Distributed X chromosome inactivation in brain circuitry is associated with X-linked disease penetrance of behavior.

The precise anatomical degree of brain X chromosome inactivation (XCI) that is sufficient to alter X-linked disorders in females is unclear. Here, we quantify whole-brain XCI at single-cell resolution to discover a prevalent activation ratio of maternal to paternal X at 60:40 across all divisions of the adult brain. This modest, non-random XCI influences X-linked disease penetrance: maternal transmission of the fragile X mental retardation 1 (Fmr1)-knockout (KO) allele confers 55% of total brain cells with mutant X-active, which is sufficient for behavioral penetrance, while 40% produced from paternal transmission is tolerated. Local XCI mosaicism within affected maternal Fmr1-KO mice further specifies sensorimotor versus social anxiety phenotypes depending on which distinct brain circuitry is most affected, with only a 50%-55% mutant X-active threshold determining penetrance. Thus, our results define a model of X-linked disease penetrance in females whereby distributed XCI among single cells populating brain circuitries can regulate the behavioral penetrance of an X-linked mutation.

Neuroenergetic Changes in Patients with X-Linked Dystonia-Parkinsonism and Female Carriers.

BACKGROUND: X-linked dystonia-parkinsonism (XDP) is a rare movement disorder characterized by profound neurodegeneration in the basal ganglia. The molecular consequences and the bioenergetic state of affected individuals remain largely unexplored. OBJECTIVES: To investigate the bioenergetic state in male patients with XDP and female carriers using 31phosphorus magnetic resonance spectroscopy imaging and to correlate these findings with clinical manifestations. METHODS: We examined the levels of high-energy phosphorus-containing metabolites (HEP) in the basal ganglia and cerebellum of five male patients with XDP, 10 asymptomatic female heterozygous carriers, and 10 SVA-insertion-free controls. RESULTS: HEP levels were reduced in the basal ganglia of patients with XDP (PwXDP) compared to controls, but increased in the cerebellum of both male patients and female carriers. CONCLUSIONS: Our findings suggest a potential compensatory mechanism in the cerebellum of female carriers regardless of sex. Our study highlights alterations in HEP levels in PwXDP patients and female carriers.

X-linked congenital adrenal hypoplasia: Report of long clinical follow-up and description of a new complex variant in the NR0B1 gene.

Adrenal hypoplasia congenita, attributed to NR0B1 pathogenic variants, accounts for more than 50% of the incidence of primary adrenal insufficiency in children. Although more than 250 different deleterious variations have been described, no genotype-phenotype correlation has been defined to date. We report a case of an adopted boy who reported the onset of an adrenal crisis at 2 weeks of age, requiring replacement therapy with mineralocorticoids and glucocorticoids for 4 months. For 3 years, he did well without treatment. At almost 4 years of age, the disorder was restarted. A long follow-up showed the evolution of hypogonadotropic hypogonadism. Molecular studies on NR0B1 revealed a novel and deleterious deletion-insertion-inversion-deletion complex rearrangement sorted in the 5'-3' direction, which is described as follows: (1) deletion of the intergenic region (between TASL and NR0B1 genes) and 5' region, (2) insertion of a sequence containing 37 bp at the junction of the intergenic region of the TASL gene and a part of exon 1 of the NR0B1 gene, (3) inversion of a part of exon 1, (4) deletion of the final portion of exon 1 and exon 2 and beginning of the 3'UTR region, (5) maintenance of part of the intergenic sequence (between genes MAGEB1 and NR0B1, telomeric sense), (6) large posterior deletion, in the same sense. The path to molecular diagnosis was challenging and involved several molecular biology techniques. Evaluating the breakpoints in our patient, we assumed that it was a nonrecurrent rearrangement that had not yet been described. It may involve a repair mechanism known as nonhomologous end-joining (NHEJ), which joins two ends of DNA in an imprecise manner, generating an "information scar," represented herein by the 37 bp insertion. In addition, the local Xp21 chromosome architecture with sequences capable of modifying the DNA structure could impact the formation of complex rearrangements.

