A Case of CDKL5 Deficiency Due to an X Chromosome Pericentric Inversion: Delineation of Structural Rearrangements as an Overlooked Recurrent Pathological Mechanism.

CDKL5 deficiency disorder (CDD) is an X-linked dominant epileptic encephalopathy, characterized by early-onset and drug-resistant seizures, psychomotor delay, and slight facial features. Genomic variants inactivating CDKL5 or impairing its protein product kinase activity have been reported, making next-generation sequencing (NGS) and chromosomal microarray analysis (CMA) the standard diagnostic tests. We report a suspicious case of CDD in a female child who tested negative upon NGS and CMA and harbored an X chromosome de novo pericentric inversion. The use of recently developed genomic techniques (optical genome mapping and whole-genome sequencing) allowed us to finely characterize the breakpoints, with one of them interrupting CDKL5 at intron 1. This is the fifth case of CDD reported in the scientific literature harboring a structural rearrangement on the X chromosome, providing evidence for the hypothesis that this type of anomaly can represent a recurrent pathogenic mechanism, whose frequency is likely underestimated, with it being overlooked by standard techniques. The identification of the molecular etiology of the disorder is extremely important in evaluating the pathological outcome and to better investigate the mechanisms associated with drug resistance, paving the way for the development of specific therapies. Karyotype and genomic techniques should be considered in all cases presenting with CDD without molecular confirmation.

PATZ1-Rearranged Tumors of the Central Nervous System: Characterization of a Pediatric Series of Seven Cases.

PATZ1-rearranged sarcomas are well-recognized tumors as part of the family of round cell sarcoma with EWSR1-non-ETS fusions. Whether PATZ1-rearranged central nervous system (CNS) tumors are a distinct tumor type is debatable. We thoroughly characterized a pediatric series of PATZ1-rearranged CNS tumors by chromosome microarray analysis (CMA), DNA methylation analysis, gene expression profiling and, when frozen tissue is available, optical genome mapping (OGM). The series consisted of 7 cases (M:F=1.3:1, 1-17 years, median 12). On MRI, the tumors were supratentorial in close relation to the lateral ventricles (intraventricular or iuxtaventricular), preferentially located in the occipital lobe. Two major histologic groups were identified: one (4 cases) with an overall glial appearance, indicated as "neuroepithelial" (NET) by analogy with the corresponding methylation class (MC); the other (3 cases) with a predominant spindle cell sarcoma morphology, indicated as "sarcomatous" (SM). A single distinct methylation cluster encompassing both groups was identified by multidimensional scaling analysis. Despite the epigenetic homogeneity, unsupervised clustering analysis of gene expression profiles revealed 2 distinct transcriptional subgroups correlating with the histologic phenotypes. Interestingly, genes implicated in epithelial-mesenchymal transition and extracellular matrix composition were enriched in the subgroup associated to the SM phenotype. The combined use of CMA and OGM enabled the identification of chromosome 22 chromothripsis in all cases suitable for the analyses, explaining the physical association of PATZ1 to EWSR1 or MN1. Six patients are currently disease-free (median follow-up 30 months, range 12-92). One patient of the SM group developed spinal metastases at 26 months from diagnosis and is currently receiving multimodal therapy (42 months). Our data suggest that PATZ1-CNS tumors are defined by chromosome 22 chromothripsis as causative of PATZ1 fusion, show peculiar MRI features (eg, relation to lateral ventricles, supratentorial frequently posterior site), and, although epigenetically homogenous, encompass 2 distinct histologic and transcriptional subgroups.

Deep Intronic LINE-1 Insertions in NF1: Expanding the Spectrum of Neurofibromatosis Type 1-Associated Rearrangements.

Neurofibromatosis type 1 is an autosomal-dominant condition caused by NF1 gene inactivation. Clinical diagnosis is corroborated by genetic tests on gDNA and cDNA, which are inconclusive in approximately 3-5% of cases. Genomic DNA approaches may overlook splicing-affecting intronic variants and structural rearrangements, especially in regions enriched in repetitive sequences. On the other hand, while cDNA-based methods provide direct information about the effect of a variant on gene transcription, they are hampered by non-sense-mediated mRNA decay and skewed or monoallelic expression. Moreover, analyses on gene transcripts in some patients do not allow tracing back to the causative event, which is crucial for addressing genetic counselling, prenatal monitoring, and developing targeted therapies. We report on a familial NF1, caused by an insertion of a partial LINE-1 element inside intron 15, leading to exon 15 skipping. Only a few cases of LINE-1 insertion have been reported so far, hampering gDNA studies because of their size. Often, they result in exon skipping, and their recognition of cDNA may be difficult. A combined approach, based on Optical Genome Mapping, WGS, and cDNA studies, enabled us to detect the LINE-1 insertion and test its effects. Our results improve knowledge of the NF1 mutational spectrum and highlight the importance of custom-built approaches in undiagnosed patients.

