Publications by authors named "Caterina Lo Rizzo"

20 Publications

  • Page 1 of 1

IQSEC2 disorder: A new disease entity or a Rett spectrum continuum?

Clin Genet 2021 Mar 9;99(3):462-474. Epub 2021 Jan 9.

Medical Genetics, University of Siena, Siena, Italy.

IQSEC2 mutations are associated with IQSEC2-related intellectual disability (ID). Phenotypic spectrum has been better defined in the last few years by the increasing number of reported cases although the genotype-phenotype relationship for IQSEC2 remains overall complex. As for IQSEC2-related ID a wide phenotypic diversity has been described in Rett syndrome (RTT). Several patients harboring IQSEC2 mutations present with clinical symptoms similar to RTT and some cases meet most of the criteria for classic RTT. With the aim of establishing a genotype-phenotype correlation, we collected data of 16 patients harboring IQSEC2 point mutations (15 of them previously unreported) and of five novel patients carrying CNVs encompassing IQSEC2. Most of our patients surprisingly shared a moderate-to-mild phenotype. The similarities in the clinical course between our mild cases and patients with milder forms of atypical RTT reinforce the hypothesis that also IQSEC2 mutated patients may lay under the wide clinical spectrum of RTT and thus IQSEC2 should be considered in the differential diagnosis. Our data confirm that position, type of variant and gender are crucial for IQSEC2-associated phenotype delineation.
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http://dx.doi.org/10.1111/cge.13908DOI Listing
March 2021

AAV-mediated FOXG1 gene editing in human Rett primary cells.

Eur J Hum Genet 2020 10 15;28(10):1446-1458. Epub 2020 Jun 15.

Medical Genetics, University of Siena, Siena, Italy.

Variations in the Forkhead Box G1 (FOXG1) gene cause FOXG1 syndrome spectrum, including the congenital variant of Rett syndrome, characterized by early onset of regression, Rett-like and jerky movements, and cortical visual impairment. Due to the largely unknown pathophysiological mechanisms downstream the impairment of this transcriptional regulator, a specific treatment is not yet available. Since both haploinsufficiency and hyper-expression of FOXG1 cause diseases in humans, we reasoned that adding a gene under nonnative regulatory sequences would be a risky strategy as opposed to a genome editing approach where the mutated gene is reversed into wild-type. Here, we demonstrate that an adeno-associated viruses (AAVs)-coupled CRISPR/Cas9 system is able to target and correct FOXG1 variants in patient-derived fibroblasts, induced Pluripotent Stem Cells (iPSCs) and iPSC-derived neurons. Variant-specific single-guide RNAs (sgRNAs) and donor DNAs have been selected and cloned together with a mCherry/EGFP reporter system. Specific sgRNA recognition sequences were inserted upstream and downstream Cas9 CDS to allow self-cleavage and inactivation. We demonstrated that AAV serotypes vary in transduction efficiency depending on the target cell type, the best being AAV9 in fibroblasts and iPSC-derived neurons, and AAV2 in iPSCs. Next-generation sequencing (NGS) of mCherry/EGFP transfected cells demonstrated that the mutated alleles were repaired with high efficiency (20-35% reversion) and precision both in terms of allelic discrimination and off-target activity. The genome editing strategy tested in this study has proven to precisely repair FOXG1 and delivery through an AAV9-based system represents a step forward toward the development of a therapy for Rett syndrome.
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http://dx.doi.org/10.1038/s41431-020-0652-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7608362PMC
October 2020

Focus on progressive myoclonic epilepsy in Berardinelli-Seip syndrome.

Neurol Sci 2020 Nov 21;41(11):3345-3348. Epub 2020 May 21.

Dipartimento di Medicina Molecolare e dello Sviluppo, Universita' degli Studi di Siena, viale Bracci 16, 53100, Siena, Italy.

Introduction: Berardinelli-Seip syndrome or congenital generalized lipodystrophy type 2 is a rare genetic disorder characterized by selective loss of subcutaneous adipose tissue associated with peripheral insulin resistance and its complications. Nonprogressive mental retardation, dystonia, ataxia, and pyramidal signs are commonly present, whereas epilepsy has only occasionally been observed.

