Novel TONSL variants cause SPONASTRIME dysplasia and associate with spontaneous chromosome breaks, defective cell proliferation and apoptosis.

SPONASTRIME dysplasia is an ultrarare spondyloepimetaphyseal dysplasia featuring short stature and short limbs, platyspondyly, depressed nasal bridge with midface hypoplasia and striated metaphyses. In 2019, an autosomal recessive inheritance was demonstrated by the identification of bi-allelic hypomorphic alleles in TONSL. The encoded protein has a critical role in maintaining genome integrity by promoting the homologous recombination required for repairing spontaneous replication-associated DNA lesions at collapsed replication forks. We report a 9-year-old girl with typical SPONASTRIME dysplasia and resulted in carrier of the novel missense p.(Gln430Arg) and p.(Leu1090Arg) variants in TONSL at whole-exome sequencing. In silico analysis predicted that these variants induced thermodynamic changes with a pathogenic impact on protein function. To support the pathogenicity of the identified variants, cytogenetic analysis and microscopy assays showed that patient-derived fibroblasts exhibited spontaneous chromosomal breaks and flow cytometry demonstrated defects in cell proliferation and enhanced apoptosis. These findings contribute to our understanding of the molecular pathogenesis of SPONASTRIME dysplasia and might open the way to novel therapeutic approaches.

A novel complex genomic rearrangement affecting the KCNJ2 regulatory region causes a variant of Cooks syndrome.

Cooks syndrome (CS) is an ultrarare limb malformation due to in tandem microduplications involving KCNJ2 and extending to the 5' regulatory element of SOX9. To date, six CS families were resolved at the molecular level. Subsequent studies explored the evolutionary and pathological complexities of the SOX9-KCNJ2/Sox9-Kcnj2 locus, and suggested a key role for the formation of novel topologically associating domain (TAD) by inter-TAD duplications in causing CS. Here, we report a unique case of CS associated with a de novo 1;17 translocation affecting the KCNJ2 locus. On chromosome 17, the breakpoint mapped between KCNJ16 and KCNJ2, and combined with a ~ 5 kb deletion in the 5' of KCNJ2. Based on available capture Hi-C data, the breakpoint on chromosome 17 separated KCNJ2 from a putative enhancer. Gene expression analysis demonstrated downregulation of KCNJ2 in both patient's blood cells and cultured skin fibroblasts. Our findings suggest that a complex rearrangement falling in the 5' of KCNJ2 may mimic the developmental consequences of in tandem duplications affecting the SOX9-KCNJ2/Sox9-Kcnj2 locus. This finding adds weight to the notion of an intricate role of gene regulatory regions and, presumably, the related three-dimensional chromatin structure in normal and abnormal human morphology.

Pro-Fibrotic Phenotype in a Patient with Segmental Stiff Skin Syndrome via TGF-ß Signaling Overactivation.

Transforming growth factor ß (TGF-ß) superfamily signaling pathways are ubiquitous and essential for several cellular and physiological processes. The overexpression of TGF-ß results in excessive fibrosis in multiple human disorders. Among them, stiff skin syndrome (SSS) is an ultrarare and untreatable condition characterized by the progressive thickening and hardening of the dermis, and acquired joint limitations. SSS is distinct in a widespread form, caused by recurrent germline variants of FBN1 encoding a key molecule of the TGF-ß signaling, and a segmental form with unknown molecular basis. Here, we report a 12-year-old female with segmental SSS, affecting the right upper limb with acquired thickening of the dermis evident at the magnetic resonance imaging, and progressive limitation of the elbow and shoulder. To better explore the molecular and cellular mechanisms that drive segmental SSS, several functional studies on patient's fibroblasts were employed. We hypothesized an impairment of TGF-ß signaling and, consequently, a dysregulation of the associated downstream signaling. Lesional fibroblast studies showed a higher phosphorylation level of extracellular signal-regulated kinase 1/2 (ERK1/2), increased levels of nuclear factor-kB (NFkB), and a nuclear accumulation of phosphorylated Smad2 via Western blot and microscopy analyses. Quantitative PCR expression analysis of genes encoding key extracellular matrix proteins revealed increased levels of COL1A1, COL3A1, AGT, LTBP and ITGB1, while zymography assay reported a reduced metalloproteinase 2 enzymatic activity. In vitro exposure of patient's fibroblasts to losartan led to the partial restoration of normal transforming growth factor ß (TGF-ß) marker protein levels. Taken together, these data demonstrate that in our patient, segmental SSS is characterized by the overactivation of multiple TGF-ß signaling pathways, which likely results in altered extracellular matrix composition and fibroblast homeostasis. Our results for the first time reported that aberrant TGF-ß signaling may drive the pathogenesis of segmental SSS and might open the way to novel therapeutic approaches.

