The potential use of patient-specific induced pluripotent stem cells (hiPSCs) in the study and treatment of hematological diseases requires the setup of efficient and safe protocols for hiPSC ...generation. We aimed to adopt a reprogramming method for large-scale production of integration-free patient-specific hiPSC-lines in our stem cell processing laboratory, which supports a pediatric hematopoietic stem cell transplant unit located at a tertiary care children's hospital. We describe our 5-year experience in generation of hiPSC-lines from human bone marrow-derived mesenchymal stromal cells (BM-MSCs) using synthetic mRNAs encoding reprogramming factors. We generated hiPSC-lines from pediatric patients with β-Thalassemia, Sickle Cell Anemia, Blackfan-Diamond Anemia, Severe Aplastic Anemia, DOCK8 Immunodeficiency and 1 healthy control. After optimization of the reprogramming procedure, average reprogramming efficiency of BM-MSCs was 0.29% (range 0.25–0.4). The complete reprogramming process lasted 14–16 days. Three to five hiPSC-colonies per sample were selected, expanded to 5 culture passages and then frozen. The whole procedure took an average time of 1.8 months (range 1.6–2.2). The hiPSC-lines expressed embryonic stem cell markers and exhibited pluripotency. This mRNA reprogramming method can be applicable in a hematopoietic stem cell culture lab setting and would be useful for the clinical translation of patient-specific hiPSCs.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
46,XX male sex reversal syndrome is a rare genetic cause of male infertility. We report on two new cases of this syndrome in men presenting with hypogonadism and infertility. Cytogenetic and ...molecular analysis was performed in both patients. An extensive review of the literature for 46,XX male sex reversal syndrome cases related to infertility was also performed to fully characterise this syndrome. Genetic analyses showed translocation of the SRY on Xp chromosome and complete absence of all Azoospermia factor (AZF) genetic regions. All patients included in the review presented hypergonadotropic hypogonadism. Small testes were the most common clinical characteristic present in 90.2% of the patients, followed by small penis (31.8%), gynecomastia (26.8%) and poor hair distribution (15.4%). The presence of the SRY was identified in 130/154 (84.4%) patients: in 98.5% of cases, it was translocated on the Xp chromosome and in 1.5% on an autosome. All patients were azoospermic, due to the lack of AZF genetic regions. Males with normal phenotype and primary hypogonadism should be properly evaluated by the physicians and must be referred for cytogenetic and molecular analysis to exclude or confirm 46,XX male sex reversal syndrome. More cases of this syndrome with SRY translocated on an autosome are needed to identify if these patients have different characteristics than those with SRY translocated on Xp chromosome. Whole genome analysis of these patients is required to elucidate the genetic differences which are responsible for the phenotypic variability of the syndrome.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Both autosomal recessive and autosomal dominant inheritance have been described, but most HIES cases are sporadic.1-3 Autosomal dominant HIES (AD-HIES), the most common form, has various immunologic ...and nonimmunologic abnormalities arising as consequences of impaired cytokine signal transduction and TH17 cell deficiency caused by mutations in the signal transducer and activator of transcription 3 gene (STAT3).4,5 Although correction of the immunologic defect with allogeneic hematopoietic stem cell transplantation (HSCT) could be expected, the first transplantation attempts in 2 patients with HIES reported in 1998 and 2000 failed to show long-term benefits.6,7 We report the cases of 2 unrelated boys with sporadic HIES complicated by high-grade non-Hodgkin lymphoma, who were cured with myeloablative HLA-matched sibling bone marrow transplantation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Alternating hemiplegia of childhood (AHC) is typically distinguished from familial hemiplegic migraine (FHM) by infantile onset of the characteristic symptoms and high prevalence of associated ...neurological deficits that become increasingly obvious with age. Expansion of the clinical spectrum in FHM recently has begun to blur the distinction between these disorders. We report a novel ATP1A2 mutation in a kindred with features that bridge the phenotypic spectrum between AHC and FHM syndromes, supporting a possible common pathogenesis in a subset of such cases. Mutation analysis in classic sporadic AHC patients and in an additional five kindreds in which linkage to the ATP1A2 locus could not be excluded failed to identify additional mutations. Ann Neurol 2004;55:884–887
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Context and Objective: Plasma IL-6, the serum inflammatory markers C-reactive protein (CRP) and serum amyloid A (SAA), and the tissue destruction marker-free plasma DNA, as well as the circulating ...lipid profile, were examined in athletes participating in the ultradistance foot race of the 246-km Spartathlon.
