Multi-disciplinary Insights from the First European Forum on Visceral Myopathy 2022 Meeting.

Visceral myopathy is a rare, life-threatening disease linked to identified genetic mutations in 60% of cases. Mostly due to the dearth of knowledge regarding its pathogenesis, effective treatments are lacking. The disease is most commonly diagnosed in children with recurrent or persistent disabling episodes of functional intestinal obstruction, which can be life threatening, often requiring long-term parenteral or specialized enteral nutritional support. Although these interventions are undisputedly life-saving as they allow affected individuals to avoid malnutrition and related complications, they also seriously compromise their quality of life and can carry the risk of sepsis and thrombosis. Animal models for visceral myopathy, which could be crucial for advancing the scientific knowledge of this condition, are scarce. Clearly, a collaborative network is needed to develop research plans to clarify genotype-phenotype correlations and unravel molecular mechanisms to provide targeted therapeutic strategies. This paper represents a summary report of the first 'European Forum on Visceral Myopathy'. This forum was attended by an international interdisciplinary working group that met to better understand visceral myopathy and foster interaction among scientists actively involved in the field and clinicians who specialize in care of people with visceral myopathy.

Alazami syndrome: the first case of papillary thyroid carcinoma.

Alazami syndrome (MIM#615071) is a rare developmental disorder caused by biallelic variants in the LARP7 gene. Hallmark features include short stature, global developmental delay, and distinctive facial features. To date, 23 patients from 11 families have been reported in the literature. Here we describe a 19-year-old man who, in association with the typical features of Alazami syndrome, was diagnosed at the age of 14 years with papillary thyroid carcinoma, harboring the somatic BRAF V600E mutation. Whole exome sequencing revealed two novel LARP7 variants in compound heterozygosity, whereas only common variants were detected in genes associated with familial nonmedullary thyroid cancer (MIM#188550). LARP7 acts as a tumor suppressor in breast and gastric cancer, and possibly, according to recent studies, in thyroid tumors. Since thyroid cancer is rare among children and adolescents, we hypothesize that the LARP7 variants identified in our patient are responsible for both Alazami syndrome and tumor susceptibility. We also provide an overview of the clinical findings in all Alazami syndrome patients reported to date and discuss the possible pathogenetic mechanism that may underlie this condition, including the role of LARP7 in tumor susceptibility.

Expanding the phenotype of Wiedemann-Steiner syndrome: Craniovertebral junction anomalies.

Wiedemann-Steiner syndrome (WDSTS) is a rare autosomal dominant condition caused by heterozygous loss of function variants in the KMT2A (MLL) gene, encoding a lysine N-methyltransferase that mediates a histone methylation pattern specific for epigenetic transcriptional activation. WDSTS is characterized by a distinctive facial phenotype, hypertrichosis, short stature, developmental delay, intellectual disability, congenital malformations, and skeletal anomalies. Recently, a few patients have been reported having abnormal skeletal development of the cervical spine. Here we describe 11 such individuals, all with KMT2A de novo loss-of-function variants: 10 showed craniovertebral junction anomalies, while an 11th patient had a cervical abnormality in C7. By evaluating clinical and diagnostic imaging data we characterized these anomalies, which consist primarily of fused cervical vertebrae, C1 and C2 abnormalities, small foramen magnum and Chiari malformation type I. Craniovertebral anomalies in WDSTS patients have been largely disregarded so far, but the increasing number of reports suggests that they may be an intrinsic feature of this syndrome. Specific investigation strategies should be considered for early identification and prevention of craniovertebral junction complications in WDSTS patients.

BGN Mutations in X-Linked Spondyloepimetaphyseal Dysplasia.

Spondyloepimetaphyseal dysplasias (SEMDs) comprise a heterogeneous group of autosomal-dominant and autosomal-recessive disorders. An apparent X-linked recessive (XLR) form of SEMD in a single Italian family was previously reported. We have been able to restudy this family together with a second family from Korea by segregating a severe SEMD in an X-linked pattern. Exome sequencing showed missense mutations in BGN c.439A>G (p.Lys147Glu) in the Korean family and c.776G>T (p.Gly259Val) in the Italian family; the c.439A>G (p.Lys147Glu) mutation was also identified in a further simplex SEMD case from India. Biglycan is an extracellular matrix proteoglycan that can bind transforming growth factor beta (TGF-ß) and thus regulate its free concentration. In 3-dimensional simulation, both altered residues localized to the concave arc of leucine-rich repeat domains of biglycan that interact with TGF-ß. The observation of recurrent BGN mutations in XLR SEMD individuals from different ethnic backgrounds allows us to define "XLR SEMD, BGN type" as a nosologic entity.

