Exploring the Impact of Genetics in a Large Cohort of Moebius Patients by Trio Whole Exome Sequencing.

Moebius syndrome (MBS) is a rare congenital disorder characterized by non-progressive facial palsy and ocular abduction paralysis. Most cases are sporadic, but also rare familial cases with autosomal dominant transmission and incomplete penetrance/variable expressivity have been described. The genetic etiology of MBS is still unclear: de novo pathogenic variants in REV3L and PLXND1 are reported in only a minority of cases, suggesting the involvement of additional causative genes. With the aim to uncover the molecular causative defect and identify a potential genetic basis of this condition, we performed trio-WES on a cohort of 37 MBS and MBS-like patients. No de novo variants emerged in REV3L and PLXND1. We then proceeded with a cohort analysis to identify possible common causative genes among all patients and a trio-based analysis using an in silico panel of candidate genes. However, identified variants emerging from both approaches were considered unlikely to be causative of MBS, mainly due to the lack of clinical overlap. In conclusion, despite this large cohort, WES failed to identify mutations possibly associated with MBS, further supporting the heterogeneity of this syndrome, and suggesting the need for integrated omics approaches to identify the molecular causes underlying MBS development.

Characterization of a New Variant in ARHGAP31 Probably Involved in Adams-Oliver Syndrome in a Family with a Variable Phenotypic Spectrum.

Adams-Oliver syndrome is a rare inherited condition characterized by scalp defects and limb abnormalities. It is caused by variants in different genes such as ARHGAP31. Here, we used an interdisciplinary approach to study a family with lower limb anomalies. We identified a novel variant in the ARHGAP31 gene that is predicted to result in a truncated protein with a constitutively activated catalytic site due to the loss of 688 amino acids involved in the C-terminal domain, essential for protein auto-inhibition. Pathogenic variants in ARHGAP31 exon 12, leading to a premature protein termination, are associated with Adams-Oliver syndrome. Bioinformatic analysis was useful to elucidate the impact of the identified genetic variant on protein structure. To better understand the impact of the identified variant, 3D protein models were predicted for the ARHGAP31 wild type, the newly discovered variant, and other pathogenetic alterations already reported. Our study identified a novel variant probably involved in Adams-Oliver syndrome and increased the evidence on the phenotypic variability in patients affected by this syndrome, underlining the importance of translational research, including experimental and bioinformatics analyses. This strategy represents a successful model to investigate molecular mechanisms involved in syndrome occurrence.

Pitfalls of whole exome sequencing in undefined clinical conditions with a suspected genetic etiology.

BACKGROUND: Whole-Exome Sequencing (WES) is a valuable tool for the molecular diagnosis of patients with a suspected genetic condition. In complex and heterogeneous diseases, the interpretation of WES variants is more challenging given the absence of diagnostic handles and other reported cases with overlapping clinical presentations. OBJECTIVE: To describe candidate variants emerging from trio-WES and possibly associated with the clinical phenotype in clinically heterogeneous conditions. METHODS: We performed WES in ten patients from eight families, selected because of the lack of a clear clinical diagnosis or suspicion, the presence of multiple clinical signs, and the negative results of traditional genetic tests. RESULTS: Although we identified ten candidate variants, reaching the diagnosis of these cases is challenging, given the complexity and the rarity of these syndromes and because affected genes are already associated with known genetic diseases only partially recapitulating patients' phenotypes. However, the identification of these variants could shed light into the definition of new genotype-phenotype correlations. Here, we describe the clinical and molecular data of these cases with the aim of favoring the match with other similar cases and, hopefully, confirm our diagnostic hypotheses. CONCLUSION: This study emphasizes the major limitations associated with WES data interpretation, but also highlights its clinical utility in unraveling novel genotype-phenotype correlations in complex and heterogeneous undefined clinical conditions with a suspected genetic etiology.

Correction: Rondinone et al. Extensive Placental Methylation Profiling in Normal Pregnancies. Int. J. Mol. Sci. 2021, 22, 2136.

In the original publication [...].

Novel Pathogenetic Variants in PTHLH and TRPS1 Genes Causing Syndromic Brachydactyly.

