Keeping Up the Beat of Kleefstra Syndrome

Heart development represents a complex and tightly regulated process, which errors give rise to the most frequent malformations among newborns [1]. Emerging evidence suggests that chromatin state patterns, together with structural heart defects and early embryonic events might play a role in cardiac arrhythmias etiology [2]. Epigenetic machinery critical role during human development is also pivotal for heart development [3]. Long QT Syndrome (LQTS) represents a rare but potentially life-threatening arrhythmia characterized by delayed repolarization on Electrocardiogram (EKG) evaluation. Although LQTS is primarily related to defects in ion channel activity, the genetic cause of this condition remains unknown in approximately 20% of cases [4].

Methylation represents a major epigenetic mechanism associated with gene silencing and dependent on the activity of several proteins [5]. The impairment of Euchromatic Histone Methyltransferase 1 (EHMT1, MIM #607001, on chromosome 9q34.3) results in Kleefstra syndrome (KS, MIM #610253), one condition in the complex and overlapping spectrum of Mendelian disorders of epigenetic machinery (MDEMs). KS is a rare neurodevelopmental disorder, characterized by moderate-to-severe intellectual disability, hypotonia, autisticlike features and, often, distinctive facial traits. Seizures, sleep and behavioral anomalies are common, but signs and symptoms can potentially involve any organ and tissue. Reported cardiac abnormalities in KS include both structural defects and/or arrhythmia, but the full spectrum is yet to be elucidated [6]. Rots and colleagues have recently estimated that 31% of KS patients display an abnormality of heart morphology and 12% a rhythm disturbance. Only one case of KS with LQT was reported [7]. Here we report a further case of LQT in a KS patient, strengthening the association between these two conditions. A deeper understanding of the link between KS and heart arrhythmias, particularly LQT, is crucial not only to adequately monitor these patients but also to prevent cardiac events. Furthermore, investigating the connection between EHMT1 and heart pacemaker in larger cohorts may provide valuable insights into mechanisms underlying cardiac physiology and development.

The patient is the third child of a healthy non-consanguineous couple. Pregnancy was complicated by Hashimoto thyreopathy and polyhydramnios. He was born at 36 weeks from cesarean section, birth parameters were normal (Weight: 3100g, Length: 49.5cm, OFC: 34cm). Apgar score was 9/10. Neonatal Jaundice was treated with phototherapy. After birth, hypotonia and feeding difficulties were noticed and developed into a “slow” deglutition ability. The remaining neonatal history was uneventful, with the only exception of urinary tract infection at 3 months of age.

Psychomotor development was severely impaired: he reached seated position at 10-12 months and walked independently at 28. The first words were pronounced at 48 months of age. Last neuropsychiatric evaluation through WISC IV at the age of 11 years, confirmed a moderate degree intellectual disability (IQ:40). His neurological picture was enriched by aspecific EEG and brain MRI alterations. Particularly a thin corpus callosum, reduced thickness of frontal gyri, incomplete opercularization of sylvan valleys. Further clinical details included: hypermetropia, inguinal hernia surgically treated and dental caries. At physical examination at the age of 11 years, growth parameters were within normal range (weight: 36.5kg, height: 144cm, OFC: 52.5cm). Brachycephaly, thick eyebrows, hypertelorism, wide nasal root and midface hypoplasia were noticed. Three hypopigmented macules with Maine cost margins were detected at skin evaluation. From a cardiological point of view, the patient underwent a first EKG evaluation at the age of 7 years with the detection of a slight elonged QTc (441ms). He repeated the cardiological evaluation at the age of 11 years and was thereafter diagnosed with an exercise induced LQT. Basal EKG always remained in normal range. No structural heart defect was detectable through ultrasound examination. He reported no family history for heart disorders. His diagnostic iter passed through karyotype, performed both on blood and skin cells, CGH array and multiplex ligation probe amplification (MLPA) of RAI1 gene (MIM #* 607642), all resulted normal. In this child, KS diagnosis was reached through Next Generation Sequencing (NGS) analysis of a panel of genes related to intellectual disability and autism spectrum disorders with the evidence of a missense de novo variant c.3577G>A, p. (Gly1193Arg) in EHMT1 gene. The variant was classified as pathogenic. A targeted NGS analysis was carried out to exclude secondary diagnosis. The LQTS test detected no pathogenic variants.

Once informed consent was obtained, DNA was extracted from leukocytes using standard procedure and PCR-free whole genome sequencing (Illumina, PE 2x150) was performed. A virtual panel of 13 genes associated with cardiac arrhythmias were bioinformatically selected for analysis, namely: CACNA1C (NM_000719.7), CALM1 (NM_006888.6), CALM2 (NM_001743.6), CALM3 (NM_005184.4), CASQZ (NM_001232.4), KCNH2 (NM_000238.4), KCNJ2 (NM_000891.3), KCNQ1 (NM_000218.3), RYR2 (NM_001035.3), SCN5A (NM_001099404.2), SLC4A3 (NM_005070.4), TECRL (NM_001010874.5) and TRDN (NM_006073.4). Variants were classified on the basis of ACMG guidelines [8] and following modifications [9,10]. Genes selection and variants interpretation were performed according to disease-specific guidelines [11,12].

