Deletion of REXO1L1 locus in a patient with malabsorption syndrome, growth retardation, and dysmorphic features: a novel recognizable microdeletion syndrome?

BACKGROUND: Copy number variations (CNVs) can contribute to genetic variation among individuals and/or have a significant influence in causing diseases. Many studies consider new CNVs' effects on protein family evolution giving rise to gene duplicates or losses. "Unsuccessful" duplicates that remain in the genome as pseudogenes often exhibit functional roles. So, changes in gene and pseudogene number may contribute to development or act as susceptibility alleles of diseases. CASE PRESENTATION: We report a de novo heterozygous 271 Kb microdeletion at 8q21.2 region which includes the family of REXO1L genes and pseudogenes in a young man affected by global development delay, progeroid signs, and gastrointestinal anomalies. Molecular and cellular analysis showed that the REXO1L1 gene hemizygosity in a patient's fibroblasts induces genetic instability and increased apoptosis after treatment with different DNA damage-induced agents. CONCLUSIONS: The present results support the hypothesis that low copy gene number within REXO1L1 cluster could play a significant role in this complex clinical and cellular phenotype.

Carbon monoxide: an unusual drug.

The highly toxic gas carbon monoxide (CO) displays many physiological roles in several organs and tissues. Although many diseases, including cancer, hematological diseases, hypertension, heart failure, inflammation, sepsis, neurodegeneration, and sleep disorders, have been linked to abnormal endogenous CO metabolism and functions, CO administration has therapeutic potential in inflammation, sepsis, lung injury, cardiovascular diseases, transplantation, and cancer. Here, insights into the CO-based therapy, characterized by the induction or gene transfer of heme oxygenase-1 and either gas or CO-releasing molecule administration, are reviewed.

CO metabolism, sensing, and signaling.

CO is a colorless and odorless gas produced by the incomplete combustion of hydrocarbons, both of natural and anthropogenic origin. Several microorganisms, including aerobic and anaerobic bacteria and anaerobic archaea, use exogenous CO as a source of carbon and energy for growth. On the other hand, eukaryotic organisms use endogenous CO, produced during heme degradation, as a neurotransmitter and as a signal molecule. CO sensors act as signal transducers by coupling a "regulatory" heme-binding domain to a "functional" signal transmitter. Although high CO concentrations inhibit generally heme-protein actions, low CO levels can influence several signaling pathways, including those regulated by soluble guanylate cyclase and/or mitogen-activated protein kinases. This review summarizes recent insights into CO metabolism, sensing, and signaling.

Evidence for pH-dependent multiple conformers in iron(II) heme-human serum albumin: spectroscopic and kinetic investigation of carbon monoxide binding.

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional ligand binding capacity. HSA participates in heme scavenging by binding the macrocycle at fatty acid site 1. In turn, heme endows HSA with globin-like reactivity and spectroscopic properties. A detailed pH-dependent kinetic and spectroscopic investigation of iron(II) heme-HSA and of its carbonylated form is reported here. Iron (II) heme-HSA is a mixture of a four-coordinate intermediate-spin species (predominant at pH 5.8 and 7.0), a five-coordinate high-spin form (mainly at pH 7.0), and a six-coordinate low-spin species (predominant at pH 10.0). The acidic-to-alkaline reversible transition reflects conformational changes leading to the coordination of the heme Fe(II) atom by the His146 residue via its nitrogen atom, both in the presence and in the absence of CO. The presence of several species accounts for the complex, multiexponential kinetics observed and reflects the very slow interconversion between the different species observed both for CO association to the free iron(II) heme-HSA and for CO dissociation from CO-iron(II) heme-HSA as a function of pH.

Cancer predisposing mutations in BRCT domains.