Immune dysregulation, polyendocrinopathy and enteropathy, X-linked (IPEX) syndrome due to a mutation in FOXP3, modified by a pathogenic variant in SON (SON DNA-binding protein).

Immune dysregulation, polyendocrinopathy, enteropathy, and X-linked, known as IPEX syndrome, is a rare heterogeneous condition. Zhu-Tokita-Takenouchi-Kim Syndrome (ZTTK) is an autosomal dominant condition arising from a mutation in the SON gene, which is involved in mRNA splicing. A case showing interactions of mutations in these two genes is described in which both conditions become non-typical.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

SIMPSON-GOLABI-BEHMEL syndrome type 1: How placental immunohistochemistry can rapidly Predict the diagnosis.

INTRODUCTION: Glypican-3 (GPC3) is an oncofetal protein involved in cellular signaling, strongly expressed in the placenta, absent or diminished in postnatal life, but often increased in human malignancies. Germline loss-of-function variants of GPC3 gene are associated with Simpson-Golabi-Behmel syndrome type 1 (SGBS1), a rare recessive X-linked overgrowth disease characterized by typical facial features, congenital abnormalities, and an increased risk of developing childhood cancers. METHODS: A clinical suspicion of SGBS1 was postulated for a newborn with prenatal history of overgrowth and polyhydramnios, presenting with neonatal weight and length >99th percentile, coarse facies, iris and retinal coloboma, supernumerary nipples, and splenomegaly. While waiting for whole-genome sequencing (WGS) results, we investigated placental GPC3 immunohistochemical expression in the proband, in three additional cases of SGBS1, and disorders commonly associated with fetal macrosomia and/or placentomegaly. RESULTS: WGS in the proband identified a likely pathogenic maternally inherited missense variant in GPC3: c.1645A > G, (p.Ile549Val), and GPC3 immunohistochemistry demonstrated full-thickness loss of stain of the placental parenchyma. The same pattern ("null") was also present in the placentas of three additional cases of SGBS1, but not in those of unaffected controls. DISCUSSION: Immunohistochemical expression of GPC3 in the placenta is highly reproducible. Our findings showed that a "null pattern" of staining is predictive of SGBS1 and represents a valuable aid in the differential diagnosis of fetal macrosomias, allowing targeted genetic testing and earlier diagnosis.

A novel FOXP3 mutation in a Chinese child with IPEX-associated membranous nephropathy.

BACKGROUND: Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a monogenic immunodeficiency disease caused by forkhead box protein3 (FOXP3) mutation. The kidney is commonly involved in IPEX syndrome, but there were few studies focusing on renal involvement. METHODS: Whole-exome sequencing was used to identify the novel FOXP3 mutation. We collected clinical manifestations, kidney pathology, and gene function of the proband. All the previously published studies with IPEX-associated renal involvement were reviewed. RESULTS: We report a late-onset Chinese child with IPEX-associated membranous nephropathy (MN). Type 1 diabetes mellitus and nephrotic-range proteinuria are the main clinical manifestations. Whole-exome sequencing shows a novel c.766A > G mutation in the FOXP3 gene. The literature review indicates that renal manifestations include proteinuria, microscopic hematuria, and renal insufficiency. MN is the most common pathological type in children with IPEX, followed by tubulointerstitial nephritis, interstitial nephritis, minimal change nephrotic syndrome, and membranoproliferative glomerulonephritis. CONCLUSION: In summary, we report a novel FOXP3 mutation (c.766A > G) with MN stage II in IPEX. In a literature review, MN is the most common pathological type in children with IPEX and proteinuria is the most prevalent clinical feature. IPEX should be considered in the differential diagnosis of MN patients with related endocrine diseases and immune disorders.

Elucidating Hexanucleotide Repeat Number and Methylation within the X-Linked Dystonia-Parkinsonism (XDP)-Related SVA Retrotransposon in TAF1 with Nanopore Sequencing.

