FOXG1 variants can be associated with milder phenotypes than congenital Rett syndrome with unassisted walking and language development.

Since 2008, FOXG1 haploinsufficiency has been linked to a severe neurodevelopmental phenotype resembling Rett syndrome but with earlier onset. Most patients are unable to sit, walk, or speak. For years, FOXG1 sequencing was only prescribed in such severe cases, limiting insight into the full clinical spectrum associated with this gene. Next-generation sequencing (NGS) now enables unbiased diagnostics. Through the European Reference Network for Rare Malformation Syndromes, Intellectual and Other Neurodevelopmental Disorders, we gathered data from patients with heterozygous FOXG1 variants presenting a mild phenotype, defined as able to speak and walk independently. We also reviewed data from three previously reported patients meeting our criteria. We identified five new patients with pathogenic FOXG1 missense variants, primarily in the forkhead domain, showing varying nonspecific intellectual disability and developmental delay. These features are not typical of congenital Rett syndrome and were rarely associated with microcephaly and epilepsy. Our findings are consistent with a previous genotype-phenotype analysis by Mitter et al. suggesting the delineation of five different FOXG1 genotype groups. Milder phenotypes were associated with missense variants in the forkhead domain. This information may facilitate prognostic assessments in children carrying a FOXG1 variant and improve the interpretation of new variants identified with genomic sequencing.

p39, the primary activator for cyclin-dependent kinase 5 (Cdk5) in oligodendroglia, is essential for oligodendroglia differentiation and myelin repair.

Cyclin-dependent kinase 5 (Cdk5) plays key roles in normal brain development and function. Dysregulation of Cdk5 may cause neurodegeneration and cognitive impairment. Besides the well demonstrated role of Cdk5 in neurons, emerging evidence suggests the functional requirement of Cdk5 in oligodendroglia (OL) and CNS myelin development. However, whether neurons and OLs employ similar or distinct mechanisms to regulate Cdk5 activity remains elusive. We report here that in contrast to neurons that harbor high levels of two Cdk5 activators, p35 and p39, OLs express abundant p39 but negligible p35. In addition, p39 is selectively up-regulated in OLs during differentiation along with elevated Cdk5 activity, whereas p35 expression remains unaltered. Specific knockdown of p39 by siRNA significantly attenuates Cdk5 activity and OL differentiation without affecting p35. Finally, expression of p39, but not p35, is increased during myelin repair, and remyelination is impaired in p39(-/-) mice. Together, these results reveal that neurons and OLs harbor distinct preference of Cdk5 activators and demonstrate important functions of p39-dependent Cdk5 activation in OL differentiation during de novo myelin development and myelin repair.

MaterniCode: New Bioinformatic Pipeline to Detect Fetal Aneuploidies and Rearrangements Using Next-Generation Sequencing.

Objective: The present study is aimed at introducing and evaluating MaterniCode, a state-of-the-art bioinformatic pipeline for noninvasive prenatal testing (NIPT) that leverages the Ion Torrent semiconductor sequencing platform. The initiative strives to revolutionize prenatal diagnostics by offering a rapid and cost-effective method without sacrificing accuracy. Methods: Two distinct bioinformatic strategies were employed for fetal sex determination, one of which achieved 100% accuracy. We analyzed 1225 maternal blood samples for fetal aneuploidies, benchmarking against the industry standard Illumina VeriSeq™ NIPT Solution v2. The capability of MaterniCode to detect and characterize complex chromosomal anomalies was also assessed. Results: MaterniCode achieved near-perfect accuracy in fetal sex determination through chromosome Y (chrY )-specific gene analysis, whereas the alternative method, employing the ratio of high-quality mapped reads on chrY relative to all reads, delivered 100% accuracy. For fetal aneuploidy detection, both the integrated WisecondorX and NIPTeR algorithms demonstrated a 100% sensitivity and specificity rate, consistent with Illumina VeriSeq™ NIPT Solution v2. The pipeline also successfully identified and precisely mapped significant chromosomal abnormalities, exemplified by a 2.4 Mb deletion on chromosome 13 and a 3 Mb duplication on chromosome 2. Conclusion: MaterniCode has proven to be an innovative and highly efficient tool in the domain of NIPT, demonstrating excellent sensitivity and specificity. Its robust capability to effectively detect a wide range of complex chromosomal aberrations, including rare and subtle variations, positions it as a promising and valuable addition to prenatal diagnostic technologies. This enhancement to diagnostic precision significantly aids clinicians in making informed decisions during pregnancy management.