Lowry-Wood syndrome: further evidence of association with RNU4ATAC, and correlation between genotype and phenotype.

Lowry-Wood syndrome (LWS) is a skeletal dysplasia characterized by multiple epiphyseal dysplasia associated with microcephaly, developmental delay and intellectual disability, and eye involvement. Pathogenic variants in RNU4ATAC, an RNA of the minor spliceosome important for the excision of U12-dependent introns, have been recently associated with LWS. This gene had previously also been associated with microcephalic osteodysplastic primordial dwarfism (MOPD) and Roifman syndrome (RS), two distinct conditions which share with LWS some skeletal and neurological anomalies. We performed exome sequencing in two individuals with Lowry-Wood syndrome. We report RNU4ATAC pathogenic variants in two further patients. Moreover, an analysis of all RNU4ATAC variants reported so far showed that FitCons scores for nucleotides mutated in the more severe MOPD are higher than RS or LWS and that they were more frequently located in the 5' Stem-Loop of the RNA critical for the formation of the U4/U6.U5 tri-snRNP complex, whereas the variants are more dispersed in the other conditions. We are thus confirming that RNU4ATAC is the gene responsible for LWS and provide a genotype-phenotype correlation analysis.

Phenotype and genotype of 87 patients with Mowat-Wilson syndrome and recommendations for care.

PURPOSE: Mowat-Wilson syndrome (MWS) is a rare intellectual disability/multiple congenital anomalies syndrome caused by heterozygous mutation of the ZEB2 gene. It is generally underestimated because its rarity and phenotypic variability sometimes make it difficult to recognize. Here, we aimed to better delineate the phenotype, natural history, and genotype-phenotype correlations of MWS. METHODS: In a collaborative study, we analyzed clinical data for 87 patients with molecularly confirmed diagnosis. We described the prevalence of all clinical aspects, including attainment of neurodevelopmental milestones, and compared the data with the various types of underlying ZEB2 pathogenic variations. RESULTS: All anthropometric, somatic, and behavioral features reported here outline a variable but highly consistent phenotype. By presenting the most comprehensive evaluation of MWS to date, we define its clinical evolution occurring with age and derive suggestions for patient management. Furthermore, we observe that its severity correlates with the kind of ZEB2 variation involved, ranging from ZEB2 locus deletions, associated with severe phenotypes, to rare nonmissense intragenic mutations predicted to preserve some ZEB2 protein functionality, accompanying milder clinical presentations. CONCLUSION: Knowledge of the phenotypic spectrum of MWS and its correlation with the genotype will improve its detection rate and the prediction of its features, thus improving patient care.

Neuroimaging findings in Mowat-Wilson syndrome: a study of 54 patients.

PURPOSE: Mowat-Wilson syndrome (MWS) is a genetic disease characterized by distinctive facial features, moderate to severe intellectual disability, and congenital malformations, including Hirschsprung disease, genital and eye anomalies, and congenital heart defects, caused by haploinsufficiency of the ZEB2 gene. To date, no characteristic pattern of brain dysmorphology in MWS has been defined. METHODS: Through brain magnetic resonance imaging (MRI) analysis, we delineated a neuroimaging phenotype in 54 MWS patients with a proven ZEB2 defect, compared it with the features identified in a thorough review of published cases, and evaluated genotype-phenotype correlations. RESULTS: Ninety-six percent of patients had abnormal MRI results. The most common features were anomalies of corpus callosum (79.6% of cases), hippocampal abnormalities (77.8%), enlargement of cerebral ventricles (68.5%), and white matter abnormalities (reduction of thickness 40.7%, localized signal alterations 22.2%). Other consistent findings were large basal ganglia, cortical, and cerebellar malformations. Most features were underrepresented in the literature. We also found ZEB2 variations leading to synthesis of a defective protein to be favorable for psychomotor development and some epilepsy features but also associated with corpus callosum agenesis. CONCLUSION: This study delineated the spectrum of brain anomalies in MWS and provided new insights into the role of ZEB2 in neurodevelopment.Genet Med advance online publication 10 November 2016.