Case Report: We report the case of two sisters, 11 and 18 years old respectively, with an overlapping clinical phenotype compatible with Berardinelli-Seip syndrome and progressive myoclonic epilepsy. Molecular analysis identified an autosomal recessive c.1048C > t;(p(Arg350*)) pathogenic mutation of exon 8 of the BSCL2 gene, which was present in a homozygous state in both patients.

Conclusions: Our paper contributes to further delineate a complex phenotype associated with BSCL2 mutation, underlining how seipin has a central and partially still unknown role that goes beyond adipose tissue metabolism, with a prominent involvement in central nervous system pathology.
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http://dx.doi.org/10.1007/s10072-020-04418-1DOI Listing
November 2020

High rate of HDR in gene editing of p.(Thr158Met) MECP2 mutational hotspot.

Eur J Hum Genet 2020 09 24;28(9):1231-1242. Epub 2020 Apr 24.

Medical Genetics, University of Siena, Siena, Italy.

Rett syndrome is a progressive neurodevelopmental disorder which affects almost exclusively girls, caused by variants in MECP2 gene. Effective therapies for this devastating disorder are not yet available and the need for tight regulation of MECP2 expression for brain to properly function makes gene replacement therapy risky. For this reason, gene editing with CRISPR/Cas9 technology appears as a preferable option for the development of new therapies. To study the disease, we developed and characterized a human neuronal model obtained by genetic reprogramming of patient-derived primary fibroblasts into induced Pluripotent Stem Cells. This cellular model represents an important source for our studies, aiming to correct MECP2 variants in neurons which represent the primarily affected cell type. We engineered a gene editing toolkit composed by a two-plasmid system to correct a hotspot missense variant in MECP2, c.473 C > T (p.(Thr158Met)). The first construct expresses the variant-specific sgRNA and the Donor DNA along with a fluorescent reporter system. The second construct brings Cas9 and targets for auto-cleaving, to avoid long-term Cas9 expression. NGS analysis on sorted cells from four independent patients demonstrated an exceptionally high editing efficiency, with up to 80% of HDR and less than 1% of indels in all patients, outlining the relevant potentiality of the approach for Rett syndrome therapy.
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http://dx.doi.org/10.1038/s41431-020-0624-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609331PMC
September 2020

Analysis of the Phenotypes in the Rett Networked Database.

Int J Genomics 2019 27;2019:6956934. Epub 2019 Mar 27.

Tri-State Rett Syndrome Center, Montefiore Medical Center, Albert Einstein College of Medicine, New York City, NY, USA.

Rett spectrum disorder is a progressive neurological disease and the most common genetic cause of intellectual disability in females. is the major causative gene. In addition, and mutations have been reported in Rett patients, especially with the atypical presentation. Each gene and different mutations within each gene contribute to variability in clinical presentation, and several groups worldwide performed genotype-phenotype correlation studies using cohorts of patients with classic and atypical forms of Rett spectrum disorder. The Rett Networked Database is a unified registry of clinical and molecular data of Rett patients, and it is currently one of the largest Rett registries worldwide with several hundred records provided by Rett expert clinicians from 13 countries. Collected data revealed that the majority of -mutated patients present with the classic form, the majority of -mutated patients with the early-onset seizure variant, and the majority of -mutated patients with the congenital form. A computation of severity scores further revealed significant differences between groups of patients and correlation with mutation types. The highly detailed phenotypic information contained in the Rett Networked Database allows the grouping of patients presenting specific clinical and genetic characteristics for studies by the Rett community and beyond. These data will also serve for the development of clinical trials involving homogeneous groups of patients.
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http://dx.doi.org/10.1155/2019/6956934DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6458890PMC
March 2019

Usefulness and Limitations of Comprehensive Characterization of mRNA Splicing Profiles in the Definition of the Clinical Relevance of Variants of Uncertain Significance.

Cancers (Basel) 2019 Mar 1;11(3). Epub 2019 Mar 1.

Medical Genetics, University of Siena, 53100 Siena, Italy.