Topical Antiseptics in Minimizing Ocular Surface Bacterial Load Before Ophthalmic Surgery: A Randomized Controlled Trial.

PURPOSE: To investigate the reduction of the ocular surface bacterial load induced by 2 commercially available ophthalmic antiseptic formulations, povidone-iodine (PVI) 0.6% and chlorhexidine (CLX) 0.02%, before ocular surgery. DESIGN: Randomized controlled trial. METHODS: Seventy adult patients undergoing intraocular surgery (phacoemulsification) were randomized to receive in the index eye PVI (group A) 4 times a day for 3 days or CLX (group B) 4 times a day for 3 days before surgery. The untreated eye was used as control. A conjunctival swab was taken in both eyes before (T0) and after (T1) therapy. Microbial DNA was quantified with real-time polymerase chain reaction (PCR) analysis. The Mick algorithm was used to compare the abundance of each genus/genera against the distribution of abundances from the reference. At T1, patients filled a questionnaire to evaluate therapy-induced symptoms. Primary outcome was the reduction of bacterial DNA at T1 (microbial load), vs control arm, expressed as mean number of real-time PCR cycle times (CTs). Secondary outcomes were taxonomic composition, differential abundance, and therapy-induced ocular symptoms. RESULTS: The T0-T1 difference in CT was significant in group B, but not in group A (mean [95% CI], 0.99 [0.33] vs 0.26 [0.15], P < .001, and 0.65 [0.3] vs 0.45 [0.41], P = .09, respectively). The taxonomic composition, alpha, and beta diversity remained consistent at all time points in both groups. The rate of patients reporting therapy-induced ocular symptoms and the mean discomfort grade were greater in group A than in group B (97% vs 26% and 4.97±2.48 vs 0.66±1.53, respectively). CONCLUSIONS: Compared with PVI 0.6%, CLX 0.02% induced a greater reduction of ocular surface bacterial load, with no significant alterations of the taxonomic composition. Moreover, CLX was better tolerated than PVI.

Infectious Keratitis: Characterization of Microbial Diversity through Species Richness and Shannon Diversity Index.

Purpose: To characterize microbial keratitis diversity utilizing species richness and Shannon Diversity Index. Methods: Corneal impression membrane was used to collect samples. All swabs were processed and analyzed by Biolab Laboratory (level V-SSN Excellence: ISO 9001:2015), Biolab Srl (Ascoli Piceno, Italy). DNA extraction, library preparation, and sequencing were performed in all samples. After sequencing, low-quality and polyclonal sequences were filtered out by the Ion software. At this point, we employed Kraken2 for microbial community analysis in keratitis samples. Nuclease-free water and all the reagents included in the experiment were used as a negative control. The primary outcome was the reduction in bacterial DNA (microbial load) at T1, expressed as a percentage of the baseline value (T0). Richness and Shannon alpha diversity metrics, along with Bray-Curtis beta diversity values, were calculated using the phyloseq package in R. Principal coordinate analysis was also conducted to interpret these metrics. Results: 19 samples were included in the study. The results exhibited a motley species richness, with the highest recorded value surpassing 800 species. Most of the samples displayed richness values ranging broadly from under 200 to around 600, indicating considerable variability in species count among the keratitis samples. Conclusions: A significant presence of both typical and atypical bacterial phyla in keratitis infections, underlining the complexity of the disease's microbial etiology.

Combinatorial activation of the WNT-dependent fibrogenic program by distinct complement subunits in dystrophic muscle.

Fibrosis is associated with compromised muscle functionality in Duchenne muscular dystrophy (DMD). We report observations with tissues from dystrophic patients and mice supporting a model to explain fibrosis in DMD, which relies on the crosstalk between the complement and the WNT signaling pathways and the functional interactions of two cellular types. Fibro-adipogenic progenitors and macrophages, which populate the inflamed dystrophic muscles, act as a combinatorial source of WNT activity by secreting distinct subunits of the C1 complement complex. The resulting aberrant activation of the WNT signaling in responsive cells, such as fibro-adipogenic progenitors, contributes to fibrosis. Indeed, pharmacological inhibition of the C1r/s subunits in a murine model of DMD mitigated the activation of the WNT signaling pathway, reduced the fibrogenic characteristics of the fibro-adipogenic progenitors, and ameliorated the dystrophic phenotype. These studies shed new light on the molecular and cellular mechanisms responsible for fibrosis in muscular dystrophy and open to new therapeutic strategies.

Ocular Surface Microbiota in Naïve Keratoconus: A Multicenter Validation Study.