We also reviewed published literature searching for rhythm abnormalities in KS. We collected data from two different articles, both collecting data from wide cohorts of KS patients [6,7]. Also, we included two further cases of KS patients of our cohort displaying EKG anomalies. Data from the three KS patients of our cohort were retrieved in the contest of the Drop-by-Drop research project, dedicated to create an Italian registry of KS patients and to test possible therapies using in vitro models. The project pivots on two other centers in North Italy, the Genetic Laboratory of the ASST Papa Giovanni XXIII, Bergamo where diagnostic data have been revised, and the Department of Health sciences of the University of the Studies, in Milan. The project was approved by the local Ethics Committee and conducted according to Good Clinical Practice and Helsinki Declaration (Table 1).

This is the first report deepening the molecular investigation of LQT in a patient diagnosed with KS. In the presented case, a targeted molecular analysis excluded a second genetic diagnosis underlying LQT. To our knowledge, this is the second reported case of LQT in KS [7], although rhythm anomalies have been sporadically described in KS (Table 1). The importance of epigenetic mechanisms such as post-translational histone modification, non-coding RNA, and DNA methylation in cardiovascular development is attracting scientific interest [13]. In particular, genes involved in the regulation of histone 3 lysine 4 (H3K4) have been related to several congenital heart diseases. Histone 3 lysine 9 (H3K9) methylation complex recognizes EHMT1 as a key player [14]. The reported case, together with existing literature [6,7], suggests that, as already reported for other methyltransferase, EHMT1 may play a role in the correct function of heart pacemaker.

Table 1:Genetic and clinical data of the reported case and of patients described in literature [1=Vasireddi et al. [6]; 2=Rots et al. [7]] with EKG anomalies.

Below Abbreviations used: Abnormality=abn.; Age at Diagnosis=AgeDia; Atrial Ectopic Tachycardia=AET; Atrial Flutter=AFlu; Atrial Septal Defect=ASD; Deletion=del; Heart Palpitations=HP; Heterozygous=Het; Left Ventricular Hypertrophy=LVH; Maternal=Mat; Paternal=Pat.; PAF=Paroxysmal Atrial Fibrillation; Paroxysmal SVT=PSVT; Parental Mosaicism=ParMos; Patient=pt; Perimembranous Foramen=PF; Premature Atrial Contractions=PACs; Pulmonary Stenosis=PuSt; Sinus Tachycardia=SinT; Structural Heart Disease=SHD; Supraventricular SVA Supraventricular Tachycardia (SVT); Unknown=Unk; Unspecified Arrhythmia; UnspArr Ventricular Septal Defect=VSD; Ventricular Tachycardia=VT.

We collected data from two previous paper in order to gain a wider point of view of EKG abnormalities associated to KS. Furthermore, we included cardiological data of three KS patients displaying EKG anomalies and visited in our Medical Center (Table 1). In total we collected 22 patients with KS and an anomaly in EKG. Main rhythm anomalies reported in KS involve supraventricular structures (14/22), including supraventricular tachycardia (6/14) and atrial fibrillation (4/14). In only 2 patients a ventricular arrhythmia was reported. Rots and colleagues also reported one further patient displaying LQT, possibly related to the splicing pathogenic variant identified in EHMT1 [7]. The present report strengthens the association of KS to rhythm anomalies, in particular to anecdotally reported LQT, which might represent a life-threatening condition if underestimated and not adequately monitored. Our aim is to emphasize the importance of EKG monitoring in KS, as already depicted in specific management guidelines for KS [15]. Despite the relative rarity of cardiac pace impairment in KS (with an estimated prevalence as high as 8% [6]), it is extremely important to bear in mind that, also in adult age, these subjects may be prone to develop arrhythmias. Clinicians’ awareness about these uncommon manifestations in KS should be particularly high in reason of the severe limitations these patients may overcome in communicating any symptom [6]. In particular, the possible association of KS to long QT, although still not definitely established, is certainly an important caveat for clinicians and cardiologist dealing with KS.

In a broader perspective, it is interesting to note that KS might represent the first condition among MDEMs associated not only to morphological defects in heart development [16] but also to anomalies in its physiology. In most MDEMs, actually, once ultrasound examination rules out congenital heart defects, no further cardiological assessments might be considered. Despite the limitations given by the retrospective nature of our study, the present report, together with the review of EKG anomalies in KS and the strong phenotypical overlap that characterizes MDEMs, gives important insights into the best cardiological monitoring routine in this group of rare disorders.

The present report strengthens thsse association between LQT and KS. Data from a literature review furtherly underscore the importance of EKG monitoring in KS, providing valuable insights in management of patients affected by MDEMs.

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