Postzygotic mosaicism of a novel PTPN11 mutation in monozygotic twins discordant for metachondromatosis.

Genetic mosaicism caused by postzygotic mutations is of a great interest due to its role in human disease. Monozygotic twins arising from a single zygote are considered as genetically identical, and any differences likely to be caused by postzygotic events. Thus, phenotypically discordant monozygotic twins offer a unique opportunity to study genotype-phenotype correlation. Here, we present a three-generation family starting from a pair of monozygotic twins discordant for metachondromatosis due to postzygotic p.(Gln175His) variant in the PTPN11 gene. Both phenotypically discordant monozygotic twins harbor p.(Gln175His), however significant differences in mosaic ratio is observed not only between twins, but also within different tissue types within one individual. Phenotypic manifestation of p.(Gln175His) in examined family clearly depends on allele variant fraction (VAF). Individuals harboring constitutional mutation (VAF 50%) present typical metachondromatosis. Milder phenotype is observed in twin harboring high-level mosaicism in the tissue of ectodermal origin (VAF 45%), but not in a blood (VAF 5%). Finally, her twin sister harboring low-level mosaicism in blood (VAF 2%) and nonblood (VAF 12%) tissues is phenotypically normal. Our results provide insights into biological role of mosaicism in disease and further support the usefulness of nonblood tissues as an optimal source of DNA for the identification of postzygotic mutations in phenotypically discordant monozygotic twins.

Brain Tissue Low-Level Mosaicism for MTOR Mutation Causes Smith-Kingsmore Phenotype with Recurrent Hypoglycemia-A Novel Phenotype and a Further Proof for Testing of an Affected Tissue.

De novo somatic variants in genes encoding components of the PI3K-AKT3-mTOR pathway, including MTOR, have been linked to hemimegalencephaly or focal cortical dysplasia. Similarly to other malformations of cortical development, this condition presents with developmental delay and intractable epilepsy, often necessitating surgical treatment. We describe a first patient with the Smith-Kingsmore syndrome phenotype with recurrent hypoglycemia caused by low-level mosaic MTOR mutation restricted to the brain. We provide discussion on different aspects of somatic mosaicism. Deep exome sequencing combined with a variant search in multiple tissues and careful phenotyping may constitute a key to the diagnosis of the causes of rare brain anomalies.

Gene Expression Profile of Human Mesenchymal Stromal Cells Exposed to Hypoxic and Pseudohypoxic Preconditioning-An Analysis by RNA Sequencing.

Mesenchymal stromal cell (MSC) therapy is making its way into clinical practice, accompanied by research into strategies improving their therapeutic potential. Preconditioning MSCs with hypoxia-inducible factors-α (HIFα) stabilizers is an alternative to hypoxic priming, but there remains insufficient data evaluating its transcriptomic effect. Herein, we determined the gene expression profile of 6 human bone marrow-derived MSCs preconditioned for 6 h in 2% O2 (hypoxia) or with 40 µM Vadadustat, compared to control cells and each other. RNA-Sequencing was performed using the Illumina platform, quality control with FastQC and adapter-trimming with BBDUK2. Transcripts were mapped to the Homo_sapiens. GRCh37 genome and converted to relative expression using Salmon. Differentially expressed genes (DEGs) were generated using DESeq2 while functional enrichment was performed in GSEA and g:Profiler. Comparison of hypoxia versus control resulted in 250 DEGs, Vadadustat versus control 1071, and Vadadustat versus hypoxia 1770. The terms enriched in both phenotypes referred mainly to metabolism, in Vadadustat additionally to vesicular transport, chromatin modifications and interaction with extracellular matrix. Compared with hypoxia, Vadadustat upregulated autophagic, phospholipid metabolism, and TLR cascade genes, downregulated those of cytoskeleton and GG-NER pathway and regulated 74 secretory factor genes. Our results provide valuable insight into the transcriptomic effects of these two methods of MSCs preconditioning.

Epithelial Cells of Deep Infiltrating Endometriosis Harbor Mutations in Cancer Driver Genes.

Endometriosis is an inflammatory condition manifested by the presence of endometrial-like tissue outside of the uterine cavity. The most common clinical presentations of endometriosis are dysmenorrhea, infertility, and severe pelvic pain. Few hypotheses attempt to explain the pathogenesis of endometriosis; however, none of the theories have been fully confirmed or considered universal. We examined somatic mutations in eutopic endometrium samples, deep endometriotic nodules and peripheral blood from 13 women with deep endometriosis of the rectovaginal space. Somatic variants were identified in laser microdissected samples using next-generation sequencing. A custom panel of 1296 cancer-related genes was employed, and selected genes representing cancer drivers and non-drivers for endometrial and ovarian cancer were thoroughly investigated. All 59 detected somatic variants were of low mutated allele frequency (<10%). In deep ectopic lesions, detected variants were significantly more often located in cancer driver genes, whereas in eutopic endometrium, there was no such distribution. Our results converge with other reports, where cancer-related mutations were found in endometriosis without cancer, particularly recurrent KRAS mutations. Genetic alterations located in ectopic endometriotic nodules could contribute to their formation; nevertheless, to better understand the pathogenesis of this disease, more research in this area must be performed.