Brief report: isogenic induced pluripotent stem cell lines from an adult with mosaic down syndrome model accelerated neuronal ageing and neurodegeneration.

Trisomy 21 (T21), Down Syndrome (DS) is the most common genetic cause of dementia and intellectual disability. Modeling DS is beginning to yield pharmaceutical therapeutic interventions for amelioration of intellectual disability, which are currently being tested in clinical trials. DS is also a unique genetic system for investigation of pathological and protective mechanisms for accelerated ageing, neurodegeneration, dementia, cancer, and other important common diseases. New drugs could be identified and disease mechanisms better understood by establishment of well-controlled cell model systems. We have developed a first nonintegration-reprogrammed isogenic human induced pluripotent stem cell (iPSC) model of DS by reprogramming the skin fibroblasts from an adult individual with constitutional mosaicism for DS and separately cloning multiple isogenic T21 and euploid (D21) iPSC lines. Our model shows a very low number of reprogramming rearrangements as assessed by a high-resolution whole genome CGH-array hybridization, and it reproduces several cellular pathologies seen in primary human DS cells, as assessed by automated high-content microscopic analysis. Early differentiation shows an imbalance of the lineage-specific stem/progenitor cell compartments: T21 causes slower proliferation of neural and faster expansion of hematopoietic lineage. T21 iPSC-derived neurons show increased production of amyloid peptide-containing material, a decrease in mitochondrial membrane potential, and an increased number and abnormal appearance of mitochondria. Finally, T21-derived neurons show significantly higher number of DNA double-strand breaks than isogenic D21 controls. Our fully isogenic system therefore opens possibilities for modeling mechanisms of developmental, accelerated ageing, and neurodegenerative pathologies caused by T21.

Generation of IGGi003-A induced pluripotent stem cell line from a patient with Sotos Syndrome carrying c.1633delA NSD1 variant in exon 5.

Sotos syndrome (SoS) is a neurodevelopmental disorder that results from NSD1 mutations that cause haploinsufficiency of NSD1. Here, we generated an induced pluripotent stem cell (iPSC) line from fibroblasts of a SoS patient carrying the pathogenic variant (c.1633delA). The cell line shows typical iPSC morphology, high expression of pluripotent markers, normal karyotype, and it differentiates into three germ layers in vitro. This line is a valuable resource for studying pathological pathways involved in SoS.

Translating plasma citrulline concentration in clinical practice: Role of cross-sectional assessment in adult patients with short bowel syndrome.

BACKGROUND: Cross-sectional plasma citrulline concentration (CIT) is considered a marker of enterocyte mass. The role of CIT in clinical practice in patients with short bowel syndrome (SBS) is not clearly defined. AIM: To assess the accuracy of CIT to discriminate SBS from healthy controls (HC) and SBS with intestinal failure (SBS-IF), requiring intravenous supplementation (IVS), from SBS with intestinal insufficiency (SBS-II). METHODS: Cross-sectional study on unselected outpatients (31 SBS-II, 113 SBS-IF) and 19 healthy controls (HC). Demographic data, SBS characteristics, nutritional status, oral intake, intestinal fat absorption, renal function and IF severity, categorized by the volume of the required IVS, were collected at time of CIT evaluation (µmol/L). Data as mean±SD. RESULTS: CIT was 36.6 ± 6.0 in HC, 30.2 ± 14.0 in SBS-II and 18.8 ± 12.3 in SBS-IF (p < 0.001). CIT cutoff was 31 for the diagnosis of SBS (sensitivity 79 %, specificity 89 %), and 14 for the discrimination between SBS-IF and SBS-II (sensitivity 100 %, specificity 51 %). Wide ranges of CIT were observed in all SBS-IF severity categories. CONCLUSIONS: In unselected SBS patients, CIT was accurate to diagnose SBS, had high sensitivity to diagnose SBS-IF but showed low specificity for SBS-II. In SBS-IF, CIT was not an accurate marker of IF severity.