Highly penetrant variants of genes are involved in hereditary predisposition to breast and ovarian cancer. The detection of pathogenic BRCA variants has a considerable clinical impact, allowing appropriate cancer-risk management. However, a major drawback is represented by the identification of variants of uncertain significance (VUS). Many VUS potentially affect mRNA splicing, making transcript analysis an essential step for the definition of their pathogenicity. Here, we characterize the impact on splicing of ten variants. Aberrant splicing patterns were demonstrated for eight variants whose alternative transcripts were fully characterized. Different events were observed, including exon skipping, intron retention, and usage of de novo and cryptic splice sites. Transcripts with premature stop codons or in-frame loss of functionally important residues were generated. Partial/complete splicing effect and quantitative contribution of different isoforms were assessed, leading to variant classification according to Evidence-based Network for the Interpretation of Mutant Alleles (ENIGMA) consortium guidelines. Two variants could be classified as pathogenic and two as likely benign, while due to a partial splicing effect, six variants remained of uncertain significance. The association with an undefined tumor risk justifies caution in recommending aggressive risk-reduction treatments, but prevents the possibility of receiving personalized therapies with potential beneficial effect. This indicates the need for applying additional approaches for the analysis of variants resistant to classification by gene transcript analyses.
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http://dx.doi.org/10.3390/cancers11030295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468917PMC
March 2019

Phenotype and genotype of 87 patients with Mowat-Wilson syndrome and recommendations for care.

Genet Med 2018 09 4;20(9):965-975. Epub 2018 Jan 4.

Neuropsychiatric Department, Spedali Civili Brescia, Brescia, Italy.

Purpose: Mowat-Wilson syndrome (MWS) is a rare intellectual disability/multiple congenital anomalies syndrome caused by heterozygous mutation of the ZEB2 gene. It is generally underestimated because its rarity and phenotypic variability sometimes make it difficult to recognize. Here, we aimed to better delineate the phenotype, natural history, and genotype-phenotype correlations of MWS.

Methods: In a collaborative study, we analyzed clinical data for 87 patients with molecularly confirmed diagnosis. We described the prevalence of all clinical aspects, including attainment of neurodevelopmental milestones, and compared the data with the various types of underlying ZEB2 pathogenic variations.

Results: All anthropometric, somatic, and behavioral features reported here outline a variable but highly consistent phenotype. By presenting the most comprehensive evaluation of MWS to date, we define its clinical evolution occurring with age and derive suggestions for patient management. Furthermore, we observe that its severity correlates with the kind of ZEB2 variation involved, ranging from ZEB2 locus deletions, associated with severe phenotypes, to rare nonmissense intragenic mutations predicted to preserve some ZEB2 protein functionality, accompanying milder clinical presentations.

Conclusion: Knowledge of the phenotypic spectrum of MWS and its correlation with the genotype will improve its detection rate and the prediction of its features, thus improving patient care.
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http://dx.doi.org/10.1038/gim.2017.221DOI Listing
September 2018

Urine-derived podocytes-lineage cells: A promising tool for precision medicine in Alport Syndrome.

Hum Mutat 2018 02 22;39(2):302-314. Epub 2017 Nov 22.

Medical Genetics, University of Siena, Siena, Italy.

Alport Syndrome (ATS) is a rare genetic disorder caused by collagen IV genes mutations, leading to glomerular basement membrane damage up to end-stage renal disease. Podocytes, the main component of the glomerular structure, are the only cells able to produce all the three collagens IV alpha chains associated with ATS and thus, they are key players in ATS pathogenesis. However, podocytes-targeted therapeutic strategies have been hampered by the difficulty of non-invasively isolating them and transcripts-based diagnostic approaches are complicated by the inaccessibility of other COL4 chains-expressing cells. We firstly isolated podocyte-lineage cells from ATS patients' urine samples, in a non-invasive way. RT-PCR analysis revealed COL4A3, COL4A4, and COL4A5 expression. Transcripts analysis on RNA extracted from patient's urine derived podocyte-lineage cells allowed defining the pathogenic role of intronic variants, namely one mutation in COL4A3 (c.3882+5G>A), three mutations in COL4A4 (c.1623+2T>A, c.3699_3706+1del, c.2545+143T>A), and one mutation in COL4A5 (c.3454+2T>C). Therefore, our cellular model represents a novel tool, essential to unequivocally prove the effect of spliceogenic intronic variants on transcripts expressed exclusively at a glomerular level. This process is a key step for providing the patient with a definite molecular diagnosis and with a proper recurrence risk. The established system also opens up the possibility of testing personalized therapeutic approaches on disease-relevant cells.
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http://dx.doi.org/10.1002/humu.23364DOI Listing
February 2018

Sporadic hereditary motor and sensory neuropathies: Advances in the diagnosis using next generation sequencing technology.