Higher Mutation Burden in High Proliferation Compartments of Heterogeneous Melanoma Tumors.

Melanoma tumors are the most heterogeneous of all tumor types. Tumor heterogeneity results in difficulties in diagnosis and is a frequent cause of failure in treatment. Novel techniques enable accurate examination of the tumor cells, considering their heterogeneity. The study aimed to determine the somatic variations among high and low proliferating compartments of melanoma tumors. In this study, 12 archival formalin-fixed paraffin-embedded samples of previously untreated primary cutaneous melanoma were stained with Ki-67 antibody. High and low proliferating compartments from four melanoma tumors were dissected using laser-capture microdissection. DNA was isolated and analyzed quantitatively and qualitatively. Libraries for amplicon-based next-generation sequencing (NGS) were prepared using NEBNext Direct Cancer HotSpot Panel. NGS detected 206 variants in 42 genes in melanoma samples. Most of them were located within exons (135, 66%) and were predominantly non-synonymous single nucleotide variants (99, 73.3%). The analysis showed significant differences in mutational profiles between high and low proliferation compartments of melanoma tumors. Moreover, a significantly higher percentage of variants were detected only in high proliferation compartments (39%) compared to low proliferation regions (16%, p < 0.05). Our results suggest a significant functional role of genetic heterogeneity in melanoma.

AP4B1-associated hereditary spastic paraplegia: expansion of phenotypic spectrum related to homozygous p.Thr387fs variant.

Biallelic mutations in the AP4B1 gene, encoding adaptor-related protein complex 4 beta-1 subunit, have been recognized as an important cause of a group of conditions leading to adaptor-related protein complex 4 (AP4)-associated hereditary spastic paraplegia (SPG47). We describe a homozygous, known variant c.1160_1161delCA (p.Thr387fs) that was found in the largest ever group of patients coming from four families. The patients exhibited early hypotonia progressing to spastic paraplegia, microcephaly, epilepsy, and central nervous system (CNS) defects and global developmental delay that are consistent with the nature of SPG47. Our findings expand phenotypic spectrum of SPG47 to include polymorphic seizures, mild/moderate intellectual disability, and intracerebral cysts as well as point to founder mutation in AP4 deficiency disorders in apparently non-consanguineous Polish families without shared ancestry.

FARSA mutations mimic phenylalanyl-tRNA synthetase deficiency caused by FARSB defects.

Pathogenic variants in genes encoding aminoacyl-tRNA synthetases cause numerous disorders characterized by involvement of neurons, muscles, lungs and liver. Recently, biallelic FARSB defects have been shown to cause severe growth restriction with combined brain, liver and lung involvement (Rajab interstitial lung disease [ILD] with brain calcifications). Herein, for the first time, we present a patient with similar condition associated with biallelic mutations in FARSA (NM_004461.3: c.766T>C:p.Phe256Leu and c.1230C>A:p.Asn410Lys). Both detected FARSA variants are ultrarare and predicted to be damaging by in silico programs. Furthermore, they are both located in the active site of phenylalanyl-tRNA synthetase (PheRS) with Asn410Lys directly affecting a residue forming the wall of the phenylalanine-binding pocket. Clinical features shared between our patient and the FARSB syndrome include ILD with cholesterol pneumonitis, growth delay, hypotonia, brain calcifications with cysts and liver dysfunction. Our findings indicate that a disease similar to a syndrome associated with FARSB defects can also be caused by biallelic FARSA mutations. These findings are consistent with molecular structure of PheRS which is a tetramer including both FARSA and FARSB proteins.

A Novel Monoallelic Nonsense Mutation in the NFKB2 Gene Does Not Cause a Clinical Manifestation.