Malnutrition independently predicts mortality at 18 months in patients hospitalised for severe acute respiratory syndrome corona virus 2 (SARS-coV-2).

RATIONALE: To investigate the association between malnutrition and patient outcome following hospitalisation for Corona Virus Disease 2019 (COVID-19). METHODS: In April 2020, 268 adult patients (235 included in the follow-up) hospitalised for COVID-19 infection were evaluated for malnutrition risk and diagnosis using modified Nutritional Risk Screening 2002 and modified Global Leadership Initiative on Malnutrition criteria (GLIM), respectively. An 18-month follow-up was carried out to assess the incidence and the associated risk factors for death and re-hospitalization. RESULTS: The outcome was unknown for 33 patients. Death occurred in 39% of the 235 patients included in the follow-up. The risk of death was independently associated with malnutrition risk or diagnosis of malnutrition, whereas the male sex showed a protective association. The Kaplan-Meier survival curves showed that patients with diagnosis of malnutrition had lower survival rate. The re-hospitalization rate was 31% and was negatively associated with BMI≥25, and positively associated with length of hospitalisation for COVID-19 and with cancer comorbidity. CONCLUSIONS: In hospitalized patients for SARS-CoV-2 disease, both malnutrition risk (p = 0.050) and diagnosis of malnutrition (p = 0.047 with modified GLIM and C-reactive protein >0.5 mg/dL; p = 0.024 with modified GLIM and C-reactive protein >5 mg/dL) were predictive risk factors for mortality, whereas male sex was associated with lower risk of death. Overweight at time of hospitalization and the length of hospitalisation were respectively protective and risk factor for re-hospitalization after discharge.

Impact of NSD1 Alternative Transcripts in Actin Filament Formation and Cellular Division Pathways in Fibroblasts.

Germline variants in the NSD1 gene are responsible for Sotos syndrome, while somatic variants promote neoplastic cell transformation. Our previous studies revealed three alternative RNA isoforms of NSD1 present in fibroblast cell lines (FBs): the canonical full transcript and 2 alternative transcripts, termed AT2 (NSD1 Δ5Δ7) and AT3 (NSD1 Δ19-23 at the 5' end). The precise molecular pathways affected by each specific isoform of NSD1 are uncharacterized to date. To elucidate the role of these isoforms, their expression was suppressed by siRNA knockdown in FBs and protein expression and transcriptome data was explored. We demonstrate that one gene target of NSD1 isoform AT2 is ARP3 actin-related protein 3 homolog B (ACTR3B). We show that loss of both canonical NSD1 and AT2 isoforms impaired the ability of fibroblasts to regulate the actin cytoskeleton, and we observed that this caused selective loss of stress fibers. Our findings provide novel insights into NSD1 function by distinguishing isoform function and demonstrating an essential role of NSD1 in regulating the actin cytoskeleton and stress fiber formation in fibroblasts.

Identification of the DNA methylation signature of Mowat-Wilson syndrome.

Mowat-Wilson syndrome (MOWS) is a rare congenital disease caused by haploinsufficiency of ZEB2, encoding a transcription factor required for neurodevelopment. MOWS is characterized by intellectual disability, epilepsy, typical facial phenotype and other anomalies, such as short stature, Hirschsprung disease, brain and heart defects. Despite some recognizable features, MOWS rarity and phenotypic variability may complicate its diagnosis, particularly in the neonatal period. In order to define a novel diagnostic biomarker for MOWS, we determined the genome-wide DNA methylation profile of DNA samples from 29 individuals with confirmed clinical and molecular diagnosis. Through multidimensional scaling and hierarchical clustering analysis, we identified and validated a DNA methylation signature involving 296 differentially methylated probes as part of the broader MOWS DNA methylation profile. The prevalence of hypomethylated CpG sites agrees with the main role of ZEB2 as a transcriptional repressor, while differential methylation within the ZEB2 locus supports the previously proposed autoregulation ability. Correlation studies compared the MOWS cohort with 56 previously described DNA methylation profiles of other neurodevelopmental disorders, further validating the specificity of this biomarker. In conclusion, MOWS DNA methylation signature is highly sensitive and reproducible, providing a useful tool to facilitate diagnosis.

