1 INTRODUCTION

KBG syndrome (OMIM 158050) is an autosomal dominant condition, characterized by developmental delay, behavioral problems, facial anomalies including characteristic macrodontia of the upper central incisor of the permanent teeth, skeletal anomalies, and short stature (Brancati et al., 2004; Herrmann et al., 1975). Heterozygous variants in the ANKRD11 gene, encoding a member of the family of ankyrin repeat-containing cofactors, have been identified as the cause of KBG syndrome in 2011 (Sirmaci et al., 2011). Patients with a KBG-like syndrome carrying 16q24.3 deletion containing ANKRD11 have been reported (Miyatake et al., 2013; Sacharow et al., 2012).

The prevalence of CHD in KBG syndrome is estimated around 9%–15% in clinical reports (Brancati et al., 2006), and around 20%–25% in the recent series confirmed by molecular analysis (Low et al., 2016; Murray et al., 2017; Ockeloen et al., 2015; Scarano et al., 2019). A great variety of anatomic types of CHD are reported (Brancati et al., 2004, 2006; Crippa et al., 2015; Devriendt et al., 1998; Goldenberg et al., 2016; Low et al., 2016; Rivera-Vega et al., 1996).

In this study we analyzed the prevalence of CHDs, their anatomic types and the genetic characteristics of 46 personal patients with KBG syndrome, carrying pathogenetic variants in ANKRD11 or 16q24.3 deletion, and reviewed CHDs in patients with molecular diagnosis of KBG syndrome from the literature.

2 METHODS

Between June 2013 and December 2020, 46 patients with KBG syndrome carrying a pathogenetic variant in ANKRD11 gene (24 females, 22 males) and 6 patients with a 16q24.3 deletion (4 males, 2 females) were evaluated at our hospitals. Average age at time of last evaluation was 12 years (range 0.9–34 years) in patients with ANKRD11 variants, 9.9 years (range 1.9–13.9 years) in those with 16q24 deletion.

Genomic DNA was extracted from circulating leukocytes using QIAampH DNA Blood Kit (QIAGEN Sciences, Germantown, MD) according to the manufacturer's instructions.

Mutational analysis of ANKRD11 gene (16p24.3; chr16:89332038–89558969, 9301bp) was performed using customized panels, SeqCap EZ Choice Enrichment Kit (Roche) or Twist Human Clinical Exome Panel (Twist Bioscience), and analyzed on NextSeq550 or NovaSeq6000 sequencing platform (Illumina, San Diego, CA). Sequences were aligned to the reference genome UCSC GRCh37 by DRAGEN Germline Pipeline and analyzed with Geneyx Analysis – Geneyx, variant prioritization software. All genetic variants detected in index cases were validated on re-extracted DNA and verified by bidirectional Sanger sequencing in available family members. Potential pathogenicity was predicted using the software tools SIFT, PolyPhen-2, PROVEAN, MutationAssessor, MutationTaster, and CADD. (NCBI reference sequences: NG_032003.1, NM_013275.5, NP_037407.4) and the American College of Medical Genetics and Genomics (ACMG) guidelines (Richards et al., 2015).

Standard chromosome and microarray analysis at a practical average resolution of 100 kb were performed in all patients. Array-CGH (4x180K oligo array, Agilent) or SNP-array (Beadchip 850K, Illumina) platforms were used. Images were obtained using Agilent DNA Microarray Scanner and Agilent Scan control Software (v A.8.4.1) and data were analyzed by Agilent CytoGenomics (v 4.0.3.12) or Illumina BlueFuse Multi (v 4.4), depending on the platform. Confirmation and parental segregation analysis was performed by Real-Time PCR on ANKRD11 gene, using a SYBR Green assay (Livak & Schmittgen, 2001).

Informed consent was obtained from the patients' parents. The study was approved by the institutional scientific board of the hospital and conducted in accordance with the Helsinki Declaration.

All patients were evaluated by pediatric cardiologists. Cardiac evaluation included chest x-ray, electrocardiogram, and two-dimensional and color Doppler echocardiography in all patients. Review of anatomic types of CHD diagnosed in patients with molecularly confirmed KBG syndrome from the literature was also performed. Cited literature reports included those detailing information about CHD. Cardiac malformations have been classified accordingly to the grouping suggested by Botto et al. (2007).

3 RESULTS

4 DISCUSSION

This is the first review regarding specifically CHDs in molecularly studied KBG syndrome patients. The prevalence of CHD in our series of patients with pathogenetic variants in ANKRD11 gene is 38%, against the 9%–25% reported in literature reports (Goldenberg et al., 2016; Low et al., 2016; Murray et al., 2017; Ockeloen et al., 2015; Scarano et al., 2019). The extensive use of exome and genome sequencing in series of patients with CHD can also lead to an increased diagnosis of KBG syndrome with cardiac malformations. The number of patients with 16q24.3 deletion in the present report is low, but CHD has been diagnosed in 1/6 (17%) patients.

Left ventricular outflow tract obstructions and septal defects are the most frequent CHDs in our series and in literature reports. Valvar anomalies are frequently diagnosed, isolated or associated with septal defects, prevalently involving the left side of the heart. Additional diagnosed cardiac malformations include AVSD, patent ductus arteriosus, and situs inversus totalis with dextrocardia.

No specific hot spot for CHD can be identified. The recurrent c.1903_1907del variant has been diagnosed in 6 patients, and CHD was present in 5 of them, including ventricular septal defect in two (Low et al., 2016; Murray et al., 2017), AVSD in one (Low et al., 2016), situs inversus totalis with dextrocadia in one (present series), aortic dilation and persistent left superior vena cava in one (present series). Variable interindividual cardiac expression of the same variant has been observed in several instances (Tables 1 and 2). Intrafamilial discordance of cardiac expression has also been documented (patient 17 in present series, Ockeloen et al., 2015; Miyatake et al., 2017).

The patient with KBG syndrome and mosaic variant in ANKRD11 in our series has normal heart. The family reported by Crippa et al. (2015) is showing two siblings with atrioventricular canal defect carrying an intragenic duplication of ANKRD11, born from a mother with low mosaicism of the same molecular defect and normal heart.

Two third of patients with CHD are female, although literature reports about phenotypical expression show that females with KBG syndrome are often mildly affected in comparison to males (Brancati et al., 2006).

5 CONCLUSIONS

The prevalence of CHD in our patients is about 35% and seems to be higher than previously expected, due to systematic cardiological screening and with the more precise diagnosis possible by molecular testing. Anatomic types are including most commonly left ventricular outflow tract obstructions and septal defects. Additionally, a distinctive association with AVSD is identifiable, so that likely patients with KBG syndrome and AVSD could be underdiagnosed and AVSD could represent a marker to make diagnosis in younger children. Clinical management of KBG syndrome should include accurate and detailed echocardiogram at the time of diagnosis.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

AUTHOR CONTRIBUTIONS

Maria Cristina Digilio, Giulio Calcagni, and Maria Gnazzo elaborated the text and overviewed the literature reports. Maria Lisa Dentici, Rossella Capolino, Lorenzo Sinibaldi, Anwar Baban, and Paolo Alfieri contributed to the collection of patients and clinical data. Francesca R. Lepri, Viola Alesi, and Silvia Genovese contributed to the collection of cytogenetic-molecular data. Paolo Versacci, Carolina Putotto, and Marta Unolt contributed to the collection of cardiac data. Antonio Novelli contributed to the critical cytogenetic and molecular overview of the text. Bruno Marino contributed to the critical cardiac overview of the text. Bruno Dallapiccola contributed to the critical clinical overview of the text.