BACKGROUND: X-linked dystonia-parkinsonism (XDP) is an adult-onset neurodegenerative disorder characterized by progressive dystonia and parkinsonism. It is caused by a SINE-VNTR-Alu (SVA) retrotransposon insertion in the TAF1 gene with a polymorphic (CCCTCT)n domain that acts as a genetic modifier of disease onset and expressivity. METHODS: Herein, we used Nanopore sequencing to investigate SVA genetic variability and methylation. We used blood-derived DNA from 96 XDP patients for amplicon-based deep Nanopore sequencing and validated it with fragment analysis which was performed using fluorescence-based PCR. To detect methylation from blood- and brain-derived DNA, we used a Cas9-targeted approach. RESULTS: High concordance was observed for hexanucleotide repeat numbers detected with Nanopore sequencing and fragment analysis. Within the SVA locus, there was no difference in genetic variability other than variations of the repeat motif between patients. We detected high CpG methylation frequency (MF) of the SVA and flanking regions (mean MF = 0.94, SD = ±0.12). Our preliminary results suggest only subtle differences between the XDP patient and the control in predicted enhancer sites directly flanking the SVA locus. CONCLUSIONS: Nanopore sequencing can reliably detect SVA hexanucleotide repeat numbers, methylation and, lastly, variation in the repeat motif.

[A Novel Pathogenic variant in NR0B1 gene associated with Congenital Adrenal Hypoplasia]. / Nueva Variante Patogénica en el Gen NR0B1 Asociada a Hipoplasia Adrenal Congénita.

X-linked adrenal hypoplasia congenita is a rare cause of primary adrenal insufficiency. Mutations in the NR0B1 gene cause a loss of function in the DAX1 receptor, which activates genes involved in the development and function of the hypothalamic-pituitary-gonadal axis. Objective: To describe a case of adrenal hypoplasia congenita secondary to a mutation in the NR0B1 gene and identified the differential diagnoses of the pediatric patient with adrenal insufficiency and hypogonadotropic hypogonadism. Clinical Case: A 4-year-old male patient with no relevant history and from a rural area was admitted to the emergency room due to a 15-days of emesis, asthenia, adynamia, myalgia, and ataxic gait. On the physical examination, hypotension, hyponatremia, and hyperkalemia, as well as mucosal hyperpigmentation and bilateral cryptorchidism were observed, therefore, adrenal crisis was diagnosed, starting fluid resuscitation with saline solution, hydrocortisone, and fludrocortisone, which stabilized the patient. Adrenal hyperplasia congenita, innate metabolic error, and infectious or autoimmune etiology were ruled out as etiology. A clinical exome test was performed which iden tified the variant c.1275A > T; p.Arg425Ser (Transcript ENST00000378970.5) in the NR0B1 gene consistent with X-linked adrenal hypoplasia congenita. Management of the patient continued with glucocorticoids and mineralocorticoids with favorable clinical course at 7 years of follow-up. Con clusion: A novel pathogenic variant associated with X-linked adrenal hypoplasia is described. Variants in the NR0B1 gene should be a differential diagnosis in a male patient with the association of primary adrenal insufficiency and hypogonadism.

Nodular regenerative hyperplasia in X-linked agammaglobulinemia: An underestimated and severe complication.