Identification of alternative transcripts of NSD1 gene in Sotos Syndrome patients and healthy subjects.

NSD1 gene (Nuclear Receptor Binding SET Domain Protein 1) encodes a methyltransferase that plays an important role in embryonic development. NSD1 is implicated in the transcription and methylation of histone H3 at lysine 36 (H3-K36), but the molecular mechanisms involved in these processes remain largely unknown. Pathogenic variants of NSD1 gene lead to Sotos syndrome, and have also been detected in some type of cancers, such as acute myeloid leukemia. In this study we have investigated NSD1 mRNA expression in fibroblast cell lines obtained from 14 Sotos patients and from 8 healthy controls. In addition to the expected NSD1 canonical transcript (isoform 1), we identified two additional, not yet reported, short NSD1 mRNA isoforms: NSD1 Δ5Δ7 (isoform 2) and NSD1 Δ19-23 (isoform 3), both in healthy subjects and in Sotos patients. We also show that NSD1 mutations in patients can be associated with a decreased level of NSD1 mRNA, as expected. Moreover, one patient, bearing the NSD1 variant c.6010-10G > A, expressed an additional shorter transcript derived from an aberrant splicing event. These results may provide a basis to elucidate the impact of different NSD1 pathogenic variants on the heterogeneity of phenotype associated with Sotos syndrome.

Generation of induced pluripotent stem cell lines from a patient with Sotos syndrome carrying 5q35 microdeletion.

Sotos syndrome (SoS) is a neurodevelopmental disorder caused by haploinsufficiency of the NSD1 gene located on chromosome 5 region q35.3. In order to understand the pathogenesis of Sotos syndrome and in view of future therapeutic approaches for its efficient treatment, we generated two human induced pluripotent stem cells (iPSCs) lines from one SoS patient carrying a 5q35 microdeletion. The established iPSCs expressed pluripotency markers, showing the capacity to differentiate into the three germ layers.

Molecular Analysis and Reclassification of NSD1 Gene Variants in a Cohort of Patients with Clinical Suspicion of Sotos Syndrome.

Sotos syndrome is a rare genetic disorder caused by haploinsufficiency of the NSD1 (nuclear receptor binding SET domain containing protein 1) gene. No clinical diagnostic consensus criteria are published yet, and molecular analysis reduces the clinical diagnostic uncertainty. We screened 1530 unrelated patients enrolled from 2003 to 2021 at Galliera Hospital and Gaslini Institute in Genoa. NSD1 variants were identified in 292 patients including nine partial gene deletions, 13 microdeletions of the entire NSD1 gene, and 115 novel intragenic variants never previously described. Thirty-two variants of uncertain significance (VUS) out of 115 identified were re-classified. Twenty-five missense NSD1 VUS (25/32, 78.1%) changed class to likely pathogenic or likely benign, showing a highly significant shift in class (p < 0.01). Apart from NSD1, we identified variants in additional genes (NFIX, PTEN, EZH2, TCF20, BRWD3, PPP2R5D) in nine patients analyzed by the NGS custom panel. We describe the evolution of diagnostic techniques in our laboratory to ascertain molecular diagnosis, the identification of 115 new variants, and the re-classification of 25 VUS in NSD1. We underline the utility of sharing variant classification and the need to improve communication between the laboratory staff and the referring physician.

NSD1 Mutations in Sotos Syndrome Induce Differential Expression of Long Noncoding RNAs, miR646 and Genes Controlling the G2/M Checkpoint.

An increasing amount of evidence indicates the critical role of the NSD1 gene in Sotos syndrome (SoS), a rare genetic disease, and in tumors. Molecular mechanisms affected by NSD1 mutations are largely uncharacterized. In order to assess the impact of NSD1 haploinsufficiency in the pathogenesis of SoS, we analyzed the gene expression profile of fibroblasts isolated from the skin samples of 15 SoS patients and of 5 healthy parents. We identified seven differentially expressed genes and five differentially expressed noncoding RNAs. The most upregulated mRNA was stratifin (SFN) (fold change, 3.9, Benjamini−Hochberg corrected p < 0.05), and the most downregulated mRNA was goosecoid homeobox (GSC) (fold change, 3.9, Benjamini−Hochberg corrected p < 0.05). The most upregulated lncRNA was lnc-C2orf84-1 (fold change, 4.28, Benjamini−Hochberg corrected p < 0.001), and the most downregulated lncRNA was Inc-C15orf57 (fold change, −0.7, Benjamini−Hochberg corrected p < 0.05). A gene set enrichment analysis reported the enrichment of genes involved in the KRAS and E2F signaling pathways, splicing regulation and cell cycle G2/M checkpoints. Our results suggest that NSD1 is involved in cell cycle regulation and that its mutation can induce the down-expression of genes involved in tumoral and neoplastic differentiation. The results contribute to defining the role of NSD1 in fibroblasts for the prevention, diagnosis and control of SoS.

