variants in the autosomal genes KCNB1, CTNNB1, YY1, and ANKRD11. Three of the autosomal genes had no obvious correlation with the observed XCI skewing. However, YY1 is a known transcriptional repressor that acts in the binding of the XIST long noncoding RNA on the inactive X chromosome, providing a mechanistic link between the pathogenic variant and the detected skewed XCI in the carrier. These data confirm that extreme XCI skewing in females with ID is highly indicative of causative X-linked pathogenic variants, and point to the possibility of identifying causative variants in autosomal genes with a XCI role.

Skewed X-chromosome inactivation in unsolved neurodevelopmental disease cases can guide re-evaluation For X-linked genes.

Despite major advances in genome technology and analysis, >50% of patients with a neurodevelopmental disorder (NDD) remain undiagnosed after extensive evaluation. A point in case is our clinically heterogeneous cohort of NDD patients that remained undiagnosed after FRAXA testing, chromosomal microarray analysis and trio exome sequencing (ES). In this study, we explored the frequency of non-random X chromosome inactivation (XCI) in the mothers of male patients and affected females, the rationale being that skewed XCI might be masking previously discarded genetic variants found on the X chromosome. A multiplex fluorescent PCR-based assay was used to analyse the pattern of XCI after digestion with HhaI methylation-sensitive restriction enzyme. In families with skewed XCI, we re-evaluated trio-based ES and identified pathogenic variants and a deletion on the X chromosome. Linkage analysis and RT-PCR were used to further study the inactive X chromosome allele, and Xdrop long-DNA technology was used to define chromosome deletion boundaries. We found skewed XCI (>90%) in 16/186 (8.6%) mothers of NDD males and in 12/90 (13.3%) NDD females, far beyond the expected rate of XCI in the normal population (3.6%, OR = 4.10; OR = 2.51). By re-analyzing ES and clinical data, we solved 7/28 cases (25%) with skewed XCI, identifying variants in KDM5C, PDZD4, PHF6, TAF1, OTUD5 and ZMYM3, and a deletion in ATRX. We conclude that XCI profiling is a simple assay that targets a subgroup of patients that can benefit from re-evaluation of X-linked variants, thus improving the diagnostic yield in NDD patients and identifying new X-linked disorders.

Escape from X-inactivation in twins exhibits intra- and inter-individual variability across tissues and is heritable.

X-chromosome inactivation (XCI) silences one X in female cells to balance sex-differences in X-dosage. A subset of X-linked genes escape XCI, but the extent to which this phenomenon occurs and how it varies across tissues and in a population is as yet unclear. To characterize incidence and variability of escape across individuals and tissues, we conducted a transcriptomic study of escape in adipose, skin, lymphoblastoid cell lines and immune cells in 248 healthy individuals exhibiting skewed XCI. We quantify XCI escape from a linear model of genes' allelic fold-change and XIST-based degree of XCI skewing. We identify 62 genes, including 19 lncRNAs, with previously unknown patterns of escape. We find a range of tissue-specificity, with 11% of genes escaping XCI constitutively across tissues and 23% demonstrating tissue-restricted escape, including cell type-specific escape across immune cells of the same individual. We also detect substantial inter-individual variability in escape. Monozygotic twins share more similar escape than dizygotic twins, indicating that genetic factors may underlie inter-individual differences in escape. However, discordant escape also occurs within monozygotic co-twins, suggesting environmental factors also influence escape. Altogether, these data indicate that XCI escape is an under-appreciated source of transcriptional differences, and an intricate phenotype impacting variable trait expressivity in females.