Clinical characteristics and genetic analysis of four pediatric patients with Kleefstra syndrome

Abstract

Background: Kleefstra syndrome spectrum (KLEFS) is an autosomal dominant disorder that can lead to intellectual disability and autism spectrum disorders. KLEFS encompasses Kleefstra syndrome-1 (KLEFS1) and Kleefstra syndrome-2 (KLEFS2), with KLEFS1 accounting for more than 75%. However, limited information is available regarding KLEFS2. KLEFS1 is caused by a subtelomeric chromosomal abnormality resulting in either deletion at the end of the long arm of chromosome 9, which contains the EHMT1 gene, or by variants in the EHMT1 gene and the KMT2C gene that cause KLEFS2.

Methods: This study was a retrospective analysis of clinical data from four patients with KLEFS. Exome sequencing (ES) and Sanger sequencing techniques were used to identify and validate the candidate variants, facilitating the analysis of genotype‒phenotype correlations of the EHMT1 and KMT2C genes. Protein structure modeling was performed to evaluate the effects of the variants on the protein's three-dimensional structure. In addition, real-time quantitative reverse transcription‒polymerase chain reaction (RT‒qPCR) and western blotting were used to examine the protein and mRNA levels of the KMT2C gene.

Results: Two patients with KLEFS1 were identified: one with a novel variant (c.2382 + 1G > T) and the other with a previously reported variant (c.2426 C > T, p.Pro809Leu) in the EHMT1 gene. A De novo deletion at the end of the long arm of chromosome 9 was also reported. Furthermore, a patient with KLEFS2 was identified with a novel variant in the KMT2C gene (c.568 C > T, p.Arg190Ter). The RT‒qPCR and western blot results revealed that the expression of the KMT2C gene was downregulated in the KLEFS2 sample.

Conclusion: This study contributes to the understanding of both KLEFS1 and KLEFS2 by identifying novel variants in EHMT1 and KMT2C genes, thereby expanding the variant spectrum. Additionally, we provide the first evidence of how a KMT2C variant leads to decreased gene and protein expression, enhancing our understanding of the molecular mechanisms underlying KLEFS2. Based on these findings, children exhibiting developmental delay, hypotonia, distinctive facial features, and other neurodevelopmental abnormalities should be considered for ES to ensure early intervention and treatment.

Keywords: EHMT1 gene; KMT2C gene; Exome sequencing (ES); Kleefstra syndrome-1 (KLEFS1); Kleefstra syndrome-2 (KLEFS2); RT‒qPCR.

Fig. 1

Clinical photographs of patient A: P1 showed a head circumference of 44.8 cm (-1 SD, growth standard for children under 7 years of age, Health Industry Standards of China), with yellow hair, flat forehead, hypertelorism, joint hypermobility, a depressed nasal bridge, and hypodontia with wide interdental spacing; B: P2 showed a broad forehead, hypertelorism, low and flat nasal bridge, slightly thick lips, high-arched palate, macroglossia, small teeth, and a pigmented lesion of 1.8 cm × 2 cm on the left outer side of the lower leg; C: P3 exhibited a depressed nasal bridge, anteverted nares and hypertelorism; D: P4 had a head circumference of 43 cm (-1SD~-2SD) and pectus carinatum

Fig. 2

DNA sequence chromatograms of the EHMT1 and KMT2C variants. The arrows indicate the positions of the variants. A: A De novo variant, c.2382 + 1G > T (NM_024757.4), was found in the P1. B: A De novo variant, c.2426 C > T (p.Pro809Leu) (NM_024757), was found in the P2. C: A De novo variant, c.568 C > T (p.Arg190Ter) (NM_170606.3), was found in P4. Validation and parental origin analysis via Sanger sequencing or genetic high-throughput sequencing verified that the probands all carried De novo heterozygous variants

Fig. 3

Structural analysis of wild-type (WT, A, and C) and variant EHMT1 with variants (B and D). The residues at the missense variant sites, along with the nearby functional sites, are illustrated for both WT and variant EHMT1 via PyMol 2.5. The computed hydrogen bonds are shown as green dashed lines. P1: As shown in A-D, protein modeling revealed that the P809L variant resulted in an alteration in the number of hydrogen bonds charged from two to one. P4: Figures E-F demonstrate the p.Arg190Ter variant in the KMT2C gene, which results in the loss of protein structures (including α-helices, β-folds, and loop regions) starting from amino acid 190 onward, with the lost regions marked in gray

Fig. 4

KMT2C gene expression levels in different samples. The expression of the KMT2C gene in P4 was significantly lower than that in the parental samples

Fig. 5

WB results revealed that P4 histone methyltransferase protein expression was significantly lower than that of its parental signals