Two novel pathogenic variants in MED13L: one familial and one isolated case.

BACKGROUND: Genetic variants involving the MED13L gene can lead to an autosomal dominant syndrome characterised by intellectual disability/developmental delay and facial dysmorphism. METHODS: We investigated two cases (one familial and one isolated) of intellectual disability with speech delay and dysmorphic facial features by whole-exome sequencing analyses. Further, we performed a literature review about clinical and molecular aspects of MED13L gene and syndrome. RESULTS: Two MED13L variants have been identified [MED13L(NM_015335.5):c.4417C>T and MED13L(NM_015335.5):c.2318delC] and were classified as pathogenic according to the ACMG (American College of Medical Genetics and Genomics) guidelines. One of the variants was present in sibs. CONCLUSIONS: The two pathogenic variants identified have not been previously reported. Importantly, this is the first report of a familial case of MED13L nonsense mutation. Although the parents of the affected children were no longer available for analysis, their apparently normal phenotypes were surmised from familial verbal descriptions corresponding to normal mental behaviour and phenotype. In this situation, the familial component of mutation transmission might be caused by gonadal mosaicism of a MED13L mutation in a gonad from either the father or the mother. The case reports and the literature review presented in this manuscript can be useful for genetic counselling.

Genetic diversity of microsatellite loci in Leopardus pardalis, Leopardus wiedii and Leopardus tigrinus.

The microsatellite loci FCA045, FCA077, FCA008, and FCA096 are highly variable molecular markers which were used to determine the genetic diversity in 148 captive Leopardus sp. The PCR-amplified products of microsatellite loci were characterized in ABI Prism 310 Genetic Analyzer. Allele numbers, heterozygosity, polymorphism information content, exclusive allele number, and shared alleles were calculated. Sixty-five alleles were found and their sizes ranged from 116 to 216 bp in four microsatellite loci. The heterozygosity ranged from 0.36 to 0.81 in Leopardus pardalis, 0.57 to 0.67 in L. tigrinus and 0.80 to 0.92 in L. wiedii. The polymorphism information content was from 0.80 to 0.88 in L. pardalis, 0.76 to 0.88 in L. tigrinus and 0.77 to 0.90 in L. wiedii. The margay (L. wiedii) showed the highest index of polymorphism among the three species in this study. These results imply that microsatellite DNA markers can help in the study of the genetic diversity of Leopardus specimens.

Genetic diversity of microsatellite loci in Leopardus pardalis, Leopardus wiedii and Leopardus tigrinus

The microsatellite loci FCA045, FCA077, FCA008, and FCA096 are highly variable molecular markers which were used to determine the genetic diversity in 148 captive Leopardus sp. The PCR-amplified products of microsatellite loci were characterized in ABI Prism 310 Genetic Analyzer. Allele numbers, heterozygosity, polymorphism information content, exclusive allele number, and shared alleles were calculated. Sixty-five alleles were found and their sizes ranged from 116 to 216 bp in four microsatellite loci. The heterozygosity ranged from 0.36 to 0.81 in Leopardus pardalis, 0.57 to 0.67 in L. tigrinus and 0.80 to 0.92 in L. wiedii. The polymorphism information content was from 0.80 to 0.88 in L. pardalis, 0.76 to 0.88 in L. tigrinus and 0.77 to 0.90 in L. wiedii. The margay (L. wiedii) showed the highest index of polymorphism among the three species in this study. These results imply that microsatellite DNA markers can help in the study of the genetic diversity of Leopardus specimens.

Modifications of proteoglycans secreted into the growth medium by young and senescent human skin fibroblasts.

The properties of proteoglycans (PGs) secreted into the growth medium by normal young and senescent human skin fibroblasts (HFs) were investigated. In both cases, the incorporation per cell of radioactive precursors into total PGs was similar. The polysaccharide chains of PGs from young and senescent HFs were mainly represented by galactosaminoglycuronans and showed a similar range of size distribution. However, galactosaminoglycuronans of PGs secreted by senescent HFs had a lower content of unsulphated disaccharides and a lower proportion of D-glucuronosyl residues. Moreover, senescent HFs released into the growth medium higher relative amounts of small PGs with chondroitin sulphate, dermatan sulphate chains, such as decorin.

Modifications of proteoglycans extracted from monolayer cultures of young and senescent human skin fibroblasts.

