Family pedigree and mutation identification.

<p>(A) Electropherogram of the <i>LMNA</i> gene variant. (B) The 7 duplicated amino acids (LLNSKEA) are highly conserved among <i>LMNA</i> gene homologs in vertebrates. (C) Family pedigree of the index patient with the novel <i>LMNA</i> gene mutation.</p

Clinical characteristics of family members according to <i>LMNA</i> mutation carrier status.

<p>Mean values ± standard deviation or absolute frequencies and percentage of patients.</p><p>AF = atrial fibrillation; AVB = atrioventricular block; BMI = body mass index; CMRI = cardiac magnetic resonance imaging; DBP = diastolic blood pressure; Echo = echocardiography; LGE = late gadolinium enhancement; LVEDD = left ventricular end-diastolic diameter; LVEF = left ventricular ejection fraction; NSVT = non-sustained ventricular tachycardia; PVCs = premature ventricular complexes; RVEF = right ventricular ejection fraction; RVOT = right ventricular outflow tract; SBP = systolic blood pressure; SVT = sustained ventricular tachycardia.</p><p>*RVOT value by echocardiography was available in 19 of 20 patients, 9 of whom were <i>LMNA</i> mutation-positive subjects.</p><p>†CMRI was performed in 15 subjects, 7 of whom were <i>LMNA</i> mutation carriers.</p><p>Clinical characteristics of family members according to <i>LMNA</i> mutation carrier status.</p

Clinical characteristics of index patient and <i>LMNA</i> mutation-positive family members.

<p>Index patient’s electrocardiogram (ECG) showing, at clinical presentation, (A) sinus rhythm, first-degree AV block, and PVC of LBBB morphology and, 13 years later, (B) complete AV block. (C) Asystole documented on implantable loop recorder memory (subject III-8). (D) Sustained VT detected on telemetry monitoring, effectively terminated by internal ICD shock (subject III-1). (E) Episodes of non-sustained VT with LBBB morphology and inferior axis (subject III-5) on 12-lead Holter monitoring. (E) Episodes of non-sustained VT with RBBB morphology and superior axis (subject IV-2) on 12-lead Holter monitoring.</p

CMR imaging of <i>LMNA</i> mutation-positive family members.

<p>(A) Short-axis cine (a) and LGE sequences in the Short-axis (b) and 2-chambers long-axis views (c). Bulging (arrow in a) and LGE of the RV free wall (arrowheads in b). Linear midwall LGE is localized at the interventricular septum and LV inferior wall (arrowheads in c) (subject III-5). (B) and (C) LGE sequences in the short axis (a) and 4-chambers long axis views (b). LGE with linear midwall pattern is shown on the LV inferior wall and basal interventricular septum (arrowheads) (subjects III-8, and IV-2).</p

Analysis of nuclear envelope integrity under stressing condition in LMNA transfected HL-1 cells.

<p>Nuclear WT LMNA (A) and DUP LMNA (B) signals in control (CTR) and stressing conditions (Hyperosmolarity, Hypoxia, Oxidative stress). The merged signals of LMNA proteins and the RFP nuclear marker are shown in the insets. Confocal XY planar projections are depicted in each experimental condition.</p

Clinical characteristics of <i>LMNA</i> mutation-positive family members.

<p>AF = atrial fibrillation; ARVC = arrhythmogenic right ventricular cardiomyopathy; AVB = atrioventricular block; CMR = cardiac magnetic resonance imaging; Com = comments; Echo = echocardiography; EF = ejection fraction; F = female; FU = Follow-up; Gen = Gender; HT = heart transplantation; ICD = implantable cardioverter-defibrillator; ILR = implantable loop recorder; LBBB = left bundle-branch block; LGE = late gadolinium enhancement; LV = left ventricle; LVEDD = left ventricular end-diastolic diameter; M = male; m = minor; Mj = major; np = not performed; NSVT = non-sustained ventricular tachycardia; PM = pacemaker; PVCs = premature ventricular complexes; RBBB = right bundle-branch block; RV = right ventricle; RVOT = right ventricular outflow tract; SB = sinus bradycardia; Sub = Subject; SVT = sustained ventricular tachycardia; TFC = Task Force criteria.</p><p>*at diagnosis/clinical presentation (years);</p><p>**Morphology of NSVT was determinable in 4 of 6 <i>LMNA</i> mutation-positive family members having NSVT;</p><p>***for ARVC diagnosis Mj/m.</p><p>Clinical characteristics of <i>LMNA</i> mutation-positive family members.</p

Apoptosis assay in LMNA transfected HL-1 cells.

