Genetic and behavioral characterization of a Kmt2d mouse … · A study developed in Brazil by Massironi et al11 with the purpose of inducing new mutations in BALB/cJ mice allowed

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Genetic and behavioral characterization of a Kmt2d mouse mutant, a new

model for Kabuki Syndrome

Article in Genes Brain and Behavior · March 2019

DOI: 10.1111/gbb.12568

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OR I G I N A L A R T I C L E

Genetic and behavioral characterization of a Kmt2d mousemutant, a new model for Kabuki Syndrome

Pedro K. Yamamoto1 | Tiago A. de Souza2 | Ana T. F. B. Antiorio1 |

Dennis A. Zanatto1 | Mariana de Souza A. Garcia-Gomes1 |

Sandra R. Alexandre-Ribeiro3 | Nicassia de Souza Oliveira1 | Carlos F. M. Menck2 |

Maria M. Bernardi4 | Silvia M. G. Massironi1,3 | Claudia M. C. Mori1

1Department of Pathology, School of

Veterinary Medicine and Animal Science,

University of São Paulo (USP), Sao Paulo,

Brazil

2Department of Microbiology, Institute of

Biomedical Science, University of São Paulo

(USP), Sao Paulo, Brazil

3Department of Immunology, Institute of

Biomedical Science, University of São Paulo

(USP), Sao Paulo, Brazil

4Graduate Program in Environmental and

Experimental Pathology, Paulista University,

São Paulo, Brazil

Correspondence

Claudia M. C. Mori, DVM, PhD, University of

São Paulo, Department of Pathology, School of

Veterinary Medicine and Animal Science,

Av. Prof. Dr. Orlando Marques de Paiva, n. 87,

Cidade Universitária, CEP 05508-270. São

Paulo, Brazil.

Email: [email protected]

Funding information

Conselho Nacional de Desenvolvimento

Científico e Tecnológico (CNPq), Grant/Award

Number: 14411/2017-1; Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior

- Brasil (CAPES), Grant/Award Number:

Finance Code 001; Fundação de Amparo à

Pesquisa do Estado de São Paulo (FAPESP),

Grant/Award Numbers: 2012/25387-2,

2016/23659-6, 2017/21103-3

The recessive mutant mice bate palmas (bapa) - claps in Portuguese arose from N-

ethyl-N-nitrosourea mutagenesis. A single nucleotide, T > C, change in exon

13, leading to a Thr1289Ala substitution, was identified in the lysine (K)-specific

methyltransferase 2D gene (Kmt2d) located on chromosome 15. Mutations with a

loss-of-function in the KMT2D gene on chromosome 12 in humans are responsible

for Kabuki syndrome (KS). Phenotypic characterization of the bapa mutant was

performed using a behavioral test battery to evaluate the parameters related to

general activity, the sensory nervous system, the psychomotor system, and the

autonomous nervous system, as well as to measure motor function and spatial

memory. Relative to BALB/cJ mice, the bapa mutant showed sensory and psycho-

motor impairments, such as hypotonia denoted by a surface righting reflex impair-

ment and hindquarter fall, and a reduction in the auricular reflex, suggesting

hearing impairment. Additionally, the enhanced general activity showed by the

increased rearing and grooming frequency, distance traveled and average speed

possibly presupposes the presence of hyperactivity of bapa mice compared with

the control group. A slight motor coordination dysfunction was showed in bapa

mice, which had a longer crossing time on the balance beam compared with

BALB/cJ controls. Male bapa mice also showed spatial gait pattern changes, such

as a shorter stride length and shorter step length. In conclusion, the bapa mouse

may be a valuable animal model to study the mechanisms involved in psychomotor

and behavior impairments, such as hypotonia, fine motor coordination and hyper-

activity linked to the Kmt2d mutation.

K E YWORD S

ENU-mutagenesis, Kmt2d gene, mouse genetics, mouse phenotype, mutant behavior,

psychomotor impairment

Pedro K. Yamamoto and Tiago A. de Souza contributed equally to this study.

Received: 24 January 2019 Revised and accepted: 18 March 2019

DOI: 10.1111/gbb.12568

© 2019 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society

Genes, Brain and Behavior. 2019;e12568. wileyonlinelibrary.com/journal/gbb 1 of 12

https://doi.org/10.1111/gbb.12568

https://orcid.org/0000-0002-9393-240Xmailto:[email protected]://wileyonlinelibrary.com/journal/gbbhttps://doi.org/10.1111/gbb.12568