Setting, Design, and Participants: This race consists of continuous, prolonged, brisk exercise. Blood samples were obtained from 15 male athletes, who finished the race in less than 36 h, taken before, at the end of, and 48 h after the end of the race.
Results: IL-6, CRP, SAA, and free plasma DNA levels markedly increased (by 8000-, 152- 108-, and 10-fold, respectively) over the baseline at the end of the race. However, IL-6 levels returned to normal by 48 h, whereas CRP, SAA, and free plasma DNA remained elevated. The mean values of cholesterol, triglycerides, low-density lipoprotein, and apolipoprotein B decreased to a minimum value at the end of the race and remained low 48 h after the race. High-density lipoprotein levels, on the other hand, were mildly increased at the end of the race (P < 0.015) and decreased to normal 48 h after the race. Apolipoprotein AI levels decreased significantly during the time course of the exercise and remained low 48 h after the race (P < 0.001).
Conclusions: These observations suggest that continuous, prolonged, moderate-intensity exercise is associated with markedly elevated IL-6 and acute-phase reactant concentrations, peripheral tissue damage, and significant changes in serum lipid levels. The biochemical changes observed during the Spartathlon amount to a potent systemic inflammatory response, which might explain severe cardiovascular events that occur during prolonged exercise in compromised individuals.
The primer extension (PEXT) reaction is the most widely used approach to genotyping of single-nucleotide polymorphisms (SNPs). Current methods for analysis of PEXT reaction products are based on ...electrophoresis, fluorescence resonance energy transfer, fluorescence polarization, pyrosequencing, mass spectrometry, microarrays, and spectrally encoded microspheres. We report the first dry-reagent dipstick method that enables rapid visual detection of PEXT products without instrumentation. The method is applied to the analysis of six SNPs in the mannose-binding lectin gene (MBL2). The genomic region that spans each SNP of interest is amplified by PCR. Two primer extension reactions are performed with allele-specific primers (for one or the other variant nucleotide), which contain an oligo(dA) segment at the 5‘-end. Biotin-dUTP is incorporated in the extended strand. The product is applied to the strip followed by immersion in the appropriate buffer. As the DNA moves along the strip by capillary action, it hybridizes with oligo(dT)-functionalized gold nanoparticles, such that only extended products are captured by immobilized streptavidin at the test zone, generating a red line. A second red line is formed at the control zone of the strip by hybridization of the nanoparticles with immobilized oligo(dA). The dipstick test is complete within 10 min. We analyzed six SNPs of the mannose-binding lectin gene (MBL2) using genomic DNA from 27 patients, representing a total of 74 variant nucleotide positions. Patient genotypes showed 100% concordance with direct DNA sequencing data. The described PEXT-dipstick assay is rapid and highly accurate; it does not require specialized instrumentation or highly trained technical personnel. It is appropriate for a diagnostic laboratory where a few selected SNP markers are examined per patient with a low cost per assay.
