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1.  The Drop That Spilled the Cup: Acute Myocardial Infarction in a Young Woman with Underlying Thrombophilic Polymorphisms and Oral Contraceptive Use 
Case Reports in Cardiology  2014;2014:249715.
We present the case of a 28-year-old woman who was admitted to our cardiology unit for acute coronary syndrome. Her history was notable for cardiovascular disease familiarity, active smoking, and oral contraceptive use. On further analysis, she was noted to have thrombophilic polymorphisms involving the plasminogen activator inhibitor (PAI), angiotensin-converting enzyme (ACE), and methylenetetrahydrofolate reductase (MTHFR) genes. We discuss the implications that these cofactors may have had in the genesis of the disease.
PMCID: PMC4284985  PMID: 25587456
2.  Quantitative DNA methylation analysis improves epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome 
Epigenetics  2013;8(10):1053-1060.
Beckwith-Wiedemann syndrome (BWS) is a rare disorder characterized by overgrowth and predisposition to embryonal tumors. BWS is caused by various epigenetic and/or genetic alterations that dysregulate the imprinted genes on chromosome region 11p15.5. Molecular analysis is required to reinforce the clinical diagnosis of BWS and to identify BWS patients with cancer susceptibility. This is particularly crucial prenatally because most signs of BWS cannot be recognized in utero. We established a reliable molecular assay by pyrosequencing to quantitatively evaluate the methylation profiles of ICR1 and ICR2. We explored epigenotype-phenotype correlations in 19 patients that fulfilled the clinical diagnostic criteria for BWS, 22 patients with suspected BWS, and three fetuses with omphalocele. Abnormal methylation was observed in one prenatal case and 19 postnatal cases, including seven suspected BWS. Seven cases showed ICR1 hypermethylation, five cases showed ICR2 hypomethylation, and eight cases showed abnormal methylation of ICR1 and ICR2 indicating paternal uniparental disomy (UPD). More cases of ICR1 alterations and UPD were found than expected. This is likely due to the sensitivity of this approach, which can detect slight deviations in methylation from normal levels. There was a significant correlation (p < 0.001) between the percentage of ICR1 methylation and BWS features: severe hypermethylation (range: 75–86%) was associated with macroglossia, macrosomia, and visceromegaly, whereas mild hypermethylation (range: 55–59%) was associated with umbilical hernia and diastasis recti. Evaluation of ICR1 and ICR2 methylation by pyrosequencing in BWS can improve epigenotype-phenotype correlations, detection of methylation alterations in suspected cases, and identification of UPD.
PMCID: PMC3891686  PMID: 23917791
BWS; genomic imprinting; DNA methylation; pyrosequencing; UPD
3.  Intragenic and large NIPBL rearrangements revealed by MLPA in Cornelia de Lange patients 
Cornelia de Lange syndrome (CdLS) is a rare multisystemic congenital anomaly disorder that is characterised by intellectual disability and growth retardation, congenital heart defects, intestinal anomalies, facial dysmorphism (including synophyris and high arched eyebrows) and limb reduction defects. Mutations in three cohesin-associated genes encoding a key regulator (NIPBL, chr 5p13.2) and one structural component of the cohesin ring (SMC1A, chr Xp11) occur in about 65% of CdLS patients. NIPBL is the major causative gene, and accounts for 40–60% of CdLS patients as shown by a number of mutational screening studies that indicate a wide mutational repertoire of mainly small deletions and point mutations. Only a few data are available concerning the occurrence of large NIPBL rearrangements or intragenic deletions or duplications involving whole exons. We used multiplex ligation-dependent probe amplification (MLPA) to study 132 CdLS patients negative to the standard mutation NIPBL test out of a cohort of 200 CdLS patients. A total of 7 out of 132 patients were found to carry NIPBL alterations, including two large gene deletions extending beyond the gene, four intragenic multi- or single-exon deletions and one single-exon duplication. These findings show that MLPA leads to a 5.3% increase in the detection of mutations when used in addition to the standard NIPBL scan, and contributes per se to the molecular diagnosis of 3.5% (7/200) of clinically diagnosed CdLS patients. It is recommended that MLPA be included in the CdLS diagnostic flow chart.