Members of the breast cancer 1 (BRCA1) carboxy-terminal (BRCT) superfamily are involved in the cellular response to the DNA damage sensing and repair, as well as in the cell cycle control. All proteins are characterized by one or more BRCT domain(s), which provides a flexible framework representing scaffolding element(s) in multi-protein complexes. In particular, BRCA1, nibrin (NBN), and microcephalin (MCPH1), generally considered as molecular models for cancer-prone syndromes, contain BRCT domains able to bind phosphorylated proteins. Mutations within the BRCT domains of BRCA1, NBN, and MCPH1 are responsible for cancer susceptibility, both at the homozygous and heterozygous status. Here, we report a critical analysis of: (i) the BRCT domain structure, (ii) the role of BRCA1, NBN, and MCPH1 in DNA damage sensing and repair as well as in cell cycle control, and (iii) the pathological effects of mutations within the BRCT domains of BRCA1, NBN, and MCPH1.

O2-mediated oxidation of ferrous nitrosylated human serum heme-albumin is limited by nitrogen monoxide dissociation.

Human serum heme-albumin (HSA-heme-Fe) displays globin-like properties. Here, kinetics of O(2)-mediated oxidation of ferrous nitrosylated HSA-heme-Fe (HSA-heme-Fe(II)-NO) is reported. Values of the first-order rate constants for O(2)-mediated oxidation of HSA-heme-Fe(II)-NO (i.e., for ferric HSA-heme-Fe formation) and for NO dissociation from HSA-heme-Fe(II)-NO (i.e., for NO replacement by CO) are k=9.8 × 10(-5) and 8.3 × 10(-4) s(-1), and h=1.3 × 10(-4) and 8.5 × 10(-4) s(-1), in the absence and presence of rifampicin, respectively, at pH=7.0 and T=20.0 °C. The coincidence of values of k and h indicates that NO dissociation represents the rate limiting step of O(2)-mediated oxidation of HSA-heme-Fe(II)-NO. Mixing HSA-heme-Fe(II)-NO with O(2) does not lead to the formation of the transient adduct(s), but leads to the final ferric HSA-heme-Fe derivative. These results reflect the fast O(2)-mediated oxidation of ferrous HSA-heme-Fe and highlight the role of drugs in modulating allosterically the heme-Fe-atom reactivity.

Ibuprofen binding to secondary sites allosterically modulates the spectroscopic and catalytic properties of human serum heme-albumin.

The ibuprofen primary binding site FA3-FA4 is located in domain III of human serum albumin (HSA), the secondary clefts FA2 and FA6 being sited in domains I and II. Here, the thermodynamics of ibuprofen binding to recombinant Asp1-Glu382 truncated HSA (tHSA)-heme-Fe(III) and nitrosylated tHSA-heme-Fe(II), encompassing domains I and II only, is reported. Moreover, the allosteric effect of ibuprofen on the kinetics of tHSA-heme-Fe(III)-mediated peroxynitrite isomerization and nitrosylated tHSA-heme-Fe(II) denitrosylation has been investigated. The present data indicate, for the first time, that the allosteric modulation of tHSA-heme and HSA-heme reactivity by ibuprofen depends mainly on drug binding to the FA2 and FA6 secondary sites rather than drug association with the FA3-FA4 primary cleft. Thus, tHSA is a valuable model with which to investigate the allosteric linkage between the heme cleft FA1 and the ligand-binding pockets FA2 and FA6, all located in domains I and II of (t)HSA.

New PRSS1 and common CFTR mutations in a child with acute recurrent pancreatitis, could be considered an "Hereditary" form of pancreatitis ?