NF-κB signaling, acting through NFKB1 dependent canonical and NFKB2 dependent non-canonical pathways plays a critical role in inflammatory and immune responses. Recent studies have associated mutations in these two genes with a common variable immunodeficiency (CVID). While evaluating a female patient seeking a diagnosis explaining her recurrent infections, we found a novel heterozygous c.1831C > T (p.Arg611∗) nonsense mutation in the NFKB2 gene which introduces a Stop codon in the ankyrin repeat domain of p100. Whole exome sequencing (WES) analysis, followed by Sanger sequencing, identified this previously unknown mutation in two other family members. Penetrance of the c.1831C > T variant was assessed by flow-cytometry and protein expression in peripheral blood mononuclear cells (PBMC); whereas, activation of the NF-κB2 signaling pathway was examined through immunoblotting and real-time PCR. Heterozygous c.1831C > T variant led to the expansion of lymphocyte B subpopulations with concomitant reduction of plasmablasts, low IgG levels, and accumulation of p52 in PBMC. On the other hand, tested subjects had normal levels of IgM, IgA, IgE and no impairment in lymphocytes proliferation. Although evaluated patients did not fulfill all clinical features of CVID, their health should be monitored in the future for possible late manifestation of the disease. In conclusion, we showed that NFKB2 haplodeficiency caused by c.1831C > T nonsense mutation is asymptomatic, possibly due to the compensatory mechanisms and allele redundancy.

Mapping of breakpoints in balanced chromosomal translocations by shallow whole-genome sequencing points to EFNA5, BAHD1 and PPP2R5E as novel candidates for genes causing human Mendelian disorders.

BACKGROUND: Mapping the breakpoints in de novo balanced chromosomal translocations (BCT) in symptomatic individuals provides a unique opportunity to identify in an unbiased way the likely causative genetic defect and thus find novel human disease candidate genes. Our aim was to fine-map breakpoints of de novo BCTs in a case series of nine patients. METHODS: Shallow whole-genome mate pair sequencing (SGMPS) together with long-range PCR and Sanger sequencing. In one case (BCT disrupting BAHD1 and RET) cDNA analysis was used to verify expression of a fusion transcript in cultured fibroblasts. RESULTS: In all nine probands 11 disrupted genes were found, that is, EFNA5, EBF3, LARGE, PPP2R5E, TXNDC5, ZNF423, NIPBL, BAHD1, RET, TRPS1 and SLC4A10. Five subjects had translocations that disrupted genes with so far unknown (EFNA5, BAHD1, PPP2R5E, TXNDC5) or poorly delineated impact on the phenotype (SLC4A10, two previous reports of BCT disrupting the gene). The four genes with no previous disease associations (EFNA5, BAHD1, PPP2R5E, TXNDC5), when compared with all human genes by a bootstrap test, had significantly higher pLI (p<0.017) and DOMINO (p<0.02) scores indicating enrichment in genes likely to be intolerant to single copy damage. Inspection of individual pLI and DOMINO scores, and local topologically associating domain structure suggested that EFNA5, BAHD1 and PPP2R5E were particularly good candidates for novel disease loci. The pathomechanism for BAHD1 may involve deregulation of expression due to fusion with RET promoter. CONCLUSION: SGMPS in symptomatic carriers of BCTs is a powerful approach to delineate novel human gene-disease associations.

Novel calcineurin A (PPP3CA) variant associated with epilepsy, constitutive enzyme activation and downregulation of protein expression.

PPP3CA encodes calmodulin-binding catalytic subunit of calcineurin, a ubiquitously expressed calcium/calmodulin-regulated protein phosphatase. Recently de novo PPP3CA variants were reported as a cause of disease in 12 subjects presenting with epileptic encephalopathy and dysmorphic features. We describe a boy with similar phenotype and severe early onset epileptic encephalopathy in whom a novel de novo c.1324C>T (p.(Gln442Ter)) PPP3CA variant was found by whole exome sequencing. Western blot experiments in patient's cells (EBV transformed lymphocytes and neuronal cells derived through reprogramming) indicate that despite normal mRNA abundance the protein expression level is strongly reduced both for the mutated and wild-type protein. By in vitro studies with recombinant protein expressed in E. coli we show that c.1324C>T (p.(Gln442Ter)) results in constitutive activation of the enzyme. Our results confirm the role of PPP3CA defects in pathogenesis of a distinct neurodevelopmental disorder including severe epilepsy and dysmorphism and provide further functional clues regarding the pathogenic mechanism.

A case of severe trichothiodystrophy 3 in a neonate due to mutation in the GTF2H5 gene: Clinical report.