1 | INTRODUCTION

Mouse geneticists base a significant part of their work on natural or

induced mutants. Using forward genetics, it has been possible to dis-

cover specific genes that support a phenomenon observed in a

mutant organism.1 Even today, several tools are available to manipu-

late the mouse genome to obtain transgenic, knockout and knock-in

variants. Forward genetics is still important, as random mutagenesis

can be used to gather new information on already known genes.2 Fur-

thermore, a significant proportion of human diseases is caused by

nucleotide variants in genes that affect their function or regulation

rather than merely deactivating them.3 Considering the above, the

study of animal models obtained by mutagenesis can be used to eluci-

date the function of genes and characterize human genetic diseases.4

The induction strategy for point mutations by N-ethyl-N-nitrosourea

(ENU) together with consistent phenotypic selection can be a power-

ful tool to identify mutations responsible for complex phenotypes.2

ENU is still the most used chemical compound for mutagenesis in

mouse.1 One of the major bottlenecks for studying mutants induced

by ENU is the identification of the mutation by genetic mapping

followed by the sequencing (Sanger method) of one or more genes in

the mapped region. In addition to traditional microsatellite mapping,

hybridization panels (DNA microarrays) have been used to scan sets

of single nucleotide polymorphisms (SNPs), although they are

restricted.5,6 The use of modern sequencing techniques has increased

the chance, speed and reliability of candidate mutation identification.

Considering that approximately 75% of ENU-induced mutations have

been identified in exons,7 some research groups utilize exome

sequencing analyses to identify these mutations.6,8–10

A study developed in Brazil by Massironi et al11 with the purpose

of inducing new mutations in BALB/cJ mice allowed the identification

of 11 murine models. Among the recessive mutations identified by

the study, one of them was called claps or “bate palmas” in Portuguese

(bapa) and is characterized by abnormal movement of the hindlimbs

when the mouse is lifted by the tail. First, a bapa mutation genetic

mapping was performed using microsatellite markers scattered

throughout the mouse genome, which allowed the identification of

the candidate region carrying the mutation on chromosome 15.

By applying whole-exome sequencing, the missense mutation

NM_001033276:c.A3865G:p.T1289A was found in the lysine (K)-

specific methyltransferase 2D (Kmt2d) gene on chromosome 15, which

was confirmed by Sanger sequencing. The loss of KMT2D gene func-

tion on chromosome 12 in humans has been described as responsible

for Kabuki syndrome (KS), a rare autosomal dominant congenital dis-

order characterized by multiple anomalies involving the development

and function of various organ systems.12 Major clinical components

include typical facial dysmorphic features, postnatal growth retarda-

tion, hypotonia, skeletal abnormalities, dermatoglyphic changes and

mild to moderate intellectual disability.13–15

This study established a strategic exome sequencing methodology

to select potential causative mutations in an ENU-induced mutant

with a BALB/cJ background. Then, after the identification of a single

missense mutation in the Kmt2d candidate gene, we used behavioral

tests to evaluate the bapa mutant and its potential as a novel mouse

model to study brain-associated diseases such as KS.

2 | MATERIALS AND METHODS

2.1 | Ethics statement

The protocols for the experimental studies were approved by the

ethics committee of the Veterinary Medicine School, University of

São Paulo, Brazil (protocol number 1004070715, FMVZ-USP) and the

Institute of Biomedical Science under protocol number 053, page

32, book 3 (2015). These guidelines are similar to those in the Guide

for Care and Use of Laboratory Animals of the US National Research

Council.16 The experiments were performed under proper laboratory

practice protocols and following quality assurance methods. All efforts

were made to minimize the suffering of the animals.

2.2 | Mice

Specific pathogen-free (SPF) mice were obtained from the animal

facility of the Department of Immunology, Institute of Biomedical Sci-

ence, Universidade de São Paulo, Brazil. The animals were housed in

individually ventilated cage (Alesco Indústria e Comércio, Monte Mor,

Brazil). They had unrestricted access to filtered and autoclaved water

and autoclaved commercial pellets formulated according to the AIN-

93M rodent diet (Nuvilab, Quimtia, Paraná, Brazil). Seven days before

beginning behavior experiments, mice were transferred to the Depart-

ment of Pathology, School of the Veterinary Medicine, Universidade

de São Paulo, Brazil. Mice were housed in polypropylene cages (28 ×

17 × 12 cm) with pine shavings for bedding under controlled room

temperature (22�C ± 5�C) and humidity (55% ± 5%). The room was

kept on a 12/12 hours light/dark cycle (lights on at 07:00 AM) with

artificial light.

Mice were housed in a SPF facility for the following agents:

ectromelia virus, lymphocytic choriomeningitis virus, minute virus of

mice, mouse hepatitis virus, mouse parvovirus, pneumonia virus of

mice, reovirus, Sendai virus, Theiler murine encephalomyelitis virus,

hantaviruses, cilia-associated respiratory bacillus, Clostridium piliforme,

Klebsiella pneumonia, Mycoplasma pulmonis, Pasteurella multocida,

Pasteurella pneumotropica, Pseudomonas aeruginosa, Salmonella spp,

Staphylococcus aureus, Streptobacillus moniliformis, β-hemolytic Strep-

tococcus spp, Streptococcus pneumoniae, endoparasites and

ectoparasites.

