HRAS Arg68Trp mutation promotes GTP loading and activation of downstream signaling events.

<p>HEK 293T cells transfected with plasmids encoding the indicated HA-tagged Ras mutants were serum starved for 24 hr prior to lysis and detection of HA-tagged Ras, and endogenous ERK or phosphor ERK. Ras-GTP was isolated using GST-Raf-RBD-immobilized beads as detailed in Methods. Luciferase activity was measured from similarly-treated cells following cotransfection with reporter plasmids. Ras expression and ERK activation were confirmed by western blot of lysates used for luciferase activity (not shown).</p

A Novel HRAS Mutation Independently Contributes to Left Ventricular Hypertrophy in a Family with a Known MYH7 Mutation

<div><p>Several genetic conditions can lead to left ventricular hypertrophy (LVH). Among them, hypertrophic cardiomyopathy (HCM), caused by mutations in sarcomere genes, is the most common inherited cardiac disease. Instead, RASopathies, a rare class of disorders characterized by neuro-cardio-facial-cutaneous abnormalities and sometimes presenting with LVH, are caused by mutations in the RAS-MAPK pathway. We report on a 62-years-old male who presented isolated severe obstructive LVH but did not carry the sarcomere mutation previously identified in his affected relatives. By exome sequencing, we detected a novel mutation in <i>HRAS</i> gene (NM_005343.2:p.Arg68Trp), present also in the proband’s daughter, who showed mild LVH and severe intellectual disability. The cardiac phenotype was indistinguishable between family members carrying either mutation. <i>In silico</i> studies suggested that the mutated HRAS protein is constitutionally activated. Consistently, functional characterization <i>in vitro</i> confirmed elevated HRAS-GTP accumulation and downstream RAS-MAPK pathway activation that are known to drive cell proliferation in LVH. Our study emphasizes the role of RAS signaling in cardiac hypertrophy and highlights the complexity in differential diagnosis of RASopathies. In fact, the mild features of RASopathy and the recurrence of sarcomeric HCM in this family delayed the correct diagnosis until comprehensive genetic testing was performed.</p></div

Electropherograms of the <i>MYH7 and HRAS</i> mutations in the family.

<p>Sanger sequence analysis of family members reveals A) a transversion of nucleotide NM_000257.2: c.2795T>A in <i>MYH7</i> gene in brother and sister of proband (II.1 and II.2) and B) a transition NM_005343.2:c.202C>T in <i>HRAS</i> gene in proband (II.3) and in his daughter (III.2). The pedigree is also shown.</p

Binding free energy values calculated under MM/GBSA approximation.

<p>Binding free energy values calculated under MM/GBSA approximation.</p

Pedigree showing proband’s family.

<p>Squares and circles symbolize males and females, respectively. Closed symbols denote subject presenting with left ventricular hypertrophy. Slashed symbols indicate deceased subjects with unknown genotype and clinical status. The proband is indicated by an arrow. The asterisk indicates the presence of intellectual disability.</p

Structural analysis of HRAS protein.

<p>A) Schematic representation of HRAS structure, showing functional domains. Multiple regions participating in GTP binding are represented by blue pentagons. The switch I domain (red rectangle) is responsible for GAP interaction as well as some effector interactions, while the switch II domain (red oval) interacts with GEF. The green C-terminal box demarcates the hyper-variable regions and orange arrow demarcates CAAX box. B) Multiple-sequence alignment of Ras proteins among species is represented. The residue in position 68 is highlighted in yellow. C) and D) represent structural comparison of the most predominant conformation of HRAS wild-type and HRAS-Arg68Trp respectively from molecular dynamics simulations. In HRAS wild-type (B) the water molecule between the Arg68 and Gly60 is highlighted. In the mutant protein model (C) water molecule is weakly present.</p

Not Only Diagnostic Yield: Whole-Exome Sequencing in Infantile Cardiomyopathies Impacts on Clinical and Family Management

Whole-exome sequencing (WES) is a powerful and comprehensive tool for the genetic diagnosis of rare diseases, but few reports describe its timely application and clinical impact on infantile cardiomyopathies (CM). We conducted a retrospective analysis of patients with infantile CMs who had trio (proband and parents)-WES to determine whether results contributed to clinical management in urgent and non-urgent settings. Twenty-nine out of 42 enrolled patients (69.0%) received a definitive molecular diagnosis. The mean time-to-diagnosis was 9.7 days in urgent settings, and 17 out of 24 patients (70.8%) obtained an etiological classification. In non-urgent settings, the mean time-to-diagnosis was 225 days, and 12 out of 18 patients (66.7%) had a molecular diagnosis. In 37 out of 42 patients (88.1%), the genetic findings contributed to clinical management, including heart transplantation, palliative care, or medical treatment, independent of the patient’s critical condition. All 29 patients and families with a definitive diagnosis received specific counseling about recurrence risk, and in seven (24.1%) cases, the result facilitated diagnosis in parents or siblings. In conclusion, genetic diagnosis significantly contributes to patients’ clinical and family management, and trio-WES should be performed promptly to be an essential part of care in infantile cardiomyopathy, maximizing its clinical utility