HuR modulation counteracts lipopolysaccharide response in murine macrophages.

Lipopolysaccharide (LPS) exposure to macrophages induces an inflammatory response, which is regulated at the transcriptional and post-transcriptional levels. HuR (ELAVL1) is an RNA-binding protein that regulates cytokines and chemokines transcripts containing AU/U-rich elements (AREs) and mediates the LPS-induced response. Here, we show that small-molecule tanshinone mimics (TMs) inhibiting HuR-RNA interaction counteract LPS stimulus in macrophages. TMs exist in solution in keto-enolic tautomerism, and molecular dynamic calculations showed the ortho-quinone form inhibiting binding of HuR to mRNA targets. TM activity was lost in vitro by blocking the diphenolic reduced form as a diacetate, but resulted in prodrug-like activity in vivo. RNA and ribonucleoprotein immunoprecipitation sequencing revealed that LPS induces a strong coupling between differentially expressed genes and HuR-bound genes, and TMs reduced such interactions. TMs decreased the association of HuR with genes involved in chemotaxis and immune response, including Cxcl10, Il1b and Cd40, reducing their expression and protein secretion in primary murine bone marrow-derived macrophages and in an LPS-induced peritonitis model. Overall, TMs show anti-inflammatory properties in vivo and suggest HuR as a potential therapeutic target for inflammation-related diseases.

A Complex Genomic Rearrangement Resulting in Loss of Function of SCN1A and SCN2A in a Patient with Severe Developmental and Epileptic Encephalopathy.

Complex genomic rearrangements (CGRs) are structural variants arising from two or more chromosomal breaks, which are challenging to characterize by conventional or molecular cytogenetic analysis (karyotype and FISH). The integrated approach of standard and genomic techniques, including optical genome mapping (OGM) and genome sequencing, is crucial for disclosing and characterizing cryptic chromosomal rearrangements at high resolutions. We report on a patient with a complex developmental and epileptic encephalopathy in which karyotype analysis showed a de novo balanced translocation involving the long arms of chromosomes 2 and 18. Microarray analysis detected a 194 Kb microdeletion at 2q24.3 involving the SCN2A gene, which was considered the likely translocation breakpoint on chromosome 2. However, OGM redefined the translocation breakpoints by disclosing a paracentric inversion at 2q24.3 disrupting SCN1A. This combined genomic high-resolution approach allowed a fine characterization of the CGR, which involves two different chromosomes with four breakpoints. The patient's phenotype resulted from the concomitant loss of function of SCN1A and SCN2A.

Cardiovascular Involvement in Pediatric FLNC Variants: A Case Series of Fourteen Patients.

Filamin C is a protein specifically expressed in myocytes and cardiomyocytes and is involved in several biological functions, including sarcomere contractile activity, signaling, cellular adhesion, and repair. FLNC variants are associated with different disorders ranging from striated muscle (myofibrillar distal or proximal) myopathy to cardiomyopathies (CMPs) (restrictive, hypertrophic, and dilated), or both. The outcome depends on functional consequences of the detected variants, which result either in FLNC haploinsufficiency or in an aberrant protein, the latter affecting sarcomere structure leading to protein aggregates. Cardiac manifestations of filaminopathies are most often described as adult onset CMPs and limited reports are available in children or on other cardiac spectrums (congenital heart defects-CHDs, or arrhythmias). Here we report on 13 variants in 14 children (2.8%) out of 500 pediatric patients with early-onset different cardiac features ranging from CMP to arrhythmias and CHDs. In one patient, we identified a deletion encompassing FLNC detected by microarray, which was overlooked by next generation sequencing. We established a potential genotype-phenotype correlation of the p.Ala1186Val variant in severe and early-onset restrictive cardiomyopathy (RCM) associated with a limb-girdle defect (two new patients in addition to the five reported in the literature). Moreover, in three patients (21%), we identified a relatively frequent finding of long QT syndrome (LQTS) associated with RCM (n = 2) and a hypertrabeculated left ventricle (n = 1). RCM and LQTS in children might represent a specific red flag for FLNC variants. Further studies are warranted in pediatric cohorts to delineate potential expanding phenotypes related to FLNC.