BACKGROUND: acute recurrent pancreatitis is a complex multigenic disease, the diagnosis is even more difficult when this disease develops in a child. CASE PRESENTATION: a 6-years old boy, hospitalized with epigastric pain radiating to the back showed high serum levels of serum amylase, lipase, CRP and erythrosedimentation rate. Several similar milder episodes of pain, followed by quick recovery and complete disappearance of symptoms were reported during the previous 13 months. The child was medically treated and after 7 days with normal clinic and laboratory tests was discharged with a hypolipidic diet. All the known aetiologic hypotheses were excluded by anamnestic investigation, clinical observation and biochemical evaluation, whereas, anatomic abnormality were excluded by a secretin stimulated magnetic resonance (MRI). At the last follow-up visit, (11 months later), the child showed a normal body weight and anthropometric profile, without further abdominal pain. Mutation screening for coding regions of PRSS1, SPINK1, CFTR and the new hereditary pancreatitis-associated chymotrypsin C (CTRC) genes showed a novel variation, c.541A > G (p.S181G), in the exon 4 of PRSS1 gene and the classical CF p.F508del mutation in the CFTR. Both mutations were present in his clinically normal mother and absent in the patient's father. CONCLUSIONS: this report extend the spectrum of PRSS1 mutations, however, the absence of family history of pancreatitis leaves the present case without the hallmark of the hereditary origin of pancreatitis. At the present knowledge it can be only stated that the combined genotype CFTR (F508del)/PRSS1 (S181G) is associated to a mild phenotype of acute recurrent pancreatitis in this child without any further conclusion on its pathogenetic role or prediction on the course of the disease.

Drug binding to Sudlow's site I impairs allosterically human serum heme-albumin-catalyzed peroxynitrite detoxification.

Heme endows human serum albumin (HSA) with globin-like reactivity and spectroscopic properties. Here, the effect of chlorpropamide, digitoxin, furosemide, indomethacin, phenylbutazone, sulfisoxazole, tolbutamide, and warfarin on peroxynitrite isomerization to NO(3) (-) by ferric HSA-heme (HSA-heme-Fe(III)) is reported. Drugs binding to Sudlow's site I impair dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III). The allosteric modulation of HSA-heme-Fe(III)-mediated peroxynitrite isomerization by drugs has been ascribed to the pivotal role of Tyr150, a residue that either provides a polar environment in Sudlow's site I or protrudes into the heme cleft (i.e., the fatty acid site 1, FA1), depending on ligand occupancy of either sites.

Reductive nitrosylation of ferric human serum heme-albumin.

Heme endows human serum albumin (HSA) with heme-protein-like reactivity and spectroscopic properties. Here, the kinetics and thermodynamics of reductive nitrosylation of ferric human serum heme-albumin [HSA-heme-Fe(III)] are reported. All data were obtained at 20 degrees C. At pH 5.5, HSA-heme-Fe(III) binds nitrogen monoxide (NO) reversibly, leading to the formation of nitrosylated HSA-heme-Fe(III) [HSA-heme-Fe(III)-NO]. By contrast, at pH >or= 6.5, the addition of NO to HSA-heme-Fe(III) leads to the transient formation of HSA-heme-Fe(III)-NO in equilibrium with HSA-heme-Fe(II)-NO(+). Then, HSA-heme-Fe(II)-NO(+) undergoes nucleophilic attack by OH(-) to yield ferrous human serum heme-albumin [HSA-heme-Fe(II)]. HSA-heme-Fe(II) further reacts with NO to give nitrosylated HSA-heme-Fe(II) [HSA-heme-Fe(II)-NO]. The rate-limiting step for reductive nitrosylation of HSA-heme-Fe(III) is represented by the OH(-)-mediated reduction of HSA-heme-Fe(II)-NO(+) to HSA-heme-Fe(II). The value of the second-order rate constant for OH(-)-mediated reduction of HSA-heme-Fe(II)-NO(+) to HSA-heme-Fe(II) is 4.4 x 10(3) M(-1) s(-1). The present results highlight the role of HSA-heme-Fe in scavenging reactive nitrogen species.

Premature ovarian failure, absence of pubic and axillary hair with de novo 46,X,t(X;15)(q24;q26.3).

We report on an adolescent girl with premature ovarian failure (POF), de novo unbalanced translocation X;15(q24;q26.3) with partial Xq24 duplication, and absence of pubic and axillary hair. Endocrine assessment showed normal adrenal and ovarian function. Chromosomal abnormality was identified by standard cytogenetic methods, array-CGH, and FISH analysis. Mutation analysis showed normal androgen receptor genes. Pubic and axillary hair began developing during estrogen + progesterone therapy. Our patient demonstrates that a distal X-breakpoint involving POF1 locus is able to cause POF without virilization during adolescence.

Targeting the DNA double strand breaks repair for cancer therapy.