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IJS, KILJ, NUK, PNG, UL, UM
Sotos syndrome is characterized by tall stature, advanced bone age, typical facial abnormalities, and developmental delay. The associated gene is NSD1 . The study involved 22 patients who fulfilled ...the clinical criteria. Phenotypic characteristics, central nervous system findings, and cardiovascular and urinary tract abnormalities were evaluated. Meta-analysis on the incidence of cardinal clinical manifestations from the literature was also performed. Macrocephaly was present in all patients. Advanced bone age was noted in 14 of 22 patients (63%), and its incidence presented significant statistical difference in the meta-analysis of previous studies. Some patients had serious clinical manifestations, such as congenital heart defects, dysplastic kidneys, psychosis, and leukemia. Clinical and laboratory examinations should be performed to prevent and manage any unusual medical aspect of the syndrome. Facial gestalt and macrocephaly, rather than advanced bone age, are the strongest indications for clinical diagnosis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Abstract Background : Mutations in the MECP2 gene (methyl-CpG-binding protein-2) are responsible for 60–95% of cases of Rett syndrome (RTT), an X-linked dominant neurodevelopmental disorder affecting ...mostly girls. Classic RTT is characterized by normal early development followed by psychomotor regression and onset of microcephaly, although variant forms are also observed. MECP2 has also been implicated in variable mental retardation (MR) phenotypes, including X-linked Mental Retardation (XLMR), Fragile-X-like Syndrome (FXS) and Angelman-like (AS) phenotypes. Aim : The aim of the study was: (a) to evaluate the incidence and spectrum of MECP2 mutations in children with RTT and variant MR; (b) to evaluate phenotype-genotype correlations. Methods : Exons 3–4 were analyzed for mutations in 281 MR patients (aged 13 months–27 years old, 144 males–137 females) consisting of 88 patients referred for RTT and 193 patients referred for AS-like and FXS-like types of MR. Statistical analysis included correlation between classic MECP2 -positive and MECP2 -negative and variant RTT patients, and frequency of MECP2 mutations in the various categories. Results : Mutations were detected in ≈70% of classic and ≈21% of variant RTT, respectively. Amongst MR cases, 2.1% carried MECP2 mutations. MECP2 -positive females had more problems in ambulation, muscle tone, tremor and ataxia, respiratory disturbances, head growth, hand use and stereotypies. Classic RTT-positive versus negative had significant respiratory and sitting problems and versus variant RTT-positive females ambulatory, hand and stereotypies problems. Conclusion : The analysis of the MECP2 gene could provide a diagnostic tool for RTT and non-specific MR research.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
ABSTRACT
Objectives:
Herein we report the results of mutation‐based screening for Wilson disease (WD) in 2 isolated populations of Sardinia and the Greek island of Kalymnos.
Patients and Methods:
...Mutation analysis was performed in 110 and 9 WD families originating respectively from Sardinia and Kalymons using single‐strand conformation polymorphism and sequencing methods. In Sardinia, a limited screening was performed for −441/−427del in 5290 newborns, whereas in Kalymnos 397 newborns underwent mutation screening for H1069Q and R969Q using appropriate methods.
Results:
In Sardinia, mutation analysis showed the presence of 6 mutations accounting for 85% of chromosomes, 1 of which (−441/−427del) is present in 61.7% of alleles. The screening for −441/−427del in 5290 newborns revealed the presence of 122 heterozygotes, which is equal to an allelic frequency of 1.15%. Assuming the same distribution of WD mutations in the general Sardinian population, we also inferred an allelic frequency of 0.77% for mutations other than −441/−427del, which accounts for an overall frequency of any WD mutation of 1.92%. Assuming Hardy‐Weinberg equilibrium, these data could be translated into a WD incidence of 1 in 2707 live births. In Kalymnos, mutation analysis in 9 WD families revealed the presence of only 2 mutations. The screening of 397 newborns revealed the presence of 18 heterozygotes for H1069Q, 9 for R969Q, and 1 compound heterozygote for these mutations, which is equal to an allele frequency of 3.7%. Assuming Hardy‐Weinberg equilibrium, the expected carrier rate is 7%.
Conclusions:
These data indicate the need for health education for WD prevention in these isolated populations.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Phenotypic variation in CHARGE syndrome remains unexplained. A subcategory of CHARGE patients show overlapping phenotypic characteristics with DiGeorge syndrome (thymic hypo/aplasia, hypocalcemia, ...T-cell immunodeficiency). Very few have been tested or reported to carry a mutation of the CHD7 (chromodomain helicase DNA-binding domain) gene detected in two-thirds of CHARGE patients. In an attempt to explore the genetic background of a severe CHARGE/DiGeorge phenotype, we performed comparative genomic array hybridization in an infant carrier of a CHD7 mutation. The high-resolution comparative genomic array hybridization revealed interesting findings, including a deletion distal to the DiGeorge region and disruptions in other chromosomal regions of genes implicated in immunological and other functions possibly contributing to the patient's severe phenotype and early death.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, SIK, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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