Intragenic inversions in NF1 gene as pathogenic mechanism in neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1), an autosomal dominant disorder characterized by skin pigmentary lesions and multiple cutaneous neurofibromas, is caused by neurofibromin 1 (NF1) loss of function variants. Currently, a molecular diagnosis is frequently established using a multistep protocol based on cDNA and gDNA sequence analysis and/or Multiplex Ligation-dependent Probe Amplification (MLPA) assay on genomic DNA, providing an overall detection rate of about 95-97%. The small proportion of clinically diagnosed patients, which at present do not obtain a molecular confirmation likely are mosaic, as their pathogenic variant may remain undetected due to low sensitivity of low coverage NGS approaches, or they may carry a type of pathogenic variant refractory to currently used technologies. Here, we report two unrelated patients presenting with two different inversions that disrupt the NF1 coding sequence, resulting in an NF1 phenotype. In one subject, the inversion was associated with microdeletions spanning a few NF1 exons at both breakpoints, while in the other the rearrangement did not cause exon loss, thus testing negative by MLPA assay. Considering the high proportion of repeated regions within the NF1 sequence, we propose that intragenic structural rearrangements should be considered as possible pathogenic mechanisms in patients fulfilling the NIH diagnostic criteria of NF1 but lacking of molecular confirmation and in patients with NF1 intragenic microdeletions.

"Atypical" Krabbe disease in two siblings harboring biallelic GALC mutations including a deep intronic variant.

Krabbe disease (KD) is a rare lysosomal storage disorder caused by biallelic pathogenic variants in GALC. Most patients manifest the severe classic early-infantile form, while a small percentage of cases have later-onset types. We present two siblings with atypical clinical and neuroimaging phenotypes, compared to the classification of KD, who were found to carry biallelic loss-of-function GALC variants, including a recurrent 30 kb deletion and a previously unreported deep intronic variant that was identified by mRNA sequencing. This family represents a unique description in the KD literature and contributes to expanding the clinical and molecular spectra of this rare disorder.

Reciprocal Xp11.4p11.3 microdeletion/microduplication spanning USP9X, DDX3X, and CASK genes in two patients with syndromic intellectual disability.

Only a few patients with deletions or duplications at Xp11.4, bridging USP9X, DDX3X, and CASK genes, have been described so far. Here, we report on a female harboring a de novo Xp11.4p11.3 deletion and a male with an overlapping duplication inherited from an unaffected mother, presenting with syndromic intellectual disability. We discuss the role of USP9X, DDX3X, and CASK genes in human development and describe the effects of Xp11.4 deletion and duplications in female and male patients, respectively.

Congenital heart defects in molecularly confirmed KBG syndrome patients.

Congenital heart defects (CHDs) are known to occur in 9%-25% of patients with KBG syndrome. In this study we analyzed the prevalence and anatomic types of CHDs in 46 personal patients with KBG syndrome, carrying pathogenetic variants in ANKRD11 or 16q24.3 deletion, and reviewed CHDs in patients with molecular diagnosis of KBG syndrome from the literature. CHD was diagnosed in 15/40 (38%) patients with ANKRD11 variant, and in one patient with 16q24.3 deletion. Left ventricular outflow tract obstructions have been diagnosed in 9/15 (60%), subaortic or muscular ventricular septal defect in 5/15 (33%), dextrocardia in 1/15 (8%). The single patient with 16q24.3 deletion and CHD had complete atrioventricular septal defect (AVSD) with aortic coarctation. Review of KBG patients from the literature and present series showed that septal defects have been diagnosed in 44% (27/61) of the cases, left ventricular tract obstructions in 31% (19/61), AVSD in 18% (11/61). Septal defects have been diagnosed in 78% of total patients with 16q24.3 deletion. Valvar anomalies are frequently diagnosed, prevalently involving the left side of the heart. A distinctive association with AVSD is identifiable and could represent a marker to suggest the diagnosis in younger patients. In conclusion, after precise molecular diagnosis and systematic cardiological screening the prevalence of CHD in KBG syndrome seems to be higher than previously reported in clinical articles. In addition to septal defects, left-sided anomalies and AVSD should be considered. Clinical management of KBG syndrome should include accurate and detailed echocardiogram at time of diagnosis.