Skeletal disorders, including both isolated and syndromic brachydactyly type E, derive from genetic defects affecting the fine tuning of the network of pathways involved in skeletogenesis and growth-plate development. Alterations of different genes of this network may result in overlapping phenotypes, as exemplified by disorders due to the impairment of the parathyroid hormone/parathyroid hormone-related protein pathway, and obtaining a correct diagnosis is sometimes challenging without a genetic confirmation. Five patients with Albright's hereditary osteodystrophy (AHO)-like skeletal malformations without a clear clinical diagnosis were analyzed by whole-exome sequencing (WES) and novel potentially pathogenic variants in parathyroid hormone like hormone (PTHLH) (BDE with short stature [BDE2]) and TRPS1 (tricho-rhino-phalangeal syndrome [TRPS]) were discovered. The pathogenic impact of these variants was confirmed by in vitro functional studies. This study expands the spectrum of genetic defects associated with BDE2 and TRPS and demonstrates the pathogenicity of TRPS1 missense variants located outside both the nuclear localization signal and the GATA ((A/T)GATA(A/G)-binding zinc-containing domain) and Ikaros-like binding domains. Unfortunately, we could not find distinctive phenotypic features that might have led to an earlier clinical diagnosis, further highlighting the high degree of overlap among skeletal syndromes associated with brachydactyly and AHO-like features, and the need for a close interdisciplinary workout in these rare patients. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

A novel de novo DDX3X missense variant in a female with brachycephaly and intellectual disability: a case report.

BACKGROUND: De novo pathogenic variants in the DDX3X gene are reported to account for 1-3% of unexplained intellectual disability (ID) in females, leading to the rare disease known as DDX3X syndrome (MRXSSB, OMIM #300958). Besides ID, these patients manifest a variable clinical presentation, which includes neurological and behavioral defects, and abnormal brain MRIs. CASE PRESENTATION: We report a 10-year-old girl affected by delayed psychomotor development, delayed myelination, and polymicrogyria (PMG). We identified a novel de novo missense mutation in the DDX3X gene (c.625C > G) by whole exome sequencing (WES). The DDX3X gene encodes a DEAD-box ATP-dependent RNA-helicase broadly implicated in gene expression through regulation of mRNA metabolism. The identified mutation is located just upstream the helicase domain and is suggested to impair the protein activity, thus resulting in the altered translation of DDX3X-dependent mRNAs. The proband, presenting with the typical PMG phenotype related to the syndrome, does not show other clinical signs frequently reported in presence of missense DDX3X mutations that are associated with a most severe clinical presentation. In addition, she has brachycephaly, never described in female DDX3X patients, and macroglossia, that has never been associated with the syndrome. CONCLUSIONS: This case expands the knowledge of DDX3X pathogenic variants and the associated DDX3X syndrome phenotypic spectrum.

Extensive Placental Methylation Profiling in Normal Pregnancies.

The placental methylation pattern is crucial for the regulation of genes involved in trophoblast invasion and placental development, both key events for fetal growth. We investigated LINE-1 methylation and methylome profiling using a methylation EPIC array and the targeted methylation sequencing of 154 normal, full-term pregnancies, stratified by birth weight percentiles. LINE-1 methylation showed evidence of a more pronounced hypomethylation in small neonates compared with normal and large for gestational age. Genome-wide methylation, performed in two subsets of pregnancies, showed very similar methylation profiles among cord blood samples while placentae from different pregnancies appeared very variable. A unique methylation profile emerged in each placenta, which could represent the sum of adjustments that the placenta made during the pregnancy to preserve the epigenetic homeostasis of the fetus. Investigations into the 1000 most variable sites between cord blood and the placenta showed that promoters and gene bodies that are hypermethylated in the placenta are associated with blood-specific functions, whereas those that are hypomethylated belong mainly to pathways involved in cancer. These features support the functional analogies between a placenta and cancer. Our results, which provide a comprehensive analysis of DNA methylation profiling in the human placenta, suggest that its peculiar dynamicity can be relevant for understanding placental plasticity in response to the environment.

(Epi)genetic profiling of extraembryonic and postnatal tissues from female monozygotic twins discordant for Beckwith-Wiedemann syndrome.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is an overgrowth disorder caused by defects at the 11p15.5 imprinted region. Many cases of female monozygotic (MZ) twins discordant for BWS have been reported, but no definitive conclusions have been drawn regarding the link between epigenetic defects, twinning process, and gender. Here, we report a comprehensive characterization and follow-up of female MZ twins discordant for BWS. METHODS: Methylation pattern at 11p15.5 and multilocus methylation disturbance (MLID) profiling were performed by pyrosequencing and MassARRAY in placental/umbilical cord samples and postnatal tissues. Whole-exome sequencing was carried out to identify MLID causative mutations. X-chromosome inactivation (XCI) was determined by HUMARA test. RESULTS: Both twins share KCNQ1OT1:TSS-DMR loss of methylation (LOM) and MLID in blood and the epigenetic defect remained stable in the healthy twin over time. KCNQ1OT1:TSS-DMRLOM was nonhomogeneously distributed in placental samples and the twins showed the same severely skewed XCI pattern. No MLID-causative mutations were identified. CONCLUSION: This is the first report on BWS-discordant twins with methylation analyses extended to extraembryonic tissues. The results suggest that caution is required when attempting prenatal diagnosis in similar cases. Although the causative mechanism underlying LOM remains undiscovered, the XCI pattern and mosaic LOM suggest that both twinning and LOM/MLID occurred after XCI commitment.