In the field of Ophthalmology, the mNGS 16S rRNA sequencing method of studying the microbiota and ocular microbiome is gaining more and more weight in the scientific community. This study aims to characterize the ocular microbiota of patients diagnosed with keratoconus who have not undergone any prior surgical treatment using the mNGS 16S rRNA sequencing method. Samples of naïve keratoconus patients were collected with an eNAT with 1 mL of Liquid Amies Medium (Copan Brescia, Italy), and DNA was extracted and analyzed with 16S NGS. The microbiota analysis showed a relative abundance of microorganisms at the phylum level in each sample collected from 38 patients with KC and 167 healthy controls. A comparison between healthy control and keratoconus samples identified two genera unique to keratoconus, Pelomonas and Ralstonia. Our findings suggest that alterations in the microbiota may play a role in the complex scenario of KC development.

Investigation of modifier genes within copy number variations in Rett syndrome.

MECP2 mutations are responsible for two different phenotypes in females, classical Rett syndrome and the milder Zappella variant (Z-RTT). We investigated whether copy number variants (CNVs) may modulate the phenotype by comparison of array-CGH data from two discordant pairs of sisters and four additional discordant pairs of unrelated girls matched by mutation type. We also searched for potential MeCP2 targets within CNVs by chromatin immunopreceipitation microarray (ChIP-chip) analysis. We did not identify one major common gene/region, suggesting that modifiers may be complex and variable between cases. However, we detected CNVs correlating with disease severity that contain candidate modifiers. CROCC (1p36.13) is a potential MeCP2 target, in which a duplication in a Z-RTT and a deletion in a classic patient were observed. CROCC encodes a structural component of ciliary motility that is required for correct brain development. CFHR1 and CFHR3, on 1q31.3, may be involved in the regulation of complement during synapse elimination, and were found to be deleted in a Z-RTT but duplicated in two classic patients. The duplication of 10q11.22, present in two Z-RTT patients, includes GPRIN2, a regulator of neurite outgrowth and PPYR1, involved in energy homeostasis. Functional analyses are necessary to confirm candidates and to define targets for future therapies.

13q Deletion Syndrome Involving RB1: Characterization of a New Minimal Critical Region for Psychomotor Delay.

Retinoblastoma (RB) is an ocular tumor of the pediatric age caused by biallelic inactivation of the RB1 gene (13q14). About 10% of cases are due to gross-sized molecular deletions. The deletions can involve the surrounding genes delineating a contiguous gene syndrome characterized by RB, developmental anomalies, and peculiar facial dysmorphisms. Overlapping deletions previously found by traditional and/or molecular cytogenetic analysis allowed to define some critical regions for intellectual disability (ID) and multiple congenital anomalies, with key candidate genes. In the present study, using array-CGH, we characterized seven new patients with interstitial 13q deletion involving RB1. Among these cases, three patients with medium or large 13q deletions did not present psychomotor delay. This allowed defining a minimal critical region for ID that excludes the previously suggested candidate genes (HTR2A, NUFIP1, PCDH8, and PCDH17). The region contains 36 genes including NBEA, which emerged as the candidate gene associated with developmental delay. In addition, MAB21L1, DCLK1, EXOSC8, and SPART haploinsufficiency might contribute to the observed impaired neurodevelopmental phenotype. In conclusion, this study adds important novelties to the 13q deletion syndrome, although further studies are needed to better characterize the contribution of different genes and to understand how the haploinsufficiency of this region can determine ID.

Exome sequencing in BRCA1-2 candidate familias: the contribution of other cancer susceptibility genes

Hereditary Breast and Ovarian Cancer (HBOC) syndrome is a condition in which the risk of breast and ovarian cancer is higher than in the general population. The prevalent pathogenesis is attributable to inactivating variants of the BRCA1-2 highly penetrant genes, however, other cancer susceptibility genes may also be involved. By Exome Sequencing (WES) we analyzed a series of 200 individuals selected for genetic testing in BRCA1-2 genes according to the updated National Comprehensive Cancer Network (NCCN) guidelines. Analysis by MLPA was performed to detect large BRCA1-2 deletions/duplications. Focusing on BRCA1-2 genes, data analysis identified 11 cases with pathogenic variants (4 in BRCA1 and 7 in BRCA1-2) and 12 with uncertain variants (7 in BRCA1 and 5 in BRCA2). Only one case was found with a large BRCA1 deletion. Whole exome analysis allowed to characterize pathogenic variants in 21 additional genes: 10 genes more traditionally associated to breast and ovarian cancer (ATM, BRIP1, CDH1, PALB2, PTEN, RAD51C, and TP53) (5% diagnostic yield) and 11 in candidate cancer susceptibility genes (DPYD, ERBB3, ERCC2, MUTYH, NQO2, NTHL1, PARK2, RAD54L, and RNASEL). In conclusion, this study allowed a personalized risk assessment and clinical surveillance in an increased number of HBOC families and to broaden the spectrum of causative variants also to candidate non-canonical genes.