J Neurol Sci 2015 Dec 9;359(1-2):409-17. Epub 2015 Oct 9.

Medical Genetics, University of Siena, Siena, Italy; Genetica Medica, Azienda Ospedaliera Universitaria Senese, Siena, Italy. Electronic address:

Hereditary motor and sensory neuropathies (HMSN) are genetically heterogeneous disorders affecting peripheral motor and sensory functions. Many different pathogenic variants in several genes involved in the demyelinating, the axonal and the intermediate HMSN forms have been identified, for which all inheritance patterns have been described. The mutation screening currently available is based on Sanger sequencing and is time-consuming and relatively expensive due to the high number of genes involved and to the absence of mutational hot spots. To overcome these limitations, we have designed a custom panel for simultaneous sequencing of 28 HMSN-related genes. We have applied this panel to three representative patients with variable HMSN phenotype and uncertain diagnostic classifications. Using our NGS platform we rapidly identified three already described pathogenic heterozygous variants in MFN2, MPZ and DNM2 genes. Here we show that our pre-custom platform allows a fast, specific and low-cost diagnosis in sporadic HMSN cases. This prompt diagnosis is useful for providing a well-timed treatment, establishing a recurrence risk and preventing further investigations poorly tolerated by patients and expensive for the health system. Importantly, our study illustrates the utility and successful application of NGS to mutation screening of a Mendelian disorder with extreme locus heterogeneity.
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http://dx.doi.org/10.1016/j.jns.2015.09.377DOI Listing
December 2015

Imbalance of excitatory/inhibitory synaptic protein expression in iPSC-derived neurons from FOXG1(+/-) patients and in foxg1(+/-) mice.

Eur J Hum Genet 2016 06 7;24(6):871-80. Epub 2015 Oct 7.

Medical Genetics, University of Siena, Siena, Italy.

Rett syndrome (RTT) is a severe neurodevelopmental disorder associated with mutations in either MECP2, CDKL5 or FOXG1. The precise molecular mechanisms that lead to the pathogenesis of RTT have yet to be elucidated. We recently reported that expression of GluD1 (orphan glutamate receptor δ-1 subunit) is increased in iPSC-derived neurons obtained from patients with mutations in either MECP2 or CDKL5. GluD1 controls synaptic differentiation and shifts the balance between excitatory and inhibitory synapses toward the latter. Thus, an increase in GluD1 might be a critical factor in the etiology of RTT by affecting the excitatory/inhibitory balance in the developing brain. To test this hypothesis, we generated iPSC-derived neurons from FOXG1(+/-) patients. We analyzed mRNA and protein levels of GluD1 together with key markers of excitatory and inhibitory synapses in these iPSC-derived neurons and in Foxg1(+/-) mouse fetal (E11.5) and adult (P70) brains. We found strong correlation between iPSC-derived neurons and fetal mouse brains, where GluD1 and inhibitory synaptic markers (GAD67 and GABA AR-α1) were increased, whereas the levels of a number of excitatory synaptic markers (VGLUT1, GluA1, GluN1 and PSD-95) were decreased. In adult mice, GluD1 was decreased along with all GABAergic and glutamatergic markers. Our findings further the understanding of the etiology of RTT by introducing a new pathological event occurring in the brain of FOXG1(+/-) patients during embryonic development and its time-dependent shift toward a general decrease in brain synapses.
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http://dx.doi.org/10.1038/ejhg.2015.216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4820038PMC
June 2016

Epilepsy in Rett syndrome--lessons from the Rett networked database.

Epilepsia 2015 Apr 19;56(4):569-76. Epub 2015 Mar 19.

Pediatric Neurology Unit and Rett National Center, Edmond and Lily Safra Children's Hospital, Sheba Medical Center, Tel HaShomer, Israel; Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.