<p>(A) Representative XY confocal planar projections of LMNA transfected cells (green) labelled with EthD-1 (red) in control conditions are depicted. (B) Quantitative analysis of apoptotic cells in control and under hyperosmotic (Hyper), hypoxic (Hypoxia), and oxidative (H₂O₂) conditions. Data are reported as % of apoptotic cells (double-labelled cells) in overall LMNA-expressing cells (green labelled cells). Statistical analysis was performed on 3 independent experiments and significance calculated by Student’s T-test for unpaired samples. *<i>P</i>< 0.0002 is relative to CTR vs. stressing conditions in WT LMNA expressing cells and **<i>P</i> < 0.0001 is relative to CTR vs. stressing conditions in DUP LMNA expressing cells.</p

Immunofluorescence confocal analysis of LMNA transfected HL-1 cells.

<p>Cells transfected with both LMNA WT and DUP are depicted. LMNA is visualized in green, Nuclear Pores in red, and colocalization in yellow in the merge panels. In the insets, a merged image of LMNA and Phalloidin-TRITC is shown. Planar XY projections were depicted in each experimental condition.</p

Molecular and Functional Characterization of Three Different Postzygotic Mutations in <i>PIK3CA</i>-Related Overgrowth Spectrum (PROS) Patients: Effects on PI3K/AKT/mTOR Signaling and Sensitivity to PIK3 Inhibitors

<div><p>Background</p><p><i>PIK3CA</i>-related overgrowth spectrum (PROS) include a group of disorders that affect only the terminal portion of a limb, such as type I macrodactyly, and conditions like fibroadipose overgrowth (FAO), megalencephaly-capillary malformation (MCAP) syndrome, congenital lipomatous asymmetric overgrowth of the trunk, lymphatic, capillary, venous, and combined-type vascular malformations, epidermal nevi, skeletal and spinal anomalies (CLOVES) syndrome and Hemihyperplasia Multiple Lipomatosis (HHML). Heterozygous postzygotic <i>PIK3CA</i> mutations are frequently identified in these syndromes, while timing and tissue specificity of the mutational event are likely responsible for the extreme phenotypic variability observed.</p><p>Methods</p><p>We carried out a combination of Sanger sequencing and targeted deep sequencing of genes involved in the PI3K/AKT/mTOR pathway in three patients (1 MCAP and 2 FAO) to identify causative mutations, and performed immunoblot analyses to assay the phosphorylation status of AKT and P70S6K in affected dermal fibroblasts. In addition, we evaluated their ability to grow in the absence of serum and their response to the PI3K inhibitors wortmannin and LY294002 <i>in vitro</i>.</p><p>Results and Conclusion</p><p>Our data indicate that patients’ cells showed constitutive activation of the PI3K/Akt pathway. Of note, PI3K pharmacological blockade resulted in a significant reduction of the proliferation rate in culture, suggesting that inhibition of PI3K might prove beneficial in future therapies for PROS patients.</p></div

Clinical and mutational spectrum of the three index cases.

<p><b>a</b> Patient 1, clinically diagnosed with MCAP, showing diffuse capillary malformation at the age of 2 months and cutaneous syndactyly between the 2^nd and 3^rd toes. The <i>PIK3CA</i> c.241 G>A [p.E81K] mutation detected by Sanger sequencing in affected cells and tissues of patient 1 showed varying levels of the mutant allele depending on the tissue tested. The mutation was absent in the patient's blood and in her parents. <b>b</b> Macrodactyly of the right 4^th finger in patient 2, diagnosed with FAO, at the age of 17 years. Sequence of <i>PIK3CA</i> exon 20 in blood and cultured fibroblasts obtained from patient 2 showing that the mutation is undetectable in these samples. <b>c</b> Patient 3, at the age of 15 months before surgical intervention; note the disproportion of the left 2^nd and 3^rd fingers and the subcutaneous mass at the left deltoid region. Sanger sequencing validation of the c.3140 A>T [p.H1047L] mutation detected with targeted deep sequencing in the biopsy from the 2^nd finger of patient 3. <b>d</b> List of samples and mutations detected with targeted deep sequencing. Coverage indicates the mean average of reads on target in the regions of interest (ROI) while frequency denotes the percentage of reads with the mutation.</p