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.

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.

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.

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.

A novel truncating variant in the FGD1 gene associated with Aarskog-Scott syndrome in a family previously diagnosed with Tel Hashomer camptodactyly.

Tel Hashomer camptodactyly syndrome is a long-known entity characterized by camptodactyly with muscular hypoplasia, skeletal dysplasia, and abnormal palmar creases. Currently, the genetic basis for this disorder is unknown, thus there is a possibility that this clinical presentation may be contained within another genetic diagnosis. Here, we present a multiplex family with a previous clinical diagnosis of Tel Hashomer camptodactyly syndrome. Whole exome sequencing and pedigree-based analysis revealed a novel hemizygous truncating variant c.269_270dup (p.Phe91Alafs*34) in the FGD1 gene (NM_004463.3) in all three symptomatic patients, congruous with a diagnosis of Aarskog-Scott syndrome. Our report adds to the limited data on Aarskog-Scott syndrome, and emphasizes the importance of unbiased comprehensive molecular testing toward establishing a diagnosis for genetic syndromes with unknown genetic basis.

Pro-inflammatory effects of DEHP in SGBS-derived adipocytes and THP-1 macrophages.

In the member countries of the Organization for Economic Co-operation and Development (OECD), overweight and obesity affect the majority of the population. The use of environmental chemicals, such as the plasticizer DEHP, has largely increased simultaneously with this development. DEHP is an "obesogen" that interferes with normal adipocyte differentiation and energy homeostasis. Obesity in turn is accompanied by chronic low-grade adipose tissue inflammation, leading to metabolic disorders such as type II diabetes. The main actors in adipose tissue inflammation are adipocytes and macrophages. However, the impact of DEHP on adipose tissue inflammation and the crosstalk between adipocytes and macrophages are unknown and the subjects of the current study. The influence of DEHP on inflammation was investigated in human Simpson-Golabi-Behmel syndrome (SGBS)-derived adipocytes and human THP-1 macrophages. The proinflammatory markers IL8, MCP1, IL1ß, TNFα and others were measured (qRT-PCR, ELISA) in SGBS-derived adipocytes treated with DEHP [day 0 (d0)-d4; 50 µg/ml] and THP-1 macrophages cultured with conditioned medium (CM) from DEHP-treated adipocytes (SGBS-CM) (from d4 and d8). DEHP exposure led to a proinflammatory state in SGBS-derived adipocytes (e.g., increased secretion of IL8 and MCP1). Surprisingly, exposure of THP-1 macrophages to SGBS-CM did not show DEHP-induced effects. However, we demonstrated that medium containing (pre)adipocyte-secreted factors had a significant impact on the expression and secretion of macrophage and inflammatory markers in THP-1 macrophages in general and led to the significantly increased accumulation of intracellular lipid droplets.

Clinical application of an NGS-based method in the preimplantation genetic testing for Duchenne muscular dystrophy.

PURPOSE: To determine whether next-generation sequencing (NGS) could be used to directly detect different mutations of Duchenne muscular dystrophy (DMD) during preimplantation genetic testing (PGT). METHODS: From Sep. 2016 to Aug. 2018, a total of six couples participated in this study. Four cases carried DMD exon deletions and two carried exon duplications. Trophectoderm cells were biopsied at day 5 or 6 and NGS was used in the genetic testing of the biopsied cells after whole-genome amplification. We developed a new method-DIRected Embryonic Cell Testing of Exon Deletion/Duplication (DIRECTED) to directly detect the single-gene mutation by NGS. Linage analysis based on single-nucleotide polymorphism (SNP) was used to validate the results from DIRECTED. RESULTS: In the four deletion cases, DIRECTED was used to detect DMD exon deletion in 16 biopsied embryos. All DIRECTED results were consistent with linkage analysis, indicating this method was reliable in detecting deletions around 1 Mb. In the two cases carrying exon duplications, no blastocyst was obtained for biopsy. Nonetheless, preliminary experiment results suggested that DIRECTED could also be used for direct detection of exon duplications in embryos. CONCLUSIONS: Exon deletions or duplications in DMD of preimplantation embryos could be detected directly by NGS-based methods during PGT.

Alveolar capillary dysplasia without misalignment of pulmonary veins, hyperinflammation, megalocornea and overgrowth - Association with a homozygous 2bp-insertion in LTBP2?