Clinical Manifestations in a Girl with NAA10-Related Syndrome and Genotype-Phenotype Correlation in Females.

Since 2011, eight males with an X-linked recessive disorder (Ogden syndrome, MIM #300855) associated with the same missense variant p.(Ser37Pro) in the NAA10 gene have been described. After the advent of whole exome sequencing, many NAA10 variants have been reported as causative of syndromic or non-syndromic intellectual disability in both males and females. The NAA10 gene lies in the Xq28 region and encodes the catalytic subunit of the major N-terminal acetyltransferase complex NatA, which acetylates almost half the human proteome. Here, we present a young female carrying a de novo NAA10 [NM_003491:c.247C > T, p.(Arg83Cys)] variant. The 18-year-old girl has severely delayed motor and language development, autistic traits, postnatal growth failure, facial dysmorphisms, interventricular septal defect, neuroimaging anomalies and epilepsy. Our attempt is to expand and compare genotype-phenotype correlation in females with NAA10-related syndrome. A detailed clinical description could have relevant consequences for the clinical management of known and newly identified individuals.

8p23.2-pter Microdeletions: Seven New Cases Narrowing the Candidate Region and Review of the Literature.

To date only five patients with 8p23.2-pter microdeletions manifesting a mild-to-moderate cognitive impairment and/or developmental delay, dysmorphisms and neurobehavioral issues were reported. The smallest microdeletion described by Wu in 2010 suggested a critical region (CR) of 2.1 Mb including several genes, out of which FBXO25, DLGAP2, CLN8, ARHGEF10 and MYOM2 are the main candidates. Here we present seven additional patients with 8p23.2-pter microdeletions, ranging from 71.79 kb to 4.55 Mb. The review of five previously reported and nine Decipher patients confirmed the association of the CR with a variable clinical phenotype characterized by intellectual disability/developmental delay, including language and speech delay and/or motor impairment, behavioral anomalies, autism spectrum disorder, dysmorphisms, microcephaly, fingers/toes anomalies and epilepsy. Genotype analysis allowed to narrow down the 8p23.3 candidate region which includes only DLGAP2, CLN8 and ARHGEF10 genes, accounting for the main signs of the broad clinical phenotype associated to 8p23.2-pter microdeletions. This region is more restricted compared to the previously proposed CR. Overall, our data favor the hypothesis that DLGAP2 is the actual strongest candidate for neurodevelopmental/behavioral phenotypes. Additional patients will be necessary to validate the pathogenic role of DLGAP2 and better define how the two contiguous genes, ARHGEF10 and CLN8, might contribute to the clinical phenotype.

FXS-Like Phenotype in Two Unrelated Patients Carrying a Methylated Premutation of the FMR1 Gene.

Fragile X syndrome (FXS) is mostly caused by two distinct events that occur in the FMR1 gene (Xq27.3): an expansion above 200 repeats of a CGG triplet located in the 5'UTR of the gene, and methylation of the cytosines located in the CpG islands upstream of the CGG repeats. Here, we describe two unrelated families with one FXS child and another sibling presenting mild intellectual disability and behavioral features evocative of FXS. Genetic characterization of the undiagnosed sibling revealed mosaicism in both the CGG expansion size and the methylation levels in the different tissues analyzed. This report shows that in the same family, two siblings carrying different CGG repeats, one in the full-mutation range and the other in the premutation range, present methylation mosaicism and consequent decreased FMRP production leading to FXS and FXS-like features, respectively. Decreased FMRP levels, more than the number of repeats seem to correlate with the severity of FXS clinical phenotypes.