BACKGROUND: Late-onset complications in X-linked agammaglobulinemia (XLA) are increasingly recognized. Nodular regenerative hyperplasia (NRH) has been reported in primary immunodeficiency but data in XLA are limited. OBJECTIVES: This study sought to describe NRH prevalence, associated features, and impact in patients with XLA. METHODS: Medical records of all patients with XLA referred to the National Institutes of Health between October 1994 and June 2019 were reviewed. Liver biopsies were performed when clinically indicated. Patients were stratified into NRH+ or NRH- groups, according to their NRH biopsy status. Fisher exact test and Mann-Whitney test were used for statistical comparisons. RESULTS: Records of 21 patients with XLA were reviewed, with a cumulative follow-up of 129 patient-years. Eight patients underwent ≥1 liver biopsy of whom 6 (29% of the National Institutes of Health XLA cohort) were NRH+. The median age at NRH diagnosis was 20 years (range, 17-31). Among patients who had liver biopsies, alkaline phosphatase levels were only increased in patients who were NRH+ (P = .04). Persistently low platelet count (<100,000 per µL for >6 months), mildly to highly elevated hepatic venous pressure gradient and either hepatomegaly and/or splenomegaly were present in all patients who were NRH+. In opposition, persistently low platelet counts were not seen in patients who were NRH-, and hepatosplenomegaly was observed in only 1 patient who was NRH-. Hepatic venous pressure gradient was normal in the only patient tested who was NRH-. All-cause mortality was higher among patients who were NRH+ (5 of 6, 83%) than in the rest of the cohort (1 of 15, 7% among patients who were NRH- and who were classified as unknown; P = .002). CONCLUSIONS: NRH is an underreported, frequent, and severe complication in XLA, which is associated with increased morbidity and mortality.

A novel NSDHL variant in CHILD syndrome with gastrointestinal manifestations and localized skin involvement.

BACKGROUND: CHILD syndrome is an X-linked dominant disorder associated with pathogenic mutations in the NSDHL gene. The condition is predominantly found in females as it is lethal in males. Most cases present at birth with extensive unilateral ichthyosiform erythroderma involving the trunk and limbs. Milder and less extensive presentations have been reported, leading to misdiagnosis especially during early childhood. METHODS AND RESULTS: We report an adult female of Malay ancestry who presented with minimal skin and limb involvement. She was only diagnosed in adulthood when she presented with gastrointestinal symptoms and worsening of skin manifestations. The clinical diagnosis was suspected after a combination of clinical, pathological and immunohistochemistry correlation, and molecularly confirmed with the discovery of a frameshift variant in NSDHL. The novel variant was inherited from her mother who had some linear hypopigmented patches over the medial aspects of both her arms and right forearm. CONCLUSION: We uncovered a novel frameshift variant associated with presentations that cast a new light on the clinical features of CHILD syndrome.

Molecular Biomarkers for Adrenoleukodystrophy: An Unmet Need.

X-linked adrenoleukodystrophy (ALD) is an inherited progressive neurometabolic disease caused by mutations in the ABCD1 gene and the accumulation of very long-chain fatty acids in plasma and tissues. Patients present with heterogeneous clinical manifestations which can include adrenal insufficiency, myelopathy, and/or cerebral demyelination. In the absence of a genotype-phenotype correlation, the clinical outcome of an individual cannot be predicted and currently there are no molecular markers available to quantify disease severity. Therefore, there is an unmet clinical need for sensitive biomarkers to monitor and/or predict disease progression and evaluate therapy efficacy. The increasing amount of biological sample repositories ('biobanking') as well as the introduction of newborn screening creates a unique opportunity for identification and evaluation of new or existing biomarkers. Here we summarize and review the many studies that have been performed to identify and improve knowledge surrounding candidate molecular biomarkers for ALD. We also highlight several shortcomings of ALD biomarker studies, which often include a limited sample size, no collection of longitudinal data, and no validation of findings in an external cohort. Nonetheless, these studies have generated a list of interesting biomarker candidates and this review aspires to direct future biomarker research.

Fine Breakpoint Mapping by Genome Sequencing Reveals the First Large X Inversion Disrupting the NHS Gene in a Patient with Syndromic Cataracts.