Objective: Rett syndrome is an X-linked dominant neurodevelopmental disorder caused by mutations in the MECP2 gene, and characterized by cognitive and communicative regression, loss of hand use, and midline hand stereotypies. Epilepsy is a core symptom, but literature is controversial regarding genotype-phenotype correlation. Analysis of data from a large cohort should overcome this shortcoming.

Methods: Data from the Rett Syndrome Networked Database on 1,248 female patients were included. Data on phenotypic and genotypic parameters, age of onset, severity of epilepsy, and type of seizures were collected. Statistical analysis was done using the IBM SPSS Version 21 software, logistic regression, and Kaplan-Meier survival curves.

Results: Epilepsy was present in 68.1% of the patients, with uncontrolled seizures in 32.6% of the patients with epilepsy. Mean age of onset of epilepsy was 4.68 ± (standard deviation) 3.5 years. Younger age of onset was correlated to severity of epilepsy (Spearman correlation r = 0.668, p < 0.01). Patients with late truncating deletions had lower prevalence of epilepsy. Compared to them, the p.R133C mutation, associated with a milder Rett phenotype, increased the risk for epilepsy (odds ratio [OR] 2.46, confidence interval [CI] 95% 1.3-4.66), but not for severe epilepsy. The p.R255X mutation conferred an increased risk for epilepsy (OR 2.07, CI 95% 1.2-3.59) as well as for severe epilepsy (OR 3.4, CI 95% 1.6-7.3). The p.T158M and p.C306C mutations relatively increased the risk for severe epilepsy (OR 3.09 and 2.69, CI 95% 1.48-6.4 and 1.19-6.05, respectively), but not for epilepsy occurrence.

Significance: Various mutations in the MECP2 gene have a different influence on epilepsy, unrelated to the severity of the general Rett phenotype. This might suggest a site-specific effect of MeCp2 on epileptic pathways. Further investigation of these mechanisms should promote better understanding of epileptogenesis in Rett syndrome.
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http://dx.doi.org/10.1111/epi.12941DOI Listing
April 2015

Next generation sequencing in sporadic retinoblastoma patients reveals somatic mosaicism.

Eur J Hum Genet 2015 Nov 25;23(11):1523-30. Epub 2015 Feb 25.

Medical Genetics, Department of Medical Biotechnologies, University of Siena, Policlinico 'Santa Maria alle Scotte', Siena, Italy.

In about 50% of sporadic cases of retinoblastoma, no constitutive RB1 mutations are detected by conventional methods. However, recent research suggests that, at least in some of these cases, there is somatic mosaicism with respect to RB1 normal and mutant alleles. The increased availability of next generation sequencing improves our ability to detect the exact percentage of patients with mosaicism. Using this technology, we re-tested a series of 40 patients with sporadic retinoblastoma: 10 of them had been previously classified as constitutional heterozygotes, whereas in 30 no RB1 mutations had been found in lymphocytes. In 3 of these 30 patients, we have now identified low-level mosaic variants, varying in frequency between 8 and 24%. In 7 out of the 10 cases previously classified as heterozygous from testing blood cells, we were able to test additional tissues (ocular tissues, urine and/or oral mucosa): in three of them, next generation sequencing has revealed mosaicism. Present results thus confirm that a significant fraction (6/40; 15%) of sporadic retinoblastoma cases are due to postzygotic events and that deep sequencing is an efficient method to unambiguously distinguish mosaics. Re-testing of retinoblastoma patients through next generation sequencing can thus provide new information that may have important implications with respect to genetic counseling and family care.
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http://dx.doi.org/10.1038/ejhg.2015.6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4613478PMC
November 2015

First identification of a triple corneal dystrophy association: keratoconus, epithelial basement membrane corneal dystrophy and fuchs' endothelial corneal dystrophy.

Case Rep Ophthalmol 2014 Sep 17;5(3):281-8. Epub 2014 Sep 17.

Medical Genetics, University of Siena, Siena, Italy.

Purpose: To report the observation of a triple corneal dystrophy association consisting of keratoconus (KC), epithelial basement membrane corneal dystrophy (EBMCD) and Fuchs' endothelial corneal dystrophy (FECD).