Corrigendum: Exome Sequencing in BRCA1-2 Candidate Familias: The Contribution of Other Cancer Susceptibility Genes.

[This corrects the article DOI: 10.3389/fonc.2021.649435.].

Syndromic mental retardation with thrombocytopenia due to 21q22.11q22.12 deletion: Report of three patients.

During the last few years, an increasing number of microdeletion/microduplication syndromes have been delineated. This rapid evolution is mainly due to the availability of microarray technology as a routine diagnostic tool. Microdeletions of the 21q22.11q22.12 region encompassing the RUNX1 gene have been reported in nine patients presenting with syndromic thrombocytopenia and mental retardation. RUNX1 gene is responsible for an autosomal dominant platelet disorder with predisposition to acute myelogenous leukemia. We report on three novel patients with an overlapping "de novo" interstitial deletion involving the band 21q22 characterized by array-CGH. All our patients presented with severe developmental delay, dysmorphic features, behavioral problems, and thrombocytopenia. Comparing the clinical features of our patients with the overlapping ones already reported two potential phenotypes related to 21q22 microdeletion including RUNX1 were highlighted: thrombocytopenia with +/- mild dysmorphic features and syndromic thrombocytopenia with growth and developmental delay.

MEIS2 gene is responsible for intellectual disability, cardiac defects and a distinct facial phenotype.

Myeloid ecotropic insertion site 2 (MEIS2) gene, encoding a homeodomain-containing transcription factor, has been recently related to syndromic intellectual disability with cleft palate and cardiac defects. Here, we present a male patient, aged 10, with cardiac defects, intellectual disability, facial dysmorphisms and gastroesophageal reflux. Whole exome sequencing revealed a novel de novo nonsense mutation in the MEIS2 gene. This patient represents another reported case with a de novo MEIS2 point mutation and helps to characterize a distinct facial phenotype consisting in low anterior hairline, thin eyebrows, anteverted nares, hypoplastic alae nasi, and M-shape upper lip. Furthermore, these data confirm the role of this gene in cardiac, nervous system development and gastrointestinal function.

AAV-mediated FOXG1 gene editing in human Rett primary cells.

Variations in the Forkhead Box G1 (FOXG1) gene cause FOXG1 syndrome spectrum, including the congenital variant of Rett syndrome, characterized by early onset of regression, Rett-like and jerky movements, and cortical visual impairment. Due to the largely unknown pathophysiological mechanisms downstream the impairment of this transcriptional regulator, a specific treatment is not yet available. Since both haploinsufficiency and hyper-expression of FOXG1 cause diseases in humans, we reasoned that adding a gene under nonnative regulatory sequences would be a risky strategy as opposed to a genome editing approach where the mutated gene is reversed into wild-type. Here, we demonstrate that an adeno-associated viruses (AAVs)-coupled CRISPR/Cas9 system is able to target and correct FOXG1 variants in patient-derived fibroblasts, induced Pluripotent Stem Cells (iPSCs) and iPSC-derived neurons. Variant-specific single-guide RNAs (sgRNAs) and donor DNAs have been selected and cloned together with a mCherry/EGFP reporter system. Specific sgRNA recognition sequences were inserted upstream and downstream Cas9 CDS to allow self-cleavage and inactivation. We demonstrated that AAV serotypes vary in transduction efficiency depending on the target cell type, the best being AAV9 in fibroblasts and iPSC-derived neurons, and AAV2 in iPSCs. Next-generation sequencing (NGS) of mCherry+/EGFP+ transfected cells demonstrated that the mutated alleles were repaired with high efficiency (20-35% reversion) and precision both in terms of allelic discrimination and off-target activity. The genome editing strategy tested in this study has proven to precisely repair FOXG1 and delivery through an AAV9-based system represents a step forward toward the development of a therapy for Rett syndrome.

Array comparative genomic hybridization in retinoma and retinoblastoma tissues.