Inversions are structural variants that are generally balanced. However, they could lead to gene disruptions or have positional effects leading to diseases. Mutations in the NHS gene cause Nance-Horan syndrome, an X-linked disorder characterised by congenital cataracts and dental anomalies. Here, we aimed to characterise a balanced pericentric inversion X(p22q27), maternally inherited, in a child with syndromic bilateral cataracts by breakpoint mapping using whole-genome sequencing (WGS). 30× Illumina paired-end WGS was performed in the proband, and breakpoints were confirmed by Sanger sequencing. EdU assays and FISH analysis were used to assess skewed X-inactivation patterns. RNA expression of involved genes in the breakpoint boundaries was evaluated by droplet-digital PCR. We defined the breakpoint position of the inversion at Xp22.13, with a 15 bp deletion, disrupting the unusually large intron 1 of the canonical NHS isoform, and also perturbing topologically-associated domains (TADs). Moreover, a microhomology region of 5 bp was found on both sides. RNA analysis confirmed null and reduced NHS expression in the proband and his unaffected mother, respectively. In conclusion, we report the first chromosomal inversion disrupting NHS, fine-mapped by WGS. Our data expand the clinical spectrum and the pathogenic mechanisms underlying the NHS defects.

Sertoli Cells Improve Myogenic Differentiation, Reduce Fibrogenic Markers, and Induce Utrophin Expression in Human DMD Myoblasts.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in DMD gene translating in lack of functional dystrophin and resulting in susceptibility of myofibers to rupture during contraction. Inflammation and fibrosis are critical hallmarks of DMD muscles, which undergo progressive degeneration leading to loss of independent ambulation in childhood and death by early adulthood. We reported that intraperitoneal injection of microencapsulated Sertoli cells (SeC) in dystrophic mice translates into recovery of muscle morphology and performance thanks to anti-inflammatory effects and induction of the dystrophin paralogue, utrophin at the muscle level, opening new avenues in the treatment of DMD. The aim of this study is to obtain information about the direct effects of SeC on myoblasts/myotubes, as a necessary step in view of a translational application of SeC-based approaches to DMD. We show that (i) SeC-derived factors stimulate cell proliferation in the early phase of differentiation in C2C12, and human healthy and DMD myoblasts; (ii) SeC delay the expression of differentiation markers in the early phase nevertheless stimulating terminal differentiation in DMD myoblasts; (iii) SeC restrain the fibrogenic potential of fibroblasts, and inhibit myoblast-myofibroblast transdifferentiation; and, (iv) SeC provide functional replacement of dystrophin in preformed DMD myotubes regardless of the mutation by inducing heregulin ß1/ErbB2/ERK1/2-dependent utrophin expression. Altogether, these results show that SeC are endowed with promyogenic and antifibrotic effects on dystrophic myoblasts, further supporting their potential use in the treatment of DMD patients. Our data also suggest that SeC-based approaches might be useful in improving the early phase of muscle regeneration, during which myoblasts have to adequately proliferate to replace the damaged muscle mass.

A functional genomics pipeline identifies pleiotropy and cross-tissue effects within obesity-associated GWAS loci.

Genome-wide association studies (GWAS) have identified many disease-associated variants, yet mechanisms underlying these associations remain unclear. To understand obesity-associated variants, we generate gene regulatory annotations in adipocytes and hypothalamic neurons across cellular differentiation stages. We then test variants in 97 obesity-associated loci using a massively parallel reporter assay and identify putatively causal variants that display cell type specific or cross-tissue enhancer-modulating properties. Integrating these variants with gene regulatory information suggests genes that underlie obesity GWAS associations. We also investigate a complex genomic interval on 16p11.2 where two independent loci exhibit megabase-range, cross-locus chromatin interactions. We demonstrate that variants within these two loci regulate a shared gene set. Together, our data support a model where GWAS loci contain variants that alter enhancer activity across tissues, potentially with temporally restricted effects, to impact the expression of multiple genes. This complex model has broad implications for ongoing efforts to understand GWAS.

Small RNA sequencing evaluation of renal microRNA biomarkers in dogs with X-linked hereditary nephropathy.