Methods: A 55-year-old male patient was referred to our cornea service for blurred vision and recurrent foreign body sensation. He reported bilateral recurrent corneal erosions with diurnal visual fluctuations. He underwent corneal biomicroscopy, Scheimpflug tomography, in vivo HRT confocal laser scanning microscopy and genetic testing for TGFBI and ZEB1 mutations using direct DNA sequencing.

Results: Biomicroscopic examination revealed the presence of subepithelial central and paracentral corneal opacities. The endothelium showed a bilateral flecked appearance, and the posterior corneal curvature suggested a possible concomitant ectatic disorder. Corneal tomography confirmed the presence of a stage II KC in both eyes. In vivo confocal laser scanning microscopy revealed a concomitant bilateral EBMCD with hyperreflective deposits in basal epithelial cells, subbasal Bowman's layer microfolds and ridges with truncated subbasal nerves as pseudodendritic elements. Stromal analysis revealed honeycomb edematous areas, and the endothelium showed a strawberry surface configuration typical of FECD. The genetic analysis resulted negative for TGFBI mutations and positive for a heterozygous mutation in exon 7 of the gene ZEB1.

Conclusion: This is the first case reported in the literature in which KC, EBMCD and FECD are present in the same patient and associated with ZEB1 gene mutation. The triple association was previously established by means of morphological analysis of the cornea using corneal Scheimpflug tomography and in vivo HRT II confocal laser scanning microscopy.
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http://dx.doi.org/10.1159/000367937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4209271PMC
September 2014

Coffin-Siris and Nicolaides-Baraitser syndromes are a common well recognizable cause of intellectual disability.

Brain Dev 2015 May 22;37(5):527-36. Epub 2014 Sep 22.

Medical Genetics, University of Siena, Siena, Italy; Genetica Medica, Azienda Ospedaliera Universitaria Senese, Siena, Italy.

Background: Nicolaides-Baraitser and Coffin-Siris syndromes are emerging conditions with overlapping clinical features including intellectual disability and typical somatic characteristics, especially sparse hair, low frontal hairline, large mouth with thick and everted lips, and hands and feet anomalies. Since 2012, mutations in genes encoding six proteins of the BAF complex were identified in both conditions.

Methods And Results: We have clinically evaluated a cohort of 1161 patients with intellectual disability from three different Italian centers. A strong clinical suspicion of either Nicolaides-Baraitser syndrome or Coffin-Siris syndrome was proposed in 11 cases who were then molecularly confirmed: 8 having de novo missense mutations in SMARCA2, two frame-shift mutations in ARID1B and one missense mutation in SMARCB1. Given the high frequency of the condition we set up a one-step deep sequencing test for all 6 genes of the BAF complex.

Conclusions: These results prove that the frequency of these conditions may be as high as the most common syndromes with intellectual deficit (about 1%). Clinical geneticists should be well aware of this group of disorders in the clinical setting when ascertaining patients with intellectual deficit, the specific facial features being the major diagnostic handle. Finally, this work adds information on the clinical differences of the two conditions and presents a fast and sensitive test for the molecular diagnosis.
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http://dx.doi.org/10.1016/j.braindev.2014.08.009DOI Listing
May 2015

GluD1 is a common altered player in neuronal differentiation from both MECP2-mutated and CDKL5-mutated iPS cells.

Eur J Hum Genet 2015 Feb 11;23(2):195-201. Epub 2014 Jun 11.

Medical Genetics, University of Siena, Siena, Italy.

Rett syndrome is a monogenic disease due to de novo mutations in either MECP2 or CDKL5 genes. In spite of their involvement in the same disease, a functional interaction between the two genes has not been proven. MeCP2 is a transcriptional regulator; CDKL5 encodes for a kinase protein that might be involved in the regulation of gene expression. Therefore, we hypothesized that mutations affecting the two genes may lead to similar phenotypes by dysregulating the expression of common genes. To test this hypothesis we used induced pluripotent stem (iPS) cells derived from fibroblasts of one Rett patient with a MECP2 mutation (p.Arg306Cys) and two patients with mutations in CDKL5 (p.Gln347Ter and p.Thr288Ile). Expression profiling was performed in CDKL5-mutated cells and genes of interest were confirmed by real-time RT-PCR in both CDKL5- and MECP2-mutated cells. The only major change in gene expression common to MECP2- and CDKL5-mutated cells was for GRID1, encoding for glutamate D1 receptor (GluD1), a member of the δ-family of ionotropic glutamate receptors. GluD1 does not form AMPA or NMDA glutamate receptors. It acts like an adhesion molecule by linking the postsynaptic and presynaptic compartments, preferentially inducing the inhibitory presynaptic differentiation of cortical neurons. Our results demonstrate that GRID1 expression is downregulated in both MECP2- and CDKL5-mutated iPS cells and upregulated in neuronal precursors and mature neurons. These data provide novel insights into disease pathophysiology and identify possible new targets for therapeutic treatment of Rett syndrome.
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http://dx.doi.org/10.1038/ejhg.2014.81DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172451PMC
February 2015