Trichothiodystrophy (TTD) is a group of predominantly autosomal recessive disorders characterized by sulfur-deficient brittle hair. Clinical features of TTD consist of variable neuroectodermal symptoms including ichthyosis, nail abnormalities, mental retardation, short stature, decreased fertility and proneness to infections. Approximately half of the reported patients with TTD have clinical and cellular photosensitivity associated with mutations in three subunits (ERCC3, ERCC2, GTF2H5) of the basal transcription factor TFHII, which is involved in transcription and nucleotide excision repair. We report on a case of a male neonate with a novel GTF2H5 gene mutation, detected by whole exome sequencing. The GTF2H5 gene's role is to provide stability to the entire TFHII complex. The reported patient was born at 33 weeks' gestation from a pregnancy complicated by intrauterine growth restriction and premature rupture of membranes. His main clinical problems included severe congenital ichthyosis and proneness to infections with episodes of multiorgan failure. The infant's history displays the most severe clinical manifestations among patients with GTF2H5 gene mutations that have so far been reported.

Evidence for HNRNPH1 being another gene for Bain type syndromic mental retardation.

The HNRNPH2-associated disease (mental retardation, X-linked, syndromic, Bain type [MRXSB, MIM #300986]) is caused by de novo mutations in the X-linked HNRNPH2 gene. MRXSB has been described in six female patients with dysmorphy, developmental delay, intellectual disability, autism, hypotonia and seizures. The reported HNRNPH2 mutations were clustered in the small domain encoding nuclear localization signal; in particular, the p.Arg206Trp was found in four independent de novo events. HNRNPH1 is a conserved autosomal paralogue of HNRNPH2 with a similar function in regulation of pre-mRNAs splicing but so far it has not been associated with human disease. We describe a boy with a disease similar to MRXSB in whom a novel de novo mutation c.616C>T (p.Arg206Trp) in HNRNPH1 was found (ie, the exact paralogue of the recurrent HNRNPH2 mutation). We propose that defective function of HNRNPH2 and HNRNPH1 nuclear localization signal has similar clinical consequences. An important difference between the two diseases is that the HNRNPH1-associated syndrome may occur in boys (as in the case of our proband) which is well explained by the autosomal (chr5q35.3) rather than X-linked localization of the HNRNPH2 gene.

Novel de novo mutation affecting two adjacent aminoacids in the EED gene in a patient with Weaver syndrome.

Overgrowth, macrocephaly, accelerated osseous maturation, variable intellectual disability, and characteristic facial features are the main symptoms of Weaver syndrome, a rare condition caused by mutations in EZH2 gene. Recently, in four patients with Weaver-like symptoms without mutations in EZH2 gene, pathogenic variants in EED were described. We present another patient clinically diagnosed with Weaver syndrome in whom WES revealed an EED de novo mutation affecting two neighboring aminoacids, NM_003797.3:c.917_919delinsCGG/p.(Arg306_Asn307delinsThrAsp) located in one allele (in cis). Our observation, together with previous reports suggests that EED gene testing is warranted in patients with the overgrowth syndrome features and suspicion of Weaver syndrome with normal results of EZH2 gene sequencing.

Clinico-pathological correlation in case of BRAT1 mutation.

The clinical picture of BRCA1-associated protein required for ATM activation-1 (BRAT1) comprises retractable early-onset epileptic encephalopathy, progressive microcephaly, and early demise. Both, inter- and intrafamilial variations of features of BRAT1-associated disease have been described. Here, the familial case of a brother and sister with homozygous pathogenic variants in BRAT1 is presented with special emphasis on differences in seizure type/onset and central nervous system lesions. The neuropathology is extensively discussed and hypotheses put forward that may shed light on etiology of brain symptomatology within the context of BRAT1 mutations.

A novel de novo COL6A1 mutation emphasizes the role of intron 14 donor splice site defects as a cause of moderate-progressive form of ColVI myopathy - a case report and review of the genotype-phenotype correlation.

Collagen VI-related myopathy is a group of disorders affecting skeletal muscles and connective tissue. The most common symptoms are muscle weakness and joint deformities which limit the movement and progress over time. Several forms of collagen VI-related myopathies have been described: Bethlem myopathy, an intermediate form and Ullrich congenital muscular dystrophy, which is the most severe. Here we report a novel de novo c.1056+3A>C substitution in intron 14 of the COL6A1 gene encoding alpha-chains of collagen VI in a 13-year-old girl suffering from collagen VI (ColVI) myopathy. Analysis performed on cDNA generated from the RNA obtained from the patient's blood cells showed that the reported variant leads to the entire exon 14 skipping and probably results in an in-frame deletion of 18 amino acids of the COL6A1 protein. Clinical presentation, abnormal secretion of the collagen demonstrated in muscle biopsy and the COL6A1 c.1056+3A>C mutation justify classification of the presented case as ColVI myopathy with moderate-progressive course. Analysis of the literature indicates that the donor splice site of COL6A1 intron 14, associated with the phenotype of Bethlem myopathy or intermediate form, is a hot spot for ColVI myopathies.