Dogs with X-linked hereditary nephropathy (XLHN) are an animal model for Alport syndrome in humans and progressive chronic kidney disease (CKD). Using mRNA sequencing (mRNA-seq), we have characterized the gene expression profile affecting the progression of XLHN; however, the microRNA (miRNA, miR) expression remains unknown. With small RNA-seq and quantitative RT-PCR (qRT-PCR), we used 3 small RNA-seq analysis tools (QIAGEN OmicSoft Studio, miRDeep2, and CPSS 2.0) to profile differentially expressed renal miRNAs, top-ranked miRNA target genes, and enriched biological processes and pathways in CKD progression. Twenty-three kidney biopsies were collected from 5 dogs with XLHN and 4 age-matched, unaffected littermates at 3 clinical time points (T1: onset of proteinuria, T2: onset of azotemia, and T3: advanced azotemia). We identified up to 23 differentially expressed miRNAs at each clinical time point. Five miRNAs (miR-21, miR-146b, miR-802, miR-142, miR-147) were consistently upregulated in affected dogs. We identified miR-186 and miR-26b as effective reference miRNAs for qRT-PCR. This study applied small RNA-seq to identify differentially expressed miRNAs that might regulate critical pathways contributing to CKD progression in dogs with XLHN.

Reducing body myopathy - A new pathogenic FHL1 variant and literature review.

Reducing body myopathy (RBM) is a rare disease marked by progressive muscle weakness caused by a mutation in FHL1 gene. We describe a new pathogenic variant and contrasted it with 44 other cases identified in the literature. A male child presented at age 3 suffering frequent falls and progressive muscular weakness. At age 8, he was wheelchair-bound and required ventilatory support. His mother and sister died due to the same problem. Creatine kinase was 428 IU/L (<190). Muscle biopsy showed typical reducing bodies, and genetic analysis identified a novel pathogenic hemizygous variant, c.370_375del. We identified 44 previous reported cases separated in two groups: 28 cases with mean age onset 7.6 ±â€¯5 years and 16 with 26.7 ±â€¯4.2 years. The time for the diagnosis was shorter to younger group. The initial symptoms, rigid spine, contractures, scoliosis and axial and neck weaknesses, dysphagia, cardiac involvement, were predominant in younger group. The variant c.369C > G predominated in younger group and c.448T > C in older one. Pathogenic variants positions seemed related to severe phenotype. Most wheelchair patients belonged to younger group. The data from this compilation and our case provided a general characterization spectrum and prognosis between two groups of age onset with RBM.

Terminal osseous dysplasia with pigmentary defects and cardiomyopathy caused by a novel FLNA variant.

Terminal osseous dysplasia with pigmentary defects (TODPD), also known as digitocutaneous dysplasia, is one of the X-linked filaminopathies caused by a variety of FLNA-variants. TODPD is characterized by skeletal defects, skin fibromata and dysmorphic facial features. So far, only a single recurrent variant (c.5217G>A;p.Val1724_Thr1739del) in FLNA has found to be responsible for TODPD. We identified a novel c.5217+5G>C variant in FLNA in a female proband with skeletal defects, skin fibromata, interstitial lung disease, epilepsy, and restrictive cardiomyopathy. This variant causes mis-splicing of exon 31 predicting the production of a FLNA-protein with an in-frame-deletion of 16 residues identical to the miss-splicing-effect of the recurrent TODPD c.5217G>A variant. This mis-spliced transcript was explicitly detected in heart tissue, but was absent from blood, skin, and lung. X-inactivation analyses showed extreme skewing with almost complete inactivation of the mutated allele (>90%) in these tissues, except for heart. The mother of the proband, who also has fibromata and skeletal abnormalities, is also carrier of the FLNA-variant and was diagnosed with noncompaction cardiomyopathy after cardiac screening. No other relevant variants in cardiomyopathy-related genes were found. Here we describe a novel variant in FLNA (c.5217+5G>C) as the second pathogenic variant responsible for TODPD. Cardiomyopathy has not been described as a phenotypic feature of TODPD before.