Case 1
Patient 1 (P1) was diagnosed with CMP prenatally at 24 weeks of gestation, when the mother (a 38-year-old G8P3 gravida 8 para 3, Egyptian woman) was referred for polyhydramnios, hydrops and severely reduced fetal biventricular systolic function and left ventricular (LV) dilation. Family history revealed parents to be 3rd -degree cousins whereas obstetric history was remarkable for two first-trimester miscarriages and two intrauterine deaths in the late second trimester; the couple also had three healthy children currently in their teens and in good health.
Prenatal ultrasound examination found no evidence of cardiac or extracardiac structural anomalies or sustained arrhythmia to explain myocardial dysfunction; screening for congenital infections (TORCH, Parvovirus, Adenovirus, Coxsackie) was negative except for SARS-CoV-2 antibodies from an asymptomatic infection. Karyotyping and array-CGH were both normal. The child was born at 34 + 4 weeks of GA by urgent cesarean section, weighing 3240 grams (99th percentile). Post-natal echocardiography confirmed the absence of structural heart disease and detected biventricular hypertrophy and LV dilation. LV end-diastolic diameter measured 24 mm (+ 3 z-score) (Fig. 1). The interventricular septum had a peculiar aspect appearing thin, almost membranous and akinetic in the proximal two thirds. Moreover, there was a severe LV dysfunction, as only the apical regions of the cardiac walls were contractile, with a biplane (Simpson) ejection fraction (LVEF) of 26% (Fig. 2). Both atrioventricular valves showed thickened leaflets with preserved mobility and severe regurgitation. No pericardial or pleural effusion were present.
The neonate was started on furosemide and captopril and, because of recurrent episodes of poorly tolerated rapid atrial ectopic tachycardia, on amiodarone. He was initially supported by nasal CPAP and then, upon discharge, he weaned to heated high flow nasal cannula but could not be weaned to spontaneous ventilation.
Postnatally, with a confirmed diagnosis of isolated CMP, metabolic screening was performed and was negative.
During the four months of hospitalization, the infant remained in good clinical conditions, echocardiography showed stable cardiac function (LVEF 25–30%) and dimensions. Medical therapy was continued and increased up to the dosage of captopril 0,5mg/kg every 6 hours and furosemide 1mg/kg every 8 hours. Full enteral feeding was reached at three weeks of life, tube feeding was occasionally maintained to prevent the baby’s fatigue. Patient’s growth was regular and satisfactory.
To exclude skeletal muscular involvement, electromyography was undertaken and resulted normal. Diaphragm and intercostal muscles were studied with noninvasive electromyography at different level of noninvasive respiratory support (10L, 5L high flow nasal cannula) and in spontaneous breathing and was also normal.
After discharge, at ten months of life, echocardiogram showed improved ventricular function with LVEF 35%-40%. He is currently twenty-four months old, he is in good clinical conditions, weaned from respiratory high flow nasal cannula support. Ventricular function at echocardiogram is stable, with good adherence and tolerability to medical therapy. Psychomotor development has been evaluated as adequate.
A trio-based whole exome sequencing (WES) performed was undertaken for the patient and both parents [13]. Briefly, the exonic and flanking splice junctions’ regions of the genome were captured using the Clinical Research Exome v.2 kit (Agilent Technologies, Santa Clara, CA). Sequencing was performed on a NextSeq500 Illumina system with 150bp paired-end reads. Reads were aligned to human genome build GRCh37/UCSC hg19 and analyzed for sequence variants using a custom-developed analysis tool. On average, coverage on target was ≥ 10X for 98% with a mean coverage of 106X. Trio-based WES identified in the proband the homozygous variant c.1156dup (p.Met386fs) in the NEXN gene (NM_144573.4); both parents were confirmed to be carriers of the same variant at the heterozygous state. To our knowledge, this NEXN variant c.1156dup has never been reported previously and is absent from population database (GnomAD). According to the American College of Medical Genetics and Genomics (ACMG) criteria [14], the variant can be interpreted as likely pathogenic (class 4).
Since the few NEXN-heterozygous CMP cases reported in adults developed DCM, HCM or overlapping forms, complete cardiac evaluation was offered to all first-degree relatives. Parents, both heterozygous for the c.1156dup variant, denied personal history of cardiac disease and symptoms of coronary artery disease (CAD) or HF; paternal grandmother died suddenly at the age of 67 for unknown reasons, and a cousin of the mother suffered Sudden Cardiac Death (SCD) at the age of 47.