Among several types of DNA lesions, the DNA double strand breaks (DSBs) are one of the most deleterious and harmful. Mammalian cells mount a coordinated response to DSBs with the aim of appropriately repair the DNA damage. Indeed, failure of the DNA damage response (DDR) can lead to the development of cancer-prone genetic diseases. The identification and development of drugs targeting proteins involved in the DDR is even more investigated, as it gives the possibility to specifically target cancer cells. Indeed, the administration of DNA repair inhibitors could be combined with chemo- and radiotherapy, thus improving the eradication of tumor cells. Here, we provide an overview about DSBs damage response, focusing on the role of the DSBs repair mechanisms, of chromatin modifications, and of the cancer susceptibility gene BRCA1 which plays a multifunctional role in controlling genome integrity. Moreover, the most investigated DSBs enzyme inhibitors tested as potential therapeutic agents for anti-cancer therapy are reported.

Determination of antituberculosis drug concentration in human plasma by MALDI-TOF/TOF.

Therapeutic drug monitoring allows to determine the best dosage regimen adapted to each patient optimizing the therapeutic benefits, while minimizing the risk for side effects. Here, the first methodological approach based on matrix-assisted laser desorption/ionization source equipped with tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry for the determination of the antituberculosis (anti-TB) drugs ethambutol, pyrazinamide, rifampicin, and streptomycin concentration in the plasma of tuberculosis-infected patients is reported. The volume of the plasma sample was 200 microL. Plasma samples were cleaned-up by protein precipitation and evaporated in a water bath under a nitrogen stream. The extracted samples were reconstituted with 200 microL of 50% methanol-0.03% formic acid solution (v/v), spiked with known amounts of anti-TB drugs, mixed (1:1) with a saturated matrix solution (4-hydroxybenzoic acid in 50% acetonitrile-0.1% trifluoracetic acid solution; v/v), and spotted onto the MALDI-TOF/TOF sample target plate. The anti-TB drug concentration was determined by standard additions analysis. Regression of standard additions was linear over the whole anti-TB drug concentration range explored (the final anti-TB drug concentration ranged from 0.20 to 200 pmol/microL). The absolute recovery of the anti-TB drugs ranged between 87 and 110%. The minimal ethambutol, pyrazinamide, rifampicin, and streptomycin concentration detectable by MALDI-TOF/TOF is 0.08, 0.20, 0.12, and 0.15 pmol/microL, respectively.

Reductive nitrosylation of ferric cyanide horse heart myoglobin is limited by cyanide dissociation.

Cyanide binds to ferric heme-proteins with a very high affinity, reflecting the very low dissociation rate constant (k(off)). Since no techniques are available to estimate k(off), we report herewith a method to determine k(off) based on the irreversible reductive nitrosylation reaction to trap ferric myoglobin (Mb(III)). The k(off) value for cyanide dissociation from ferric cyanide horse heart myoglobin (Mb(III)-cyanide) was determined at pH 9.2 and 20.0 degrees C. Mixing Mb(III)-cyanide and NO solutions brings about absorption spectral changes reflecting the disappearance of Mb(III)-cyanide with the concomitant formation of ferrous nitrosylated Mb. Since kinetics of reductive nitrosylation of Mb(III) is much faster than Mb(III)-cyanide dissociation, the k(off) value, representing the rate-limiting step, can be directly determined. The k(off) value obtained experimentally matches very well to that calculated from values of the second-order rate constant (k(on)) and of the dissociation equilibrium constant (K) for cyanide binding to Mb(III) (k(off)=k(on)xK).

Design, Construction and Validation of Targeted BAC Array-Based CGH Test for Detecting the Most Commons Chromosomal Abnormalities.