TAB2 c.1398dup variant leads to haploinsufficiency and impairs extracellular matrix homeostasis.

Transforming growth factor ß-activated kinase 1 (TAK1) mediates multiple biological processes through the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and the mitogen-activated protein kinase (MAPK) signaling pathways. TAK1 activation is tightly regulated by its binding partners (TABs). In particular, binding with TAB2 is crucial for cardiovascular development and extracellular matrix (ECM) homeostasis. In our previous work, we reported a novel multisystem disorder associated with the heterozygous TAB2 c.1398dup variant. Here, we dissect the functional effects of this variant in order to understand its molecular pathogenesis. We demonstrate that TAB2 c.1398dup considerably undergoes to nonsense-mediated messenger RNA decay and encodes a truncated protein that loses its ability to bind TAK1. We also show an alteration of the TAK1 autophosphorylation status and of selected downstream signaling pathways in patients' fibroblasts. Immunofluorescence analyses and ECM-related polymerase chain reaction-array panels highlight that patient fibroblasts display ECM disorganization and altered expression of selected ECM components and collagen-related pathways. In conclusion, we deeply dissect the molecular pathogenesis of the TAB2 c.1398dup variant and show that the resulting phenotype is well explained by TAB2 loss-of-function. Our data also offer initial insights on the ECM homeostasis impairment as a molecular mechanism probably underlying a multisystem disorder linked to TAB2.

A simultaneous next-generation sequencing approach to the diagnosis of couple infertility.

BACKGROUND: Infertility is a disorder of the male and/or female reproductive system, characterized by failure to establish a clinical pregnancy after 12 months of regular unprotected sexual intercourse. On a world basis, about one in six couples are affected by infertility during their reproductive lifespan. Despite a comprehensive diagnostic work-up, infertility in about 50% of couples remains idiopathic. In this context, a next-generation sequencing (NGS) approach has been suggested to increase diagnostic yield. Accordingly, this study aimed to evaluate the effectiveness of a custom-made NGS gene panel for the simultaneous genetic diagnosis of both partners of a large population of infertile couples. METHODS: We developed a custom-made NGS panel for 229 genes associated with male and female infertility. The panel targeted exons and their flanking regions and was used to screen 99 couples with idiopathic infertility. RESULTS: NGS sequencing revealed five pathogenic variants in six couples and 17 likely pathogenic variants or variants with uncertain significance (VUS). The pathogenic variants were identified in the following genes: GNRHR, CCDC39, DNAH5, and CCDC103; likely pathogenic variants were identified in TAC3, PROKR2, and CFTR; VUS were identified in CATSPER2, FGFR1, LRRC6, DNAH5, DNAH11, TGFBR3, and DNAI1. CONCLUSIONS: The panel of genes designed for this study allowed the identification of pathogenic gene mutations and the presence of VUS in 6.1% and 17.2%, respectively, of couples with idiopathic infertility. This is the first study to successfully apply an NGS-based genetic screening including 229 genes known to play a role in both male and female infertility.

Non-Syndromic Autosomal Dominant Hearing Loss: The First Italian Family Carrying a Mutation in the NCOA3 Gene.

Hearing loss (HL) is the most frequent sensory disorder, affecting about 1-3 per 1000 live births, with more than half of the cases attributable to genetic causes. Despite the fact that many HL causative genes have already been identified, current genetic tests fail to provide a diagnosis for about 40% of the patients, suggesting that other causes still need to be discovered. Here, we describe a four-generation Italian family affected by autosomal dominant non-syndromic hearing loss (ADNSHL), in which exome sequencing revealed a likely pathogenic variant in NCOA3 (NM_181659.3, c.2909G>C, p.(Gly970Ala)), a gene recently described as a novel candidate for ADNSHL in a Brazilian family. A comparison between the two families highlighted a series of similarities: both the identified variants are missense, localized in exon 15 of the NCOA3 gene and lead to a similar clinical phenotype, with non-syndromic, sensorineural, bilateral, moderate to profound hearing loss, with a variable age of onset. Our findings (i.e., the identification of the second family reported globally with HL caused by a variant in NCOA3) further support the involvement of NCOA3 in the etiopathogenesis of ADNSHL, which should, thus, be considered as a new gene for autosomal dominant non-syndromic hearing loss.