We present a male infant with alveolar capillary dysplasia without misalignment of pulmonary veins, hyperinflammation, megalocornea and macrosomia/macrocephaly at birth. Whole-exome sequencing revealed a homozygous 2bp-insertion in the latent transforming growth factor-beta binding protein 2 (LTBP2) (c.278_279dup, p.(Ser94Glyfs*187)). So far, LTBP2-variants have been frequently reported with an eye-restricted phenotype including primary congenital glaucoma and megalocornea/microspherphakia and ectopia lentis with/without secondary glaucoma. Hitherto reported systemic phenotypes showed, among others, features as tall stature, finger anomalies, high-arched palate and cardiovascular anomalies. The main pathophysiological finding of our patient was an alveolar capillary dysplasia (with pulmonary arterial hypertension and right ventricular impairment but without misalignment of pulmonary veins) resulting in almost continuous oxygen demand and prolonged dependence on mechanical ventilation. He died of respiratory failure at the age of seven months. This patient may extend the LTBP2-related phenotype with resulting diagnostic implications.

New Findings of Immunodysregulation, Polyendocrinopathy, and Enteropathy X-linked Syndrome (IPEX); Granulomas in Lung and Duodenum.

Immune dysregulation, polyendocrinopathy and enteropathy, X-linked (IPEX) syndrome is a rare disorder caused by loss-of-function mutations in the gene forkhead box protein 3 (FOXP3). IPEX patients frequently show chronic diarrhea (enteropathy) associated with villous atrophies in the small intestine. Our case is different from this classical information in the literature, since he presented with neonatal onset inflammatory bowel disease within the first months of life accompanied by deep ulcers throughout colonic mucosa. Moreover, he developed chronic lung disease during follow-up and histopathological examinations showed granulomas in both gastrointestinal tract and lung parenchyma. Genetic analysis revealed the diagnosis of IPEX syndrome with a germline mutation in FOXP3. Thus, our study provides an unusual presentation of IPEX syndrome with colitis and granulomas presence in histopathological examinations.

A combination of cyclophosphamide and interleukin-2 allows CD4+ T cells converted to Tregs to control scurfy syndrome.

Immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is caused by mutations in forkhead box P3 (FOXP3), which lead to the loss of function of regulatory T cells (Tregs) and the development of autoimmune manifestations early in life. The selective induction of a Treg program in autologous CD4+ T cells by FOXP3 gene transfer is a promising approach for curing IPEX. We have established a novel in vivo assay of Treg functionality, based on adoptive transfer of these cells into scurfy mice (an animal model of IPEX) and a combination of cyclophosphamide (Cy) conditioning and interleukin-2 (IL-2) treatment. This model highlighted the possibility of rescuing scurfy disease after the latter's onset. By using this in vivo model and an optimized lentiviral vector expressing human Foxp3 and, as a reporter, a truncated form of the low-affinity nerve growth factor receptor (ΔLNGFR), we demonstrated that the adoptive transfer of FOXP3-transduced scurfy CD4+ T cells enabled the long-term rescue of scurfy autoimmune disease. The efficiency was similar to that seen with wild-type Tregs. After in vivo expansion, the converted CD4FOXP3 cells recapitulated the transcriptomic core signature for Tregs. These findings demonstrate that FOXP3 expression converts CD4+ T cells into functional Tregs capable of controlling severe autoimmune disease.

AIFM1-associated X-linked spondylometaphyseal dysplasia with cerebral hypomyelination.

Spondylometaphyseal dysplasia with cerebral hypomyelination (SMD-H) is a very rare but distinctive phenotype, unusually combining spondylometaphyseal dysplasia with hypomyelinating leukodystrophy. Recently, SMD-H has been associated with variants confined to a specific intra-genic locus involving Exon 7, suggesting that AIFM1 plays an important role in bone development and metabolism as well as cerebral myelination. Here we describe two further affected boys, one with a novel intronic variant associated with skipping of Exon 7 of AIFM1 and the other a synonymous variant within Exon 7 of AIFM1. We describe their clinical course and radiological and genetic findings, providing further insight into the natural history of this condition.

A novel Xp11.22-22.33 deletion suggesting a possible mechanism of congenital cervical spinal muscular atrophy.