We designed a targeted-array called GOLD (Gain or Loss Detection) Chip consisting of 900 FISH-mapped non-overlapping BAC clones spanning the whole genome to enhance the coverage of 66 unique human genomic regions involved in well known microdeletion/microduplication syndromes. The array has a 10 Mb backbone to guarantee the detection of the aneuploidies, and has an implemented resolution for telomeres, and for regions involved in common genomic diseases. In order to evaluate clinical diagnostic applicability of GOLDChip, analytical validity was carried-out via retrospective analysis of DNA isolated from a series of cytogenetically normal amniocytes and cytogenetically abnormal DNA obtained from cultured amniocytes, peripheral blood and/or cell lines. We recruited 47 DNA samples corresponding to pathologies with significant frequencies (Cri du Chat syndrome, Williams syndrome, Prader Willi/Angelman syndromes, Smith-Magenis syndrome, DiGeorge syndrome, Miller-Dieker syndrome, chromosomes 13, 18 and 21 trisomies). We set up an experimental protocol that allowed to identify chromosomal rearrangements in all the DNA samples analyzed. Our results provide evidence that our targeted BAC array can be used for the identification of the most common microdeletion syndromes and common aneuploidies.

Deletion 2q37: an identifiable clinical syndrome with mental retardation and autism.

Terminal deletion of the long arm of chromosome 2 is a rare chromosomal disorder characterized by low birth weight, delayed somatic and mental development, craniofacial defects, short neck, heart and lung congenital defects, and autistic features. We report on a girl with 46,XX.ish del(2)(q37.1) de novo karyotype, mental retardation, dysmorphic features, gastrointestinal anomalies, and autistic traits and compare her clinical manifestations with patients with the same deletion previously described in literature.

Hyperproliferation in nasal polyposis tissues is not associated with somatic genomic instability.

BACKGROUND: Nasal polyps (NPs) are abnormal lesions that arise from any portion of the nasal mucosa or paranasal sinuses within the nasal cavities. They are characterized by the formation of inflammatory swellings of unstable respiratory mucosa, which protrude into the nasal cavity. The causes and the pathogenesis of NP development are not well understood. The current and principal hypothesis is that NPs result from allergy and inflammation, which cause an unstable respiratory mucosa with increased epithelial cell proliferation and epithelial morphologic changes. OBJECTIVE: To understand if epithelial cell hyperproliferation can lead to acquired genomic instability and if nasal polyposis is a consequence of acquired chromosomal alterations in hyperproliferative nasal epithelia. MATERIALS AND METHODS: To test this hypothesis and to perform a survey of chromosomal aberrations in nasal polyps, we applied array comparative genome hybridization (aCGH; 1 Mb resolution, made of 2464 bacterial artificial chromosome (BAC), P1 derived artificial chromosome (PAC), and P1 clones spanning the whole human genome) to deoxyribonucleic acid (DNA) obtained from the NPs of nine patients. The patients recruited in this study had been analyzed for Ki-67 expression using the MIB-1 antibody as a marker of proliferation and were chosen to represent a wide range of hyperproliferative status. RESULTS: After data analysis, no chromosomal aberrations were detected by aCGH. CONCLUSIONS: Our data do not support the hypothesis that nasal polyposis is a consequence of acquired chromosomal alterations in hyperproliferative nasal epithelia.

Compound heterozygosity for mutations in LMNA in a patient with a myopathic and lipodystrophic mandibuloacral dysplasia type A phenotype.

CONTEXT: Mandibuloacral dysplasia type A (MADA; OMIM 248370) is a rare progeroid syndrome characterized by dysmorphic craniofacial and skeletal features, lipodystrophy, and metabolic complications. Most Italian patients carry the same homozygous missense mutation (p.R527H) in the C-terminal tail domain of the LMNA gene, which encodes lamin A/C, an intermediate filament component of the nuclear envelope. OBJECTIVE: The objective of the study was to identify novel LMNA mutations in individuals with clinical characteristics (bird-like facies, mandibular and clavicular hypoplasia, acroosteolysis, lipodystrophy, alopecia) observed in other well-known patients. DESIGN: The LMNA gene was sequenced. Functional properties of the mutant alleles were investigated. PATIENT: We report a 27-yr-old Italian woman showing a MADA-like phenotype. Features include a hypoplastic mandible, acroosteolysis, pointed nose, partial loss of sc fat, and a progeric appearance. Due to the absence of clavicular dysplasia and normal metabolic profiles, generally associated with muscle hyposthenia and generalized hypotonia, this phenotype can be considered an atypical laminopathy. RESULTS: We identified a patient compound heterozygote for the p.R527H and p.V440M alleles. The patient's cells showed nuclear shape abnormalities, accumulation of pre-lamin A, and irregular lamina thickness. Lamins A and C showed normal expression and localization. The electron microscopy detected heterochromatin defects with a pattern similar to those observed in other laminopathies. However, chromatin analysis showed a normal distribution pattern of the major heterochromatin proteins: heterochromatin protein-1beta and histone H3 methylated at lysine 9. CONCLUSIONS: The clinical and cellular features of this patient show overlapping laminopathy phenotypes that could be due to the combination of p.R527H and p.V440M alleles.