In retinoblastoma, two RB1 mutations are necessary for tumor development. Recurrent genomic rearrangements may represent subsequent events required for retinoblastoma progression. Array-comparative genomic hybridization was carried out in 18 eye samples, 10 from bilateral and eight from unilateral retinoblastoma patients. Two unilateral cases also showed areas of retinoma. The most frequent imbalance in retinoblastomas was 6p gain (40%), followed by gains at 1q12-q25.3, 2p24.3-p24.2, 9q22.2, and 9q33.1 and losses at 11q24.3, 13q13.2-q22.3, and 16q12.1-q21. Bilateral cases showed a lower number of imbalances than unilateral cases (P = 0.002). Unilateral cases were divided into low-level (< or = 4) and high-level (> or = 7) chromosomal instability groups. The first group presented with younger age at diagnosis (mean 511 days) compared with the second group (mean 1606 days). In one retinoma case ophthalmoscopically diagnosed as a benign lesion no rearrangements were detected, whereas the adjacent retinoblastoma displayed seven aberrations. The other retinoma case identified by retrospective histopathological examination shared three rearrangements with the adjacent retinoblastoma. Two other gene-free rearrangements were retinoma specific. One rearrangement, dup5p, was retinoblastoma specific and included the SKP2 gene. Genomic profiling indicated that the first retinoma was a pretumoral lesion, whereas the other represents a subclone of cells bearing 'benign' rearrangements overwhelmed by another subclone presenting aberrations with higher 'oncogenic' potential. In summary, the present study shows that bilateral and unilateral retinoblastoma have different chromosomal instability that correlates with the age of tumor onset in unilateral cases. This is the first report of genomic profiling in retinoma tissue, shedding light on the different nature of lesions named 'retinoma'.

Analysis of the Phenotypes in the Rett Networked Database.

Rett spectrum disorder is a progressive neurological disease and the most common genetic cause of intellectual disability in females. MECP2 is the major causative gene. In addition, CDKL5 and FOXG1 mutations have been reported in Rett patients, especially with the atypical presentation. Each gene and different mutations within each gene contribute to variability in clinical presentation, and several groups worldwide performed genotype-phenotype correlation studies using cohorts of patients with classic and atypical forms of Rett spectrum disorder. The Rett Networked Database is a unified registry of clinical and molecular data of Rett patients, and it is currently one of the largest Rett registries worldwide with several hundred records provided by Rett expert clinicians from 13 countries. Collected data revealed that the majority of MECP2-mutated patients present with the classic form, the majority of CDKL5-mutated patients with the early-onset seizure variant, and the majority of FOXG1-mutated patients with the congenital form. A computation of severity scores further revealed significant differences between groups of patients and correlation with mutation types. The highly detailed phenotypic information contained in the Rett Networked Database allows the grouping of patients presenting specific clinical and genetic characteristics for studies by the Rett community and beyond. These data will also serve for the development of clinical trials involving homogeneous groups of patients.

Erratum to: Clinical and molecular characterization of Italian patients affected by Cohen syndrome.

The complete affiliations of two authors appeared incorrectly. The affiliations of A. Selicorni and D. Milani should be given as.

Private inherited microdeletion/microduplications: implications in clinical practice.

The introduction of array-CGH analysis is allowing the identification of novel genomic disorders. However, this new high-resolution technique is also opening novel diagnostic challenges when inherited private CNVs of unclear clinical significance are found. Oligo array-CGH analysis of 84 patients with mild to severe mental retardation associated with multiple congenital anomalies revealed 10 private CNVs inherited from a healthy parent. Three were deletions (7q31, 14q21.1, Xq25) and seven duplications (12p11.22, 12q21.31, 13q31.1, 17q12, Xp22.31, Xq28) ranging between 0.1 and 3.8Mb. Six rearrangements were not polymorphic. Four overlapped polymorphic regions to the extent of 10-61%. In one case the size was different between the proband and the healthy relative. Three small rearrangements were gene deserts. The remaining seven had a mean gene content of five (ranging from 1 to 18). None of the rearranged genes is known to be imprinted. Three disease-genes were found in three different cases: KAL1 in dupXp22.31, STS in another dupXp22.31 and TCF2 in dup17q12. The patient carrying the last duplication presents sex reversal, Peters' anomaly and renal cysts and the duplication is located 4Mb away from the HSD17B1 gene, coding a key enzyme of testosterone biosynthesis. Considering the overlap with polymorphic regions, size-identity within the family, gene content, kind of rearrangement and size of rearrangement we suggest that at least in five cases the relationship to the phenotype has not to be excluded. We recommend to maintain caution when asserting that chromosomal abnormalities inherited from a healthy parent are benign. A more complex mechanism may in fact be involved, such as a concurrent variation in the other allele or in another chromosome that influences the phenotype.