The mother, a 38-year-old woman, shows early signs of CMP, consistent with the diagnosis of possible Hypokinetic non-dilated Cardiomyopathy, already described in a relative of a patient with DCM [9]: Left bundle branch block (LBBB) with consequent ventricular dyssynchrony and mildly reduced LVEF (53%). Moreover, TTE (TransThoracic Echocardiography) showed mild tricuspid valve regurgitation allowing estimation of a mildly elevated systolic Pulmonary Artery Pressure (PAPs 35 mmHg). CMR (Cardiac Magnetic Resonance) findings confirmed a mild reduction of left ventricular systolic function (LVEF 51%), with normal left ventricular volume indexed for BSA (EDV 71 ml/mq) without evidence of Late Gadolinium Enhancement (LGE) or edema, and with normal T1 mapping values.
The only cardiac remarkable finding for the father, a man aged 45, is mild LV hypertrophy at TTE. CMR showed normal function (LVEF 57%) and morphology of the left ventricle (EDV 82 ml/mq), with no evidence of edema or fibrosis excepted for a spot of aspecific junctional LGE spot, and normal T1 mapping values. Since left ventricular hypertrophy is common in arterial hypertension, we can conclude that the patient’s father doesn’t reach the criteria for possible cardiomyopathy at this time of follow-up. All three asymptomatic siblings aged 13, 15 and 17 years had normal ECG and echocardiograms. Therefore, also considering the absence of affected relatives before adulthood, they have not been tested yet for the familial variant.
Case 2
Patient 2 (P2) was prenatally diagnosed with DCM at third trimester of pregnancy. Family history was negative for cardiovascular diseases or recurrent miscarriages. His Italian healthy parents had a remote degree of consanguinity (they both come from a small town in south of Italy). Prenatal karyotype performed on amniotic fluid revealed a paternally inherited supernumerary marker chromosome that was also present in the healthy sister.
The patient was born full term by cesarean section. Birth weight was 3600 g (50th-75th percentile), length 51 cm (25th-50th percentile) and OFC 35 cm (50th percentile). Medical treatment for HF was started since birth including diuretics and ACE inhibitors. The patient was referred to our center at the age of 4 years old. Ross functional class was II-III. Echocardiography showed moderate to severe LV dilatation and dysfunction (LVEF 31%), apical LV hypertrabeculation, moderate left atrial dilatation (Vol. 35ml; 54ml/m2) and moderate mitral valve regurgitation with tethering of posterior leaflets and focal thickening of the distal part of both mitral leaflets. Medical treatment was modified by adding carvedilol, spironolactone and acetyl salicylic acid. Due to persistent severe LV dysfunction, carvedilol was progressively increased since the age of 5 years old. At the age of 6 years, LVEF was increased to 38%. The patient has always shown good adherence and tolerance to therapy. At 8 years old, CMR showed moderate LV dilatation and dysfunction, EDV (end-diastolic volume) 104 ml (indexed 117 ml/m²), ESV (end-systolic volume) 63 ml, systolic ejection 40ml, FE 39%, mild to moderate mitral insufficiency, normal right ventricular function and dimension. At the age of 10 years, LVEF was 35%, medical therapy was supplemented with ivabradine that was well tolerated. At the following years, there was progressive increase in LVEF reaching 40–45%. Holter ECG monitoring was always normal. He was never admitted for heart failure. He is in good and stable functional class NYHA I-II at age 15 years.
In addition, the child showed moderate developmental delay and behavioral disturbances.
Next generation sequencing (NGS) analysis was performed, on genomic DNA trios by using the Twist Custom Panel (clinical exome Twist Bioscience) on NovaSeq6000 platform (Illumina, San Diego, CA, USA). The reads were aligned to human genome build GRCh37/UCSC hg19. The Dragen Enrichment application of BaseSpace (Illumina) and Geneyx Analysis (knowledge-driven NGS Analysis tool powered by the GeneCards Suite) were used for the variant calling and annotation. The coverage on target region was ≥ 10X for 99.4% with a mean coverage of 217.84X. Trio analysis identified the homozygous variant c.1579_1584del p. (Glu527_Glu528del) in NEXN; both parents were heterozygous. The variant was evaluated by VarSome (Kopanos et al., Bioinformatics 2018) and classified according to the American College of Medical Genetics and Genomics criteria [14] as variant of uncertain significance (class3). The Chromosomal Microarray Analysis was performed using the Infinium CytoSNP-850K BeadChip (SNP-array, Illumina, San Diego, CA, USA), which identified a heterozygous maternal duplication arr[hg19] 12q21.33 (89,683,180 − 89,935,072) x3 of about 251 kb, included (Online Mendelian Inheritance in Man) Disease Causing DUSP6 e POC1B and classified as of uncertain significance.
As for P1, following the diagnosis, parents underwent cardiac screening and follow-up (every 2–3 years) with normal ECG and echocardiography.