Prenatal diagnosis of genomic disorders and chromosome abnormalities using array-based comparative genomic hybridization.

Cytogenetic analysis is a crucial tool of prenatal diagnosis. The ability to rapidly detect aneuploidy and identify small structural abnormalities of foetal chromosomes has been greatly improved by the use of molecular cytogenetic technologies. Microarray-based Comparative Genomic Hybridization (aCGH) has been recently employed in postnatal diagnosis of cryptic chromosomal aberrations, but use in prenatal diagnosis is still limited.We set-up a diagnostic protocol which uses aCGH technology on genomic DNA isolated from uncultured chorionic villus sampled at 11-12 week's gestation. We used a commercially targeted microarray (MDTelArray, Technogenetics Srl - Bouty Group, Sesto S. Giovanni, Milan, Italy) constituted by 167 genomic clones corresponding to 34 critical regions frequently involved in microdeletions and microduplications and 126 subtelomeric clones. Array validation has been carried-out via retrospective analysis of DNA isolated from a series of cytogenetically normal chorionic villus samples (CVS) and of DNA isolated from cytogenetically abnormal cultured amniocytes, CVS or peripheral blood. A pilot prospective study was undertaken analyzing 25 CVS obtained from foetuses at risk for chromosomal aberrations. aCGH results both for retrospective and prospective studies were in agreement with data obtained using "classical" cytogenetic analysis, and/or FISH analysis or DNA testing. Although these preliminary data support the usefulness of aCGH in prenatal diagnosis, further prospective studies are required to verify the feasibility of introducing this technique as part of the diagnostic armamentarium for identify affected foetuses.

Use of RNA fluorescence in situ hybridization in the prenatal molecular diagnosis of myotonic dystrophy type I.

BACKGROUND: Myotonic dystrophy type 1 (DM1; OMIM #160900) is an autosomal-dominant genetic disorder with multisystemic clinical features associated with a CTG expansion in the 3' untranslated region of the DMPK gene on chromosome 19q13.3. A long-PCR protocol to detect the DM1 expansion is rapid, sensitive, and accurate, but interpretative limitations can occur when the expansion size exceeds the PCR amplification range and in cases of somatic mosaicism. METHODS: To overcome these problems, we used RNA fluorescence in situ hybridization (RNA-FISH) to study cultured cells derived from chorionic villus samples (CVS) with the DM1 mutation. The RNA-FISH method is designed to detect the distinctive DM1 cellular phenotype, characterized by the presence of nuclei with focal ribonuclear inclusions (foci) containing the DMPK expanded transcripts. We analyzed 6 CVS from DM1-predicted pregnancies and 6 CVS from DM1-negative pregnancies. RESULTS: In 4 DM1-predicted fetuses with a CTG expansion >200 CTG, varying numbers of ribonuclear inclusions were clearly visible in all cells. One case with a somatic mosaicism for the DMPK mutation showed 15% of cells with no nuclear foci. No nuclear signals were detected in all controls examined (n = 6) and in 1 DM1-positive sample with a CTG expansion <100 copies. CONCLUSION: Nuclear foci, and therefore the DM1 mutation they are caused by, can be detected efficiently on interphase nuclei of trophoblast cells with RNA-FISH when the CTG expansion is >200 copies.