PMCID: PMC3376273  PMID: 22353942
Cornelia de Lange; NIPBL; MLPA; intragenic deletion/duplication; large rearrangements; transcript analysis
4.  Genomic imbalances in patients with a clinical presentation in the spectrum of Cornelia de Lange syndrome 
BMC Medical Genetics  2013;14:41.
Cornelia de Lange syndrome (CdLS) is a rare autosomal-dominant disorder characterised by facial dysmorphism, growth and psychomotor developmental delay and skeletal defects. To date, causative mutations in the NIPBL (cohesin regulator) and SMC1A (cohesin structural subunit) genes account for > 50% and 6% of cases, respectively.
We recruited 50 patients with a CdLS clinical diagnosis or with features that overlap with CdLS, who were negative for mutations at NIPBL and SMC1A at molecular screening. Chromosomal rearrangements accounting for the clinical diagnosis were screened for using array Comparative Genomic Hybridisation (aCGH).
Four patients were shown to carry imbalances considered to be candidates for having pathogenic roles in their clinical phenotypes: patient 1 had a 4.2 Mb de novo deletion at chromosome 20q11.2-q12; patient 2 had a 4.8 Mb deletion at chromosome 1p36.23-36.22; patient 3 carried an unbalanced translocation, t(7;17), with a 14 Mb duplication of chromosome 17q24.2-25.3 and a 769 Kb deletion at chromosome 7p22.3; patient 4 had an 880 Kb duplication of chromosome 19p13.3, for which his mother, who had a mild phenotype, was also shown to be a mosaic.
Notwithstanding the variability in size and gene content of the rearrangements comprising the four different imbalances, they all map to regions containing genes encoding factors involved in cell cycle progression or genome stability. These functional similarities, also exhibited by the known CdLS genes, may explain the phenotypic overlap between the patients included in this study and CdLS. Our findings point to the complexity of the clinical diagnosis of CdLS and confirm the existence of phenocopies, caused by imbalances affecting multiple genomic regions, comprising 8% of patients included in this study, who did not have mutations at NIPBL and SMC1A. Our results suggests that analysis by aCGH should be recommended for CdLS spectrum cases with an unexplained clinical phenotype and included in the flow chart for diagnosis of cases with a clinical evaluation in the CdLS spectrum.
PMCID: PMC3626829  PMID: 23551878
5.  The Steady-State Serum Concentration of Genistein Aglycone Is Affected by Formulation: A Bioequivalence Study of Bone Products 
BioMed Research International  2012;2013:273498.
An FDA-regulated, prescription medical food (Fosteum; 27 mg natural genistein, 200 IU cholecalciferol, 20 mg citrated zinc bisglycinate (4 mg elemental zinc) per capsule) and an over-the-counter (OTC) supplement (Citracal Plus Bone Density Builder; 27 mg synthetic genistein, 600 mg elemental calcium (calcium citrate), 400 IU vitamin D3, 50 mg magnesium, 7.5 mg zinc, 1 mg copper, 75 μg molybdenum, 250 μg boron per two tablets) were compared to a clinically proven bone formulation (27 mg natural genistein, 400 IU cholecalciferol, 500 mg elemental calcium (calcium carbonate) per tablet; the Squadrito formulation) in an 8-day steady-state pharmacokinetic (PK) study of healthy postmenopausal women (n = 30) randomized to receive 54 mg of genistein per day. Trough serum samples were obtained before the final dose on the morning of the ninth day followed by sampling at 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 72, and 96 hrs. Total serum genistein, after β-glucuronidase/sulfatase digestion, was measured by time-resolved fluorometric assay. Maximal time (Tmax), concentration (Cmax), half-life (T1/2), and area under the curve (AUC) were determined for genistein in each formulation. Fosteum and the Squadrito study formulation were equivalent for genistein Tmax (2 hrs), Cmax (0.7 μM), T1/2 (18 ± 6.9 versus 21 ± 4.9 hrs), and AUC (9221 ± 413 versus 9818 ± 1370 ng·hr/mL). The OTC supplement's synthetically derived genistein, however, showed altered Tmax (6 hrs), Cmax (0.57 μM), T1/2 (8.3 ± 1.9 hrs), and AUC (6474 ± 287 ng·hr/mL). Differences in uptake may be due to multiple ingredients in the OTC supplement which interfere with genistein absorption.