BACKGROUND: Congenital cervical spinal muscular atrophy (CCSMA) is a rare, nonprogressive, neurogenic disorder characterized by symmetric arthrogryposis and motor deficits mainly confined to upper extremities. Since its first proposal by Darwish et al. 39 years ago, only few cases have ever been reported. Vascular insult to the anterior horn of cervical spinal cord during fetal development was speculated to be the cause, however, the exact pathogenesis is still not well understood. METHODS: In this study, whole-exome sequencing (WES) and copy number variation (CNV) analysis were conducted on a definitive CCSMA patient, confirmed by the clinical manifestations and other supplementary examinations. RESULTS: On physical examination, the patient was mainly characterized by symmetric, congenital, nonprogressive contractures, hypotonia, and muscle weakness mainly confined to the upper limbs, which were further supported by MRI and electromyography. Neuromuscular biopsy of the deltoid muscle demonstrated the type 1 myofiber predominance without any infiltration of inflammatory cells. The WES and CNV analysis unveiled a de novo Xp11.22-22.33 deletion. On further examination of the genes contained within this segment, we recognize UBA1 gene as the most likely pathogenic gene. Ubiquitin-like modifier activating enzyme 1 is encoded by UBA1 gene (MIM 314370) located in Xp11.3 and is a critical protein that plays a vital role in ubiquitin-proteasome system and autophagy. It is well documented that UBA1 gene mutation causes X-linked infantile spinal muscular atrophy (XL-SMA), which manifests phenotypes of arthrogryposis, hypotonia, and myopathic face. Type 2 XL-SMA, which follows a nonprogressive and nonlethal course is very similar to the presentations of CCSMA. CONCLUSION: The phenotypic similarities between this CCSMA case and XL-SMA prompt us to hypothesize a possible connection between UBA1 gene deficit and the pathogenesis of CCSMA. Our study is the first to demonstrate that CCSMA might have a genetic etiology, thus, expanding our insights into the underlying cause of CCSMA.

A Dominant C150Y Mutation in FHL1 Induces Structural Alterations in LIM2 Domain Causing Protein Aggregation In Human and Drosophila Indirect Flight Muscles.

FHL1-related myopathies are rare X-linked dominant myopathies. Though clinically classified into several subgroups, spinal and scapuloperoneal muscle involvement are common to all. In this study, we identified c.449G > A, p.C150Y mutation by clinical exome sequencing in two patients from same family (son and mother) of Indian origin who presented with multiple contractures. Muscle biopsy showed numerous intracytoplasmic aggregates intensely stained on HE and MGT. The strong reactions to M-NBT revealed aggregates to be reducing bodies and positively labeled to anti-FHL1 antibody. Ultrastructurally, Z-band streaming and granular and granulofilamentous material were seen. Further, the translational evidence of mutant peptide was confirmed using mass spectrometric analysis. To establish p.C150Y as the cause for protein aggregation, in vivo studies were carried out using transgenic Drosophila model which highlighted Z-band abnormalities and protein aggregates in indirect flight muscles with compromised physiological function. Thus, recapitulating the X-linked human disease phenotype. Additionally, the molecular dynamics simulation analysis unraveled the drastic change in α-helix of LIM2, the region immediately next to site of C150Y mutation that could be the plausible cause for protein aggregation. To the best of our knowledge, this is the first study of p.C150Y mutation in FHL1 identified in Indian patients with in vivo and in silico analysis to establish the cause for protein aggregation in muscle.

CD8-positive cutaneous lymphoproliferation associated with large granular lymphocyte leukemia in a patient with X-linked agammaglobulinemia.

Patients with primary immunodeficiency are at increased risk for malignancy, especially hematologic neoplasms. This paper reports a unique case of a 47-year-old man with X-linked agammaglobulinemia who presented with progressive asymptomatic violaceous papules and plaques on his face, hands, and trunk for 1 year. Skin biopsies revealed deep, nodular infiltrates of histiocytes and CD8-positive lymphocytes, with a CD4:CD8 ratio of 1:10. Laboratory studies showed cytopenias. Flow cytometry in the skin, blood, and bone marrow (BM) showed a CD3+/CD8+/CD57+ large granular lymphocyte population. BM biopsy showed 30% involvement with these atypical T-cells. T-cell gene rearrangement studies of skin, blood, and BM revealed identical T-cell clones. He was diagnosed with T-large granular lymphocyte leukemia (T-LGLL) with an associated CD8+ cutaneous lymphoproliferation. Skin involvement was suspected to represent infiltration by T-LGLL. However, co-existence of two lymphoproliferative disorders (LPDs), T-LGLL and CD8+ granulomatous LPD, remains a possibility. In general, cutaneous infiltrates associated with LGLL are rare and poorly understood. It has been suggested that they are markers of poor prognosis. Our case report describes skin, blood, and BM findings in an immunosuppressed patient with T-LGLL in detail. These findings have not yet been reported and their significance requires further investigation.