2.3 | Genetic mapping, exome enrichment andsequencing

BALB/cJ males treated with three ×100 mg/kg of ENU were crossed

with nontreated BALB/cJ female. G1 males were crossed again with

nontreated females and then G2 females were backcrossed to their

father generating BALB/cJ-Kmt2dbapa/Kmt2dbapa mutant in the G3

progeny.11 Fourteen mutants were used to locate the chromosome

2 of 12 YAMAMOTO ET AL.

carrying the bapa mutation. These mutants were selected from

53 mice from the outcross-intercross generation of bapa and

C57BL/6J by their phenotype, which was clapping of the hindlimbs

when held by the tail at 3 months of age. A genome scan with 21 poly-

morphic microsatellites distributed over the mouse genome was

employed, and six markers on chromosome 15 were used to define

the region carrying the mutation more precisely.

For exome analysis, tail samples from bapa mutant mice and con-

trol C57BL/6J and BALB/cJ mice were used for the extraction of

genomic DNA. Approximately 5 μg of genomic DNA from each sample

was employed for exome enrichment and the preparation of the

libraries. Exome enrichment was performed using the SureSelect

Mouse All-Exon kit (Agilent Technologies) and the libraries from bapa

and the isogenic C57BL/6J and BALB/cJ strains were sequenced

using the SOLiD 5500xl platform (ThermoFisher Scientific) in single-

end mode generating 75-bp reads.

The reads were mapped in color space mode using LifeScope 2.1

suite (ThermoFisher Scientific) to the mouse reference genome mm9

(NCBI37/mm9). Enrichment probe coordinates were obtained from

the supplier of the SureSelect Mouse All-Exon kit for the mm9

genome. SNP calling was performed with the diBayes algorithm as a

LifeScope module using standard stringency for SNP calling. Compari-

son and filtering steps were performed using VCFTools tool17 to

select only exclusive homozygous nonsynonymous or splice site vari-

ants in the bapa mutant compared with inbred strains as well as those

in the Mouse Genomes Project database (REL-1211). Exclusive vari-

ants were also restricted to the chromosomal region coordinates iden-

tified by genetic mapping as described above. Annotate Variation

(ANNOVAR)18 was used to annotate the variants using the UCSC

RefSeq/mm9 database. The impact of each point mutation was

predicted using the PolyPhen2,19 PROVEAN20 and SpliceMan21 tools.

Validation of the candidate SNVs (single nucleotide variants) was

performed by polymearse chain reaction (PCR) amplification using

specific oligonucleotide pairs designed to amplify a 244-bp region

(F-TGCTAGCAAACATCGGACTG and R-TGGGTCCCTTCCATCACTTA).

PCR products were evaluated for the expected size and purified by

E-Gel 2% electrophoresis (ThermoFisher Scientific), submitted to the

BigDye 3.1 sequencing reaction (ThermoFisher Scientific) and

sequenced using an ABI 3130XL platform (ThermoFisher Scientific).

PCR amplification products from genomic DNA samples from bapa

mutant, C57BL/6J, BALB/cJ and A/J mice as well as unrelated ENU-

mutant controls whose exome was not previously sequenced were

used in the Sanger validation procedure.

2.4 | Phenotypic characterization

A total of 30 mutant bapa (15 males and 15 females) and 30 BALB/cJ

(15 males and 15 females) mice that were 12 weeks old were used.

Females were kept in groups of five animals. Males were isolated to

reduce aggression, and one female BALB/cJ mouse was introduced to

each cage to avoid stress due to the isolation. These females did not

participate in the experiment.

The phenotypic characterization was assessed using a behavioral

test battery as described by Manes et al22 with modifications to

assess the phenotype observed in the bapa mutant. The behavior

tasks were performed from the least to most stressful with 1-week

intervals, between 8:00 and 12:00 AM. The order of tests was as fol-

lows: (a) open field test (OFT), (b) T-maze alternation, (c) balance

beam, (d) gait analyze, (e) tail suspension test (TST) and (f) forced swim

test (FST). The apparatuses were cleaned with a 5% alcohol/water

solution before placement of the animals to prevent possible bias cau-

sed by odor cues left by the previous mouse. All procedures were fil-

med for later visual evaluation by two experienced observers.

Additionally, an Ethovision video tracking system23 was used for data

acquisition of the distance traveled (cm) and speed (cm/s) in the OFT.

First, the OFT was used to evaluate the general activity by mea-

suring the distance traveled and average speed as well as the frequen-

cies of rearing and grooming; parameters related to the psychomotor,

autonomous and sensory nervous systems were also analyzed. A

score from one to five was given for each parameter (Table 1), except

for micturition and defecation, for which numbers of urine spots and

fecal boli were counted, respectively. Testing was performed in a small

room with dim lighting for 5 minutes. At the end of the observations,

the scores were summed and used for the statistical analysis.

The T-maze alternation test was performed following a protocol

described by Manes et al22 to evaluate the spatial memory of the bapa

mutant. According to Deacon & Rawlins,24