TSPEAR variants are primarily associated with ectodermal dysplasia and tooth agenesis but not hearing loss: A novel cohort study.

Biallelic loss-of-function variants in the thrombospondin-type laminin G domain and epilepsy-associated repeats (TSPEAR) gene have recently been associated with ectodermal dysplasia and hearing loss. The first reports describing a TSPEAR disease association identified this gene is a cause of nonsyndromic hearing loss, but subsequent reports involving additional affected families have questioned this evidence and suggested a stronger association with ectodermal dysplasia. To clarify genotype-phenotype associations for TSPEAR variants, we characterized 13 individuals with biallelic TSPEAR variants. Individuals underwent either exome sequencing or panel-based genetic testing. Nearly all of these newly reported individuals (11/13) have phenotypes that include tooth agenesis or ectodermal dysplasia, while three newly reported individuals have hearing loss. Of the individuals displaying hearing loss, all have additional variants in other hearing-loss-associated genes, specifically TMPRSS3, GJB2, and GJB6, that present competing candidates for their hearing loss phenotype. When presented alongside previous reports, the overall evidence supports the association of TSPEAR variants with ectodermal dysplasia and tooth agenesis features but creates significant doubt as to whether TSPEAR variants are a monogenic cause of hearing loss. Further functional evidence is needed to evaluate this phenotypic association.

A New Intronic Variant in ECEL1 in Two Patients with Distal Arthrogryposis Type 5D.

Distal Arthrogryposis type 5D (DA5D) is characterized by congenital contractures involving the distal joints, short stature, scoliosis, ptosis, astigmatism, and dysmorphic features. It is inherited in an autosomal recessive manner, and it is a result of homozygous or compound heterozygous variants in the ECEL1 gene. Here, we report two patients of Sardinian origin harboring a new intronic homozygous variant in ECEL1 (c.1507-9G>A), which was predicted to affect mRNA splicing by activating a cryptic acceptor site. The frequency of the variant is very low in the general human population, and its presence in our families can be attributed to a founder effect. This study provides an updated review of the known causative mutations of the ECEL1 gene, enriching the allelic spectrum to include the noncoding sequence.

Autism and severe clinical phenotype in a patient with 8p21.2p11.21 deletion: Case report and literature review.

Interstitial 8p deletions were previously described, in literature and databases, in approximately 30 patients with neurodevelopmental disorders. We report on a novel patient with a 8p21.2p11.21 deletion presenting a clinical phenotype that includes severe intellectual disability, microcephaly, epilepsy, and autism, the latter having been rarely associated with this genetic defect.

Homozygous HESX1 and COL1A1 Gene Variants in a Boy with Growth Hormone Deficiency and Early Onset Osteoporosis.

We report on a patient born to consanguineous parents, presenting with Growth Hormone Deficiency (GHD) and osteoporosis. SNP-array analysis and exome sequencing disclosed long contiguous stretches of homozygosity and two distinct homozygous variants in HESX1 (Q6H) and COL1A1 (E1361K) genes. The HESX1 variant was described as causative in a few subjects with an incompletely penetrant dominant form of combined pituitary hormone deficiency (CPHD). The COL1A1 variant is rare, and so far it has never been found in a homozygous form. Segregation analysis showed that both variants were inherited from heterozygous unaffected parents. Present results further elucidate the inheritance pattern of HESX1 variants and recommend assessing the clinical impact of variants located in C-terminal propeptide of COL1A1 gene for their potential association with rare recessive and early onset forms of osteoporosis.

Atypical 7q11.23 deletions excluding ELN gene result in Williams-Beuren syndrome craniofacial features and neurocognitive profile.

Williams-Beurens syndrome (WBS) is a rare genetic disorder caused by a recurrent 7q11.23 microdeletion. Clinical characteristics include typical facial dysmorphisms, weakness of connective tissue, short stature, mild to moderate intellectual disability and distinct behavioral phenotype. Cardiovascular diseases are common due to haploinsufficiency of ELN gene. A few cases of larger or smaller deletions have been reported spanning towards the centromeric or the telomeric regions, most of which included ELN gene. We report on three patients from two unrelated families, presenting with distinctive WBS features, harboring an atypical distal deletion excluding ELN gene. Our study supports a critical role of CLIP2, GTF2IRD1, and GTF2I gene in the WBS neurobehavioral profile and in craniofacial features, highlights a possible role of HIP1 in the autism spectrum disorder, and delineates a subgroup of WBS individuals with an atypical distal deletion not associated to an increased risk of cardiovascular defects.