Interstitial 22q13 deletions not involving SHANK3 gene: a new contiguous gene syndrome.

Am J Med Genet A 2014 Jul 3;164A(7):1666-76. Epub 2014 Apr 3.

Medical Genetics, University of Siena, Siena, Italy.

Phelan-McDermid syndrome (22q13.3 deletion syndrome) is a contiguous gene disorder resulting from the deletion of the distal long arm of chromosome 22. SHANK3, a gene within the minimal critical region, is a candidate gene for the major neurological features of this syndrome. We report clinical and molecular data from a study of nine patients with overlapping interstitial deletions in 22q13 not involving SHANK3. All of these deletions overlap with the largest, but not with the smallest deletion associated with Phelan-McDermid syndrome. The deletion sizes and breakpoints varied considerably among our patients, with the largest deletion spanning 6.9 Mb and the smallest deletion spanning 2.7 Mb. Eight out of nine patients had a de novo deletion, while in one patient the origin of deletion was unknown. These patients shared clinical features common to Phelan-McDermid syndrome: developmental delay (11/12), speech delay (11/12), hypotonia (9/12), and feeding difficulties (7/12). Moreover, the majority of patients (8/12) exhibited macrocephaly. In the minimal deleted region, we identified two candidate genes, SULT4A1 and PARVB (associated with the PTEN pathway), which could be associated in our cohort with neurological features and macrocephaly/hypotonia, respectively. This study suggests that the haploinsufficiency of genes in the 22q13 region beside SHANK3 contributes to cognitive and speech development, and that these genes are involved in the phenotype associated with the larger Phelan-McDermid syndrome 22q13 deletions. Moreover, because the deletions in our patients do not involve the SHANK3 gene, we posit the existence of a new contiguous gene syndrome proximal to the smallest terminal deletions in the 22q13 region.
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http://dx.doi.org/10.1002/ajmg.a.36513DOI Listing
July 2014

Ambiguous external genitalia due to defect of 5-α-reductase in seven Iraqi patients: prevalence of a novel mutation.

Gene 2013 Sep 8;526(2):490-3. Epub 2013 May 8.

Medical Genetics, University of Siena, Siena, Italy.

We report on seven Iraqi patients with 46,XY karyotype and ambiguous genitalia characterized by perineo-scrotal hypospadias, bifid scrotum, clitoris like phallus, palpable testes in inguinal canal and pseudovagina. Patients were raised five as females and two as males. They are all unrelated with the exception of two couples of brothers. The diagnosis of 5-α-reductase-2 deficiency syndrome was first hypothesized on clinical grounds and then confirmed by molecular analysis. Direct sequencing analysis of the SRD5A2 gene revealed in five patients a novel homozygous frame-shift mutation (c.453delC) and in two related patients a previous reported missense mutation. The presence of the same mutation in unrelated patients of the same population suggests a possible founder effect. This report brings the 5-α-reductase-2 deficiency syndrome to the attention of clinical geneticists and child surgeons and discusses the appropriate clinical and surgical strategies for treating these patients.
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http://dx.doi.org/10.1016/j.gene.2013.04.070DOI Listing
September 2013

Revealing the complexity of a monogenic disease: rett syndrome exome sequencing.

PLoS One 2013 28;8(2):e56599. Epub 2013 Feb 28.

Medical Genetics, University of Siena, Siena, Italy.