Complications related to in vitro reproductive techniques support the implementation of natural procreative technologies.

BACKGROUND AND AIM: Infertility affects ~20% of the couples in the world. Assisted reproductive technologies (ARTs) are currently the most common treatment option for infertility. Nevertheless, ARTs may be associated with complications for mothers and/or offspring. Natural procreative technology (NaProTechnology) is a natural treatment which minimizes these risks by seeking to identify the causes of infertility to enable better treatments. This narrative review summarizes the complications related to ARTs and clarifies how the NaProTechnology approach can help ARTs to achieve better results or be used in alternative to ARTs. METHODS: Data in the literature indicate that NaProTechnology is a natural approach for treating infertility. RESULTS: The percentage of live births obtained by NaProTechnology is similar to that of ARTs. CONCLUSIONS: An extensive search for the genetic defects causing infertility or subfertility through genetic testing can help both ARTs and NaProTechnology to achieve successful pregnancies. By discovering the underlying causes of infertility, genetic tests enable better family counseling, like the implications of transmitting risk- and disease-alleles to future generations.

Insights into the molecular pathogenesis of cardiospondylocarpofacial syndrome: MAP3K7 c.737-7A > G variant alters the TGFß-mediated α-SMA cytoskeleton assembly and autophagy.

Transforming growth factor beta-activated kinase 1 (TAK1) is a highly conserved kinase protein encoded by MAP3K7, and activated by multiple extracellular stimuli, growth factors and cytokines. Heterozygous variants in MAP3K7 cause the cardiospondylocarpofacial syndrome (CSCFS) which is characterized by short stature, dysmorphic facial features, cardiac septal defects with valve dysplasia, and skeletal anomalies. CSCFS has been described in seven patients to date and its molecular pathogenesis is only partially understood. Here, the functional effects of the MAP3K7 c.737-7A > G variant, previously identified in a girl with CSCFS and additional soft connective tissue features, were explored. This splice variant generates an in-frame insertion of 2 amino acid residues in the kinase domain of TAK1. Computational analysis revealed that this in-frame insertion alters protein dynamics in the kinase activation loop responsible for TAK1 autophosphorylation after binding with its interactor TAB1. Co-immunoprecipitation studies demonstrate that the ectopic expression of TAK1-mutated protein impairs its ability to physically bind TAB1. In patient's fibroblasts, MAP3K7 c.737-7A > G variant results in reduced TAK1 autophosphorylation and dysregulation of the downstream TAK1-dependent signaling pathway. TAK1 loss-of-function is associated with an impaired TGFß-mediated α-SMA cytoskeleton assembly and cell migration, and defective autophagy process. These findings contribute to our understanding of the molecular pathogenesis of CSCFS and might offer the rationale for the design of novel therapeutic targets.

Prenatal genetic diagnosis: Fetal therapy as a possible solution to a positive test.

BACKGROUND: Fetal abnormalities cause 20% of perinatal deaths. Advances in prenatal genetic and other types of screening offer great opportunities for identifying high risk pregnancies. METHODS: Through a literature search, here we summarise what are the prenatal diagnostic technique that are being used and how those techniques may allow for prenatal interventions. RESULTS: Next generation sequencing and non-invasive prenatal testing are fundamental for clinical diagnostics because of their sensitivity and accuracy in identifying point mutations, aneuploidies, and microdeletions, respectively. Timely identification of genetic disorders and other fetal abnormalities enables early intervention, such as in-utero gene therapy, fetal drug therapy and prenatal surgery. CONCLUSION: Prenatal intervention is mainly focused on conditions that may cause death or lifelong disabilities, like spina bifida, congenital diaphragm hernia and sacrococcygeal teratoma; and may be an alternative therapeutic option to termination of pregnancy. However, it is not yet widely available, due to lack of specialized centers.

Generation and characterization of a human iPSC line from an ALS patient carrying the Q66K-MATR3 mutation.

Fibroblasts isolated from an Amyotrophic Lateral Sclerosis (ALS)-patient carrying a mutation in Matrin-3 (p.Q66K -MATR3) gene were reprogrammed to the pluripotency stage by using non-integrating episomal plasmids. We generated the Q66K#44DRM induced pluripotent stem cell (iPSC) line that showed regular karyotype, expressed pluripotency-associated markers and were able to properly differentiate into the three germ layers. The heterozygous missense mutation in the MATR3 gene (p.Q66K), which is associated to ALS disease, was present in the generated iPSC line. Resource table.