Proteoglycans (PGs) were extracted from culture monolayers of human skin fibroblasts (HFs) at early and late passages. Total PGs from senescent cells had markedly reduced abilities to bind type I collagen and hyaluronic acid, but retained normal binding properties with fibronectin and laminin. The constituent polysaccharides of PGs were comparatively characterised. PGs recovered from young and senescent HF cultures had equivalent total polyanionic charges and similar size distributions of the glycosaminoglycan chains. This applied to both types of polysaccharide chains found in PGs, namely the galactosaminoglycuronans (GalN-GAGs) and the glucosaminoglycuronans (GlcN-GAGs). However, senescent HFs produced a greater proportion of PGs containing GlcN-GAG chains and increased the sulphation of the remaining PG fraction with GalN-GAG moieties, yielding a major gain of C6-sulphate groups in the galactosamine residues.

Inhibition of DNA polymerization and DNA transcription to RNA by seminal plasma peptides.

An oligopeptide fraction purified from the extracellular compartment of bull semen and strongly interacting with DNA was shown to hinder mononucleotide polymerizations to DNA and RNA in vitro. The fraction, collectively called seminal plasma inhibitor, was active in the endogenous DNA and RNA polymerase reactions of the nuclei from rat hepatocytes and in the analogous nucleotide polymerizations catalyzed by purified enzymes of bacterial origin. The type of the induced inhibition was studied using the RNA polymerase from Escherichia coli as a representative nucleotidyl transferase. In the enzymatic polycondensation of mononucleotides, the seminal plasma inhibitor appeared to exert its effect mainly by a competitive inhibition for the utilization of DNA templates without specificity with respect to the source and the base sequence of DNA. Concavities of the plots of V0/Vi versus the amounts of inhibitor in the nucleotide polymerizing reactions and of the Dixon plots in the assays of RNA polymerase from E. coli suggested that the isolated oligopeptide fraction contained more than one active molecular species with differential effects at low and high doses. Preliminary results on the microheterogeneity of the seminal plasma inhibitor supported this contention.

Structure of the alpha-form of poly[d(A)].poly[d(T)] and related polynucleotide duplexes.

The alpha-form of poly[d(A)].poly[d(T)], observed in fibers at high (greater than 80%) relative humidity, is a 10-fold double-helical structure of pitch 3.2 nm. This new X-ray analysis shows that the two strands of the double helix are of the same kind conformationally and both B-like in containing C-2'-endo-puckered deoxyribose rings. Nevertheless, the two strands are different enough for the overall morphology of the duplex to resemble that of the heteromerous model for the drier (beta) form of poly[d(A)].poly[d(T)] in which one strand has C-2'-endo rings and the other C-3'-endo. Since the orientations of the bases in poly[d(A)].poly[d(T)] are persistently different from those of classical B-DNA it is likely that there will be local bending (about 10 degrees) at the junctions between general sequence tracts and the oligo[d(A)].oligo[d(T)] tracts that occur in some native DNAs. The conclusions about the structure of alpha-poly[d(A)].poly[d(T)] are reinforced by independent analyses of similar X-ray diffraction patterns from poly[d(A)].poly[d(U)] and poly[d(A-I)].poly[d(C-T)].

Seminal plasma peptides inhibiting RNA synthesis.

A peptide fraction, called seminal plasma inhibitor (SPI), present in mammalian semen, was shown to block DNA transcription in vitro (Lugaro et al., 1984). Peptides responsible for this effect were partially purified from bovine seminal plasma. This report outlines the preparative procedure for the isolation of SPI and provides preliminary information on the action mechanism of the RNA synthesis inhibition.

Terminal deoxynucleotidyl transferase, TdT, as a marker for leukemia and lymphoma cells.

Terminal deoxynucleotidyl transferase, TdT, was assayed in the mononucleate cells of blood and bone marrow from 121 patients with leukemias at the onset of disease and from 95 subjects with malignant lymphomas at diagnosis. This intracellular marker was also investigated by cytoimmunofluorescent tests in 17 other cases of initial leukemias and in 3 diagnosed lymphoblastic lymphomas. Generally, the TdT levels were significantly enhanced in the blasts of the following: acute undifferentiated leukemias; the more immature types of acute lymphoblastic leukemias i.e., the null, non-T non-B, common, early T and pre-B subgroups; a fraction of blastic crises in chronic myelogenous leukemias; and many lymphoblastic lymphomas. TdT might also be slightly increased in the mononucleate blood cells obtained from the most immature forms of acute myelogenous leukemias. Relapses with changes in cell phenotypes were occasionally observed in previously TdT-positive leukemias as a result of clonal evolution of the disease. The leukemias with blasts containing high levels of TdT were usually responsive to treatment with corticosteroids and vincristine. TdT is an oligoclonal marker characterizing several populations of undifferentiated or poorly differentiated blasts that tend to develop towards or along the lymphoid pathway. Together with specific immunological markers, this enzyme is useful to define the particular type of leukemic cells. It also serves to identify the quasi-lymphoblastic nature of the malignant clone, a helpful indication for the choice of therapy.

