Mechanism of KMT5B haploinsufficiency in neurodevelopment in humans and mice

Abstract

Pathogenic variants in KMT5B, a lysine methyltransferase, are associated with global developmental delay, macrocephaly, autism, and congenital anomalies (OMIM# 617788). Given the relatively recent discovery of this disorder, it has not been fully characterized. Deep phenotyping of the largest (n = 43) patient cohort to date identified that hypotonia and congenital heart defects are prominent features that were previously not associated with this syndrome. Both missense variants and putative loss-of-function variants resulted in slow growth in patient-derived cell lines. KMT5B homozygous knockout mice were smaller in size than their wild-type littermates but did not have significantly smaller brains, suggesting relative macrocephaly, also noted as a prominent clinical feature. RNA sequencing of patient lymphoblasts and Kmt5b haploinsufficient mouse brains identified differentially expressed pathways associated with nervous system development and function including axon guidance signaling. Overall, we identified additional pathogenic variants and clinical features in KMT5B-related neurodevelopmental disorder and provide insights into the molecular mechanisms of the disorder using multiple model systems.

Fig. 1.. Visual summary of experiments.

Here, we evaluate a cohort of 43 individuals with pathogenic variants in KMT5B. We perform experiments in patient-derived fibroblasts and lymphoblasts, zebrafish, and a knockout mouse model, as well as in silico analysis of the missense variants.

Fig. 2.. Location of KMT5B variants and pictures of affected patients.

In (A), we show the location of all variants aligned to NM_017635.5 in this patient cohort. Loss-of-function (pLOF) variants are shown on top, and missense variants are shown below. Photos of patients with (B) pLOF variants and (C) missense variants are shown. Some common features are long face, arched eyebrows, wide-spaced eyes with upslanting palpebral fissures, prominent ears, and mild prognathism. The final two photos are of the same patient at two different ages.

Fig. 3.. KMT5B patient phenotype summary.

Primary phenotypes are shown for each patient (row) in the study. Individuals with pLOF variants are in (A), and individuals with missense variants are in (B). Black indicates that the feature is present; white indicates that the feature is absent, and gray indicates that the status of the feature is unknown or not applicable. All individuals for which information was available had DD or ID. Sixty-three percent (24 of 38) had macrocephaly, but only 21% (9 of 43) had tall stature. Sixty-two percent (18 of 29) were diagnosed with autism. Eighty-one percent (22 of 27) had hypotonia. Thirty-one percent (9 of 29) had seizures. Facial dysmorphism is one of the most common features in the phenotype, affecting 79% (31 of 39) of individuals in the cohort. Twenty-seven percent (8 of 30) had congenital heart defects including atrial or ventricular septal defects. A full clinical summary can be found for each patient in table S1.

Fig. 4.. Structural analyses of KMT5B variants.

In (A), we evaluated the evolutionary conservation of the residues altered by the missense variants in our cohort. Arrows indicate the locations of the missense variants. In (B), we show the location of the missense variants (pink) in the structure of the SET domain of KMT5B (light blue ribbon). The cofactor SAM is shown in orange, and the zinc atom is shown as a yellow sphere. (C to N) Local environment of each variant residue (pink). Neighbors (dark blue) are defined as those residues with at least one interatomic contact (atom-atom distance, <5 Å) with the variant residue. A homology model was built and used for each variant.

Fig. 5.. Expression of Kmt5b over mouse development.

(A) RNAscope in situ hybridization using a Kmt5b probe is shown in representative samples over an embryonic developmental time course (E11.5 to E16). (B to G) Whole-mount β-galactosidase staining of HET (B to D) or WT (E to G) embryos at E8.75 (B, C, E, and F) and E10.5 (D and G). Scale bars, 0.8 mm. Matched Kmt5b RNAscope images are shown in E14.5 (H) WT and (I) KO brains at 4× and 20× resolution. Representative regions from RNAscope on postnatal brains collected at (J) P1 to P2 and (K) P56 are shown. Qualitative Kmt5b expression is compared in tables S3 and S4. DG, dentate gyrus; CA, hippocampal fields 1, 2, and 3; CC, corpus callosum; LV, lateral ventricle; CP, caudoputamen; Mo, molecular layer; Gr, granular layer; CTX, cortex; TH, thalamus; HY, hypothalamus; Amy, amygdala.

Fig. 6.. RNA-seq results from human and mouse models of KMT5B haploinsufficiency.

(A, C, and D) Volcano plots illustrate the genes that are significantly up- (red) or down-regulated (blue) in the (A) human and (C and D) mouse datasets. HET, heterozygous; KO, homozygous knockout; black dots, nonsignificant genes. A functional enrichment summary is shown for the human dataset (hDEGs, P < 0.05) in (B) and for the KO mouse (mDEGs, q < 0.05) in (F). Orange, activated; blue, inhibited. Venn diagrams show (E) the number of mDEGs that overlap between the genotypes tested and (G) genes that overlapped between the hDEG and mDEG datasets. (F) Simulation testing for gene overlaps between the (H) mDEG and (I) hDEG down-regulated genes and high-risk autism genes from SFARI Gene. Dashed line, observed overlap. Gene in bold was found in both the human and mouse datasets (q < 0.05).

Fig. 7.. Cell growth and death in human fibroblasts and mouse brains.

(A) Cell growth was measured for primary fibroblast lines derived from unaffected individuals and two individuals with heterozygous KMT5B variants and was repeated three times for all cell lines. Cell growth was significantly different between control lines and lines from affected individuals at 48 hours (P < 0.05) and 72 hours (P < 0.001) as determined by Student’s t test. (B) Cell viability for primary fibroblast cell lines was measured using annexin V/propidium iodide staining and evaluated using flow cytometry. There was no significant difference between the lines determined by Student’s t test. (C and D) Brains from P0 mouse pups were weighed. There was no statistically significant difference in the mouse brain weight of the different genotype determined by Student’s t test.