PMCID: PMC3591111  PMID: 23484100
6.  Juxtaposition of heterochromatic and euchromatic regions by chromosomal translocation mediates a heterochromatic long-range position effect associated with a severe neurological phenotype 
The term "position effect" is used when the expression of a gene is deleteriously affected by an alteration in its chromosomal environment even though the integrity of the protein coding sequences is maintained. We describe a patient affected by epilepsy and severe neurodevelopment delay carrying a balanced translocation t(15;16)(p11.2;q12.1)dn that we assume caused a position effect as a result of the accidental juxtaposition of heterochromatin in the euchromatic region.
FISH mapped the translocation breakpoints (bkps) to 15p11.2 within satellite III and the 16q12.1 euchromatic band within the ITFG1 gene. The expression of the genes located on both sides of the translocation were tested by means of real-time PCR and three, all located on der(16), were found to be variously perturbed: the euchromatic gene NETO2/BTCL2 was silenced, whereas VPS35 and SHCBP1, located within the major heterochromatic block of chromosome 16q11.2, were over-expressed. Pyrosequencing and chromatin immunoprecipitation of NETO2/BTCL2 and VPS35 confirmed the expression findings. Interphase FISH analysis showed that der(16) localised to regions occupied by the beta satellite heterochromatic blocks more frequently than der(15).
To the best of our knowledge, this is the first report of a heterochromatic position effect in humans caused by the juxtaposition of euchromatin/heterochromatin as a result of chromosomal rearrangement. The overall results are fully in keeping with the observations in Drosophila and suggest the occurrence of a human heterochromatin position effect associated with the nuclear repositioning of the der(16) and its causative role in the patient's syndromic phenotype.
PMCID: PMC3395859  PMID: 22475481
Balanced translocation; Heterochromatin; Position effect; Gene expression perturbation; Epigenetic modification
7.  The KCNQ1OT1 imprinting control region and non-coding RNA: new properties derived from the study of Beckwith–Wiedemann syndrome and Silver–Russell syndrome cases 
Human Molecular Genetics  2011;21(1):10-25.
A cluster of imprinted genes at chromosome 11p15.5 is associated with the growth disorders, Silver–Russell syndrome (SRS) and Beckwith–Wiedemann syndrome (BWS). The cluster is divided into two domains with independent imprinting control regions (ICRs). We describe two maternal 11p15.5 microduplications with contrasting phenotypes. The first is an inverted and in cis duplication of the entire 11p15.5 cluster associated with the maintenance of genomic imprinting and with the SRS phenotype. The second is a 160 kb duplication also inverted and in cis, but resulting in the imprinting alteration of the centromeric domain. It includes the centromeric ICR (ICR2) and the most 5′ 20 kb of the non-coding KCNQ1OT1 gene. Its maternal transmission is associated with ICR2 hypomethylation and the BWS phenotype. By excluding epigenetic mosaicism, cell clones analysis indicated that the two closely located ICR2 sequences resulting from the 160 kb duplication carried discordant DNA methylation on the maternal chromosome and supported the hypothesis that the ICR2 sequence is not sufficient for establishing imprinted methylation and some other property, possibly orientation-dependent, is needed. Furthermore, the 1.2 Mb duplication demonstrated that all features are present for correct imprinting at ICR2 when this is duplicated and inverted within the entire cluster. In the individuals maternally inheriting the 160 kb duplication, ICR2 hypomethylation led to the expression of a truncated KCNQ1OT1 transcript and to down-regulation of CDKN1C. We demonstrated by chromatin RNA immunopurification that the KCNQ1OT1 RNA interacts with chromatin through its most 5′ 20 kb sequence, providing a mechanism likely mediating the silencing activity of this long non-coding RNA.
PMCID: PMC3235007  PMID: 21920939
8.  Genetic investigations on 8 patients affected by ring 20 chromosome syndrome 
BMC Medical Genetics  2010;11:146.
Mosaic Chromosome 20 ring [r(20)] is a chromosomal disorder associated with a rare syndrome characterized by a typical seizure phenotype, a particular electroclinical pattern, cognitive impairment, behavioural problems and absence of a consistent pattern of dysmorphology. The pathogenic mechanism underlying seizures disorders in r(20) syndrome is still unknown. We performed a detailed clinical and genetic study on 8 patients with r(20) chromosome, aimed at detecting the genetic mechanism underlying r(20) syndrome.