Substrate preferences of human placental DNA methyltransferase investigated with synthetic polydeoxynucleotides.

A DNA methyltransferase partly purified from human placenta has been tested on a variety of synthetic polydeoxynucleotides. The results showed that: the enzyme is most active as a 'maintenance' or 'hemi-' methylase but also has some de novo methylating activity; the presence or absence of A or T in the substrate strand has little influence on maintenance or de novo activity, while polymers containing C but not G in the same strand are poor de novo substrates and bind poorly to the enzyme; single-stranded polymers are about as good substrates as double-stranded ones, and the effects of nucleotide composition (particularly G and mC content) on enzyme activity with single strands are similar to those with double-stranded polymers; strands in which all the cytosines are methylated bind the enzyme well. A mechanism is suggested involving two different sites on the enzyme that recognize CG and mCG, and which rationalizes the activity of eukaryotic DNA methyltransferases towards single-stranded DNA.

The toxicity of tritium: the effects of tritiated amino-acids on preimplanted mouse embryos.

The effects of tritiated amino-acids, arginine, lysine, histidine and aspartic acid on the growth and development of two-cell mouse embryos, cultured in vitro, were investigated. The LD50 for the dibasic amino acids, measured on the third day of growth, ranged from 30 to 130 nCi/ml. This was compared with the DNA precursor, thymidine, for which the LD50 was 80 nCi/ml.

[Analysis of the enzyme terminal deoxyribonucleotidyl transferase in lymphoma and leukemia cells]. / Studio della presenza dell'enzima terminale dessossinucleotidil trasferase (TdT) nelle cellule dei linfomi e delle leucemie.

This work analyses the presence of terminal deoxynucleotidyl transferase (TdT) in neoplastic lymphoid cells from a series of malignant lymphomas and in leukemic cells from blood or bone marrow of patients with lymphoproliferative diseases. The studied cases were 63 patients with acute leukemias, 26 patients with chronic leukemias, 85 patients with lymphomas and 14 normal controls. The presence of TdT in the neoplastic cells was determined by optimized assays of enzymatic activity or by immunofluorescence test for TdT positive blasts. The fluorescence tests made use of anti-TdT antibodies specifically absorbed on cells containing the enzyme and then revealed by a second fluorescent antibody. Appreciable amounts of TdT were found in the white cells of blood or bone marrow from the following 25 out of 35 acute lymphoblastic leukemias (ALL) with modulations in the different phenotypes; 13 out of 14 acute undifferentiated leukemias (AUL); and 9 out of 15 blastic crises in chronic myelogenous leukemia (CMLbc). The enzyme was present in 12 out of 14 lymphomas of lymphoblastic type (LL); 2 B lymphoblastic lymphomas did not show any TdT positivity. The highest levels of TdT were detected in cells classified as immature lymphoblast or in their precursors. TdT was absent from normal lymph nodes from leukocytes of chronic lymphocytic leukemia (CLL) and chronic (CML) and acute myelogenous leukemia (AML). 2 out of 14 cases of AML showed a border-line positivity. A definite correlation between concentrations of enzymatic activity and percentage of immunofluorescent cells could not be established. We did not find a precise correlation between the TdT content of lymphoid blasts and other clinical prognostic indices.(ABSTRACT TRUNCATED AT 250 WORDS)

Properties of purified uracil-DNA glycosylase from calf thymus. An in vitro study using synthetic DNA-like substrates.

The bovine uracil-DNA glycosylase previously isolated from thymocyte nuclei was further purified by 1 order of magnitude with the aid of affinity chromatography. The final preparation was totally devoid of DNase and apurinic or apyrimidinic (AP) endonuclease activities, and it corresponded to purifications of 457-fold over the nuclear extract and of about 2000-fold over the crude tissue homogenate. Most of the general enzyme properties already described were confirmed. Furthermore, this mammalian uracil-DNA glycosylase was shown to bind specifically with polymerized and not with monomeric nucleotide compounds, while having a preference for double-stranded forms. It cleaved N-glycosyl linkages only at the deoxyuridyl units located in internal positions of polynucleotide chains. The enzyme also used RNA-DNA hybrids as functional substrates and was practically ineffective on deoxyuridyl residues at the 3'-ends of nucleic acids. The activity of the glycosylase was greatly impaired in assays with DNA substrates that contained amounts of AP sites exceeding 5 microM. The inhibitory concentrations of AP residues were about 100 times lower than those found equally effective for the other reaction product, i.e. free uracil, and were almost comparable to the Km values for deoxyuridyl nucleotides in the DNA substrates. This all appears as a modulation of the glycosylase catalysis by the relative amounts of its substrate and product structures in DNA. The data lead us to surmise that the removal of uracil from cellular DNA is functionally coupled to the expected elimination of the formed AP sites by specific endonucleases. Base-exchange and base-insertion experiments with the purified enzyme yielded negative results under various conditions. The glycosylase behaved essentially as a hydrolase which has no associated base-insertase properties and irreversibly excises uracil from DNA by a mechanism for channeling the process to the next steps of the repair pathway.