Rett syndrome (OMIM#312750) is a monogenic disorder that may manifest as a large variety of phenotypes ranging from very severe to mild disease. Since there is a weak correlation between the mutation type in the Xq28 disease-gene MECP2/X-inactivation status and phenotypic variability, we used this disease as a model to unveil the complex nature of a monogenic disorder. Whole exome sequencing was used to analyze the functional portion of the genome of two pairs of sisters with Rett syndrome. Although each pair of sisters had the same MECP2 (OMIM*300005) mutation and balanced X-inactivation, one individual from each pair could not speak or walk, and had a profound intellectual deficit (classical Rett syndrome), while the other individual could speak and walk, and had a moderate intellectual disability (Zappella variant). In addition to the MECP2 mutation, each patient has a group of variants predicted to impair protein function. The classical Rett girls, but not their milder affected sisters, have an enrichment of variants in genes related to oxidative stress, muscle impairment and intellectual disability and/or autism. On the other hand, a subgroup of variants related to modulation of immune system, exclusive to the Zappella Rett patients are driving toward a milder phenotype. We demonstrate that genome analysis has the potential to identify genetic modifiers of Rett syndrome, providing insight into disease pathophysiology. Combinations of mutations that affect speaking, walking and intellectual capabilities may represent targets for new therapeutic approaches. Most importantly, we demonstrated that monogenic diseases may be more complex than previously thought.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0056599PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3585308PMC
August 2013

Antioxidant effects of potassium ascorbate with ribose in costello syndrome.

Anticancer Res 2013 Feb;33(2):691-5

Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Viale Mario Bracci 36, 53100, Siena, Italy.

Background: Costello syndrome is a rare genetic condition characterized by coarse facies, short stature, loose folds of skin especially on hands and feet, severe feeding difficulties and failure to thrive. Other features include cardiac anomalies, developmental disability and increased risk of neoplasms. Given the link between oxidative stress (OS) and carcinogenesis, we tested the hypothesis that OS occurs in this syndrome, supposing its role both in cancer development and in other clinical features.

Patients And Methods: We describe four cases with Costello syndrome in which we verified the presence of OS by measuring a redox biomarker profile including total hydroperoxides, non-protein-bound iron, advanced oxidation protein products, thyols, carbonyl groups and isoprostanes. Thus, we introduced an antioxidant agent, namely potassium ascorbate with ribose (PAR) into the therapy and monitored the redox profile every three months to verify its efficacy.

Results: A progressive decrease in OS biomarkers occurred, together with an improvement in the clinical features of the patients.

Conclusion: OS was proven in all four cases of Costello syndrome. The antioxidant therapy with PAR demonstrated positive effects. These promising results need further research to confirm the relevance of OS and the efficacy of PAR therapy in Costello syndrome.
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February 2013

Beckwith-Wiedemann syndrome: potassium ascorbate with ribose therapy in a syndrome with high neoplastic risk.

Anticancer Res 2011 Nov;31(11):3973-6

Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, Viale Mario Bracci 36, 53100 Siena, Italy.

Background: Beckwith-Wiedemann Syndrome (BWS) is a genomic imprinting disorder characterized by overgrowth and increased risk of malignancy. We studied the oxidative stress (OS) pattern of our patients with BWS and administered, for the first time, potassium ascorbate with ribose (PAR) once a day as long-term therapy in order to correct the effects induced by free radicals.

Patients And Methods: We describe the clinical features of three patients examined every three months in our clinic. OS was ascertained by measuring a panel of OS biomarkers: non-protein-binding iron, total hydroperoxides, advanced oxidation protein products, isoprostanes, carbonyl groups and thiols. After the presence of OS was established, treatment with PAR was started at the dosage of 300 mg of Potassium Bicarbonate and 150 mg of Ascorbic Acid in aqueous solution and changes occurring in OS biomarkers were followed dosing every three months.

Results: Our patients showed higher levels of OS biomarkers than controls at the time of diagnosis. There was a reduction in OS biomarker values for all three patients with treatment. No primary or secondary neoplastic disease was observed in 9 months of follow-up.

Conclusion: This is the first report showing OS occurring in BWS. No drug until this report has been published showing efficacy against OS in any cancer. Given the limited number of patients, care must be taken to mitigate enthusiasm. We are collecting data for a large number of BWS patients to confirm these preliminary results.
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November 2011