We submitted 8 subjects with a previous diagnosis of ring 20 chromosome mosaicism to a clinical re-evaluation, followed by cytogenetic, FISH, array-CGH and molecular analyses. The genetic study was also extended to their available parents.
FISH and array-CGH experiments indicate that cryptic deletions on chromosome 20 are not the cause of the r(20) chromosome associated disease. Moreover, no evidence of chromosome 20 uniparental disomy was found. Analysis of FISH signals given by variant in size alphoid tandem repeats probes on the normal chromosome 20 and the r(20) chromosome in the mosaic carriers suggests that the r(20) chromosome is the same chromosome not circularized in the "normal" cell line.
Higher percentages of r(20) chromosome cells were observed to be related with precocious age at seizure onset and with resistance to antiepileptic drug treatment. Behavioural problems also seem to be associated with higher percentages of r(20) chromosome cells. Our results suggest that an epigenetic mechanism perturbing the expression of genes close to the telomeric regions, rather than deletion of genes located at the distal 20p and/or 20q regions, may underlie the manifestation of r(20) syndrome.
PMCID: PMC2967536  PMID: 20939888
9.  Hypomethylation at multiple maternally methylated imprinted regions including PLAGL1 and GNAS loci in Beckwith–Wiedemann syndrome 
Genomic imprinting is an epigenetic phenomenon restricting gene expression in a manner dependent on parent of origin. Imprinted gene products are critical regulators of growth and development, and imprinting disorders are associated with both genetic and epigenetic mutations, including disruption of DNA methylation within the imprinting control regions (ICRs) of these genes. It was recently reported that some patients with imprinting disorders have a more generalised imprinting defect, with hypomethylation at a range of maternally methylated ICRs. We report a cohort of 149 patients with a clinical diagnosis of Beckwith–Wiedemann syndrome (BWS), including 81 with maternal hypomethylation of the KCNQ1OT1 ICR. Methylation analysis of 11 ICRs in these patients showed that hypomethylation affecting multiple imprinted loci was restricted to 17 patients with hypomethylation of the KCNQ1OT1 ICR, and involved only maternally methylated loci. Both partial and complete hypomethylation was demonstrated in these cases, suggesting a possible postzygotic origin of a mosaic imprinting error. Some ICRs, including the PLAGL1 and GNAS/NESPAS ICRs implicated in the aetiology of transient neonatal diabetes and pseudohypoparathyroidism type 1b, respectively, were more frequently affected than others. Although we did not find any evidence for mutation of the candidate gene DNMT3L, these results support the hypotheses that trans-acting factors affect the somatic maintenance of imprinting at multiple maternally methylated loci and that the clinical presentation of these complex cases may reflect the loci and tissues affected with the epigenetic abnormalities.
PMCID: PMC2986258  PMID: 19092779
Beckwith-Wiedemann syndrome (BWS); KCNQ1OT1; methylation; imprinting; imprinting disorder; hypomethylation of imprinted loci (HIL)
10.  Molecular analysis using DHPLC of cystic fibrosis: increase of the mutation detection rate among the affected population in Central Italy 
Cystic fibrosis (CF) is a multisystem disorder characterised by mutations of the CFTR gene, which encodes for an important component in the coordination of electrolyte movement across of epithelial cell membranes. Symptoms are pulmonary disease, pancreatic exocrine insufficiency, male infertility and elevated sweat concentrations. The CFTR gene has numerous mutations (>1000) and functionally important polymorphisms (>200). Early identification is important to provide appropriate therapeutic interventions, prognostic and genetic counselling and to ensure access to specialised medical services. However, molecular diagnosis by direct mutation screening has proved difficult in certain ethnic groups due to allelic heterogeneity and variable frequency of causative mutations.
We applied a gene scanning approach using DHPLC system for analysing specifically all CFTR exons and characterise sequence variations in a subgroup of CF Italian patients from the Lazio region (Central Italy) characterised by an extensive allelic heterogeneity.
We have identified a total of 36 different mutations representing 88% of the CF chromosomes. Among these are two novel CFTR mutations, including one missense (H199R) and one microdeletion (4167delCTAAGCC).
Using this approach, we were able to increase our standard power rate of mutation detection of about 11% (77% vs. 88%).
PMCID: PMC419352  PMID: 15084222
Cystic fibrosis; CFTR mutation screening; DHPLC

Results 1-10 (10)