Design and synthesis of 2-oxindole based multi-targeted inhibitors of PDK1/Akt signaling pathway for the treatment of glioblastoma multiforme.

Aggressive behavior and diffuse infiltrative growth are the main features of Glioblastoma multiforme (GBM), together with the high degree of resistance and recurrence. Evidence indicate that GBM-derived stem cells (GSCs), endowed with unlimited proliferative potential, play a critical role in tumor development and maintenance. Among the many signaling pathways involved in maintaining GSC stemness, tumorigenic potential, and anti-apoptotic properties, the PDK1/Akt pathway is a challenging target to develop new potential agents able to affect GBM resistance to chemotherapy. In an effort to find new PDK1/Akt inhibitors, we rationally designed and synthesized a small family of 2-oxindole derivatives. Among them, compound 3 inhibited PDK1 kinase and downstream effectors such as CHK1, GS3Kα and GS3Kß, which contribute to GCS survival. Compound 3 appeared to be a good tool for studying the role of the PDK1/Akt pathway in GCS self-renewal and tumorigenicity, and might represent the starting point for the development of more potent and focused multi-target therapies for GBM.

Establishment and genetic characterization of ANGM-CSS, a novel, immortal cell line derived from a human glioblastoma multiforme.

Glioblastoma multiforme (World Health Organization, grade IV astrocytoma) is the most common and most aggressive malignant primary brain tumor. We report a novel cell line, designated as ANGM-CSS, which was established from a 56-year-old male patient with a surgically removed glioblastoma multiforme. The ANGM-CSS cell line was established in vitro and characterized using histological and immunohistochemical staining, classical and molecular cytogenetic analyses, molecular studies and functional assays using a xenograft model in immunodeficient animals. ANGM-CSS was positive for CD133, nestin and vimentin proteins, whereas GFAP showed staining only in a fraction of the cells. Cytogenetic and molecular cytogenetic analysis revealed a near-tetraploid karyotype, with a modal chromosome number from 88 to 91, and additional cytogenetic abnormalities, such as the t(6;14)(p12;q11.2), t(8;10)(q24.2;q21.1) and t(5;9)(q34;p21) unbalanced translocations. Moreover, ANGM-CSS showed amplification of the MET and EGFR genes whose overexpression was observed at the mRNA level. Interestingly, ANGM-CSS is tumorigenic when implanted in immunodeficient mice, and the cells obtained from the xenografts showed the same morphology and karyotype in vitro as the original cell line. ANGM-CSS represents a biologically relevant cell line to be used to investigate the molecular pathology of glioblastoma multiforme, also to evaluate the efficacy of novel therapeutic drugs in vitro.

Calcium-sensing receptor (CASR) mutations in hypercalcemic states: studies from a single endocrine clinic over three years.

CONTEXT: Inactivating mutations of the calcium-sensing receptor (CASR) are implicated in different hypercalcemic syndromes, including familial hypocalciuric hypercalcemia (FHH), primary hyperparathyroidism (PHPT), and familial isolated hyperparathyroidism (FIHP). However, molecular diagnostics applied to large nonselected hypercalcemic cohorts from a single center have not been reported. OBJECTIVE: Our objective was to describe the prevalence, type, and potential pathogenicity of CASR mutations in a series of cases with FHH (n = 17), PHPT (n = 165), and FIHP (n = 3) and controls (n = 198) presenting at a single endocrine clinic. SUBJECTS: All were prospectively evaluated at the "Casa Sollievo della Sofferenza" Hospital in southern Italy over a 3-yr period. METHODS: CASR screening was conducted by denaturing HPLC. The variant CASRs were functionally characterized by transient transfection studies in kidney cells in vitro. RESULTS: A single novel missense variant was identified in one PHPT case. However, in FHH probands, mutations were found in eight of 17 (47%). With a hypercalcemic family member, mutation detection rate in FHH rose to seven of eight (87%), whereas only one of nine sporadic cases was positive, and none of the three FIHP cases had detectable CASR mutations. Five missense variant CASRs, identified in control subjects, performed as wild type in functional assays, whereas the missense mutant CASRs identified in the FHH patients, and in the one PHPT case, exhibited significant impairment. A novel intronic mutation (IVS4-19a-->c) found in one FHH family, created an abnormally spliced product in an in vitro minigene assay. CONCLUSION: CASR testing, with functional analysis, provides critical confirmatory evidence in the differential diagnosis of hypercalcemic states.