Studies of terminal deoxynucleotidyl transferase in normal and neoplastic human cells.

Optimized biochemical assays and cytoimmunofluorescence tests were used to detect terminal deoxynucleotidyl transferase, TdT, in malignant cells of 36 leukemias and 75 lymphomas from patients not receiving chemotherapy. TdT was virtually absent from normal lymph nodes and from leukocytes of chronic lymphocytic leukemia, CLL, taken as controls. Its quantitative distribution in the neoplasms matched the current knowledge. Appreciable amounts of TdT were found in all the 10 lymphomas of lymphoblastic type, LL, and in the white blood cells of: 16 out of 19 acute lymphoblastic leukemia, AAL, perhaps with modulation in the various phenotypes; 2 out of 3 acute undifferentiated leukemias, AUL; and 3 out of 7 blastic crises in chronic myelogenous leukemia, b.c. CML. Biochemical and cytoimmunological analyses yielded concordant responses and even roughly comparable estimates in the same patients. TdT immunofluorescence was clearly nuclear in most cells and was cytoplasmic occasionally. Definite correlations between concentrations of enzymatic activity and percentage of immunofluorescent cells could not e established. Further detailed work will be required to identify putative subgroups in TdT-positive blast populations.

Terminal deoxynucleotidyl transferase in human lymphomas: possible existence of forms with high and low molecular weights.

Optimized methods for extraction and enzyme assay in crude tissue preparations were used to determine the amounts of terminal deoxnucleotidyl transferase (TdT) in malignant lymphomas. The TdT concentration was increased only in lymphoblastic lymphomas (LL) and was as high in these tumours as in the white blood cells from untreated patients with acute lymphoblastic leukaemia (ALL). The enzymes extracted from such lymphomas and from the leukaemic lymphoblasts had the same properties. Moreover, forms of TdT with low and high mol. wt were found in the LL tumours, similar to other reports of TdT-positive leukaemias. The overall study points at some basic biochemical identity of certain lymphoblastic malignancies, irrespective of whether the transformed cells are in solid tumours or are disseminated in the blood.

Preparation of polydeoxynucleotides linked to a solid support by coupling CNBr-activated cellulose with 5'-NH2-terminated oligo and poly(pdT)'s.

Synthetically-prepared 5'-NH2-dT(pdT)n oligomers (66,n=4 or 7) were immobilized on cyanogen bromide activated cellulose. The influence of temperature, pH, and ionic strength on the rate of the coupling process was studied. The oligomer 5'-NH2-DT(pdT)8 could be elongated enzymatically to the polymers 5'-NH2-dT(pdT)n (n=20, 51 and 84), which could be immobilized on cellulose. The cellulose-NH-dT(pdT)84 polymer thus obtained could be assembled to a new solid-state polymer e.g. poly(dA)290 . poly(/3H/dT)200, poly(dT)85-cellulose which, in turn, was a very convenient substrate for assaying DNA-ligase.

The activity of mammalian polynucleotide ligase on x-irradiated DNAs.

Selected samples of heterogeneous DNA from calf thymus with similar number-average molecular weight, Mn, and a low incidence of single-strand breaks were exposed in aqueous solutions to a mild X-ray dose of 1500 rads. The irradiation produced on the average about 0.2 bihelical and 2.2 monohelical scissions per DNA molecule of 1708 000 Mn. The percent distribution of the chemical termini released at the radiation nicks of DNA was as follows: 64.0 OH, 9.0 PO4 and 27.0 unknowns at the 3' ends: 3.8 OH, 68.2 PO4 and 28.0 unknowns at the 5' ends. A nuclease-free polynucleotide ligase I purified about 3000-fold over the crude homogenate from calf thymus succeeded in rejoining 50% of the breaks in the X-irradiated DNA. The ability of the enzyme to close radiation nicks in DNA directly was confirmed also by experiments on synthetic poly(dA).poly([3H]dT),poly(dT)-cellulose substrates with an irradiated dT chain at either the 3' or the 5' side of the functional break. The poor discrimination of mammalian ligase versus nicked DNA containing radiation damage is of practical relevance. While rejoining altered nucleotide chains in the helices of DNA, the enzyme might contribute to the fixation of premutational lesions in the genetic material.