PPP1R21-related syndromic intellectual disability: Report of an adult patient and review.

Variants in PPP1R21 were recently found to be associated with an autosomal recessive intellectual disability syndrome in 9 individuals. Our patient, the oldest among the known subjects affected by PPP1R21-related syndrome, manifested intellectual disability, short stature, congenital ataxia with cerebellar vermis hypoplasia, generalized hypertrichosis, ulcerative keratitis, muscle weakness, progressive coarse appearance, macroglossia with fissured tongue, and deep palmar and plantar creases. We provide an overview of the clinical spectrum and natural history of this newly recognized disorder, arguing the emerging notion that PPP1R21 gene mutations could result in endolysosomal functional defects. The oldest patients could display a more severe clinical outcome, due to accumulation of metabolites or damage secondary to an alteration of the autophagy pathway. Follow-up of patients with PPP1R21 mutations is recommended for improving the understanding of PPP1R21-related syndromic intellectual disability.

KBG syndrome: Common and uncommon clinical features based on 31 new patients.

KBG syndrome (MIM #148050) is an autosomal dominant disorder characterized by developmental delay, intellectual disability, distinct craniofacial anomalies, macrodontia of permanent upper central incisors, skeletal abnormalities, and short stature. This study describes clinical features of 28 patients, confirmed by molecular testing of ANKRD11 gene, and three patients with 16q24 deletion encompassing ANKRD11 gene, diagnosed in a single center. Common clinical features are reported, together with uncommon findings, clinical expression in the first years of age, distinctive associations, and familial recurrences. Unusual manifestations emerging from present series include juvenile idiopathic arthritis, dysfunctional dysphonia, multiple dental agenesis, idiopathic precocious telarche, oral frenula, motor tics, and lipoma of corpus callosum, pilomatrixoma, and endothelial corneal polymorphic dystrophy. Facial clinical markers suggesting KBG syndrome before 6 years of age include ocular and mouth conformation, wide eyebrows, synophrys, long black eyelashes, long philtrum, thin upper lip. General clinical symptoms leading to early genetic evaluation include developmental delay, congenital malformations, hearing anomalies, and feeding difficulties. It is likely that atypical clinical presentation and overlapping features in patients with multiple variants are responsible for underdiagnosis in KBG syndrome. Improved knowledge of common and atypical features of this disorder improves clinical management.

A familial chromosomal complex rearrangement confirms RUNX1T1 as a causative gene for intellectual disability and suggests that 1p22.1p21.3 duplication is likely benign.

BACKGROUND: Complex chromosomal rearrangements are constitutive structural aberrations involving three or more breaks. They can be balanced or unbalanced and result in different outcomes, depending on deletion/duplication of genomic material, gene disruption, or position effects. CASE PRESENTATION: We report on a patient presenting with severe anemia, splenomegaly, mild intellectual disability and facial dysmorphisms harboring a 4.3 Mb duplication at 1p22.1p21.3 and a 2.1 Mb deletion at 8q21.3q22.1, involving RUNX1T1 gene. The healthy brother presented the same duplication of chromosome 1p as at 1p22.1p21.3. CONCLUSIONS: The rearrangement found both these siblings resulted from malsegregation in the proband and recombination in her healthy brother of a balanced paternal complex chromosomal rearrangement. These results confirm RUNX1T1 as a causative gene for intellectual disability and suggest the 1p22.1p21.3 duplication is likely benign.

A heterozygous, intragenic deletion of CNOT2 recapitulates the phenotype of 12q15 deletion syndrome.

Only a few individuals with 12q15 deletion have been described, presenting with a disorder characterized by learning disability, developmental delay, nasal speech, and hypothyroidism. The smallest region of overlap for this syndrome was included in a genomic segment spanning CNOT2, KCNMB4, and PTPRB genes. We report on an additional patient harboring a 12q15 microdeletion encompassing only part of CNOT2 gene, presenting with a spectrum of clinical features overlapping the 12q15 deletion syndrome phenotype. We propose CNOT2 as the phenocritical gene for 12q15 deletion syndrome and its haploinsufficiency being associated with an autosomal dominant disorder, presenting with developmental delay, hypotonia, feeding problems, learning difficulties, nasal speech, skeletal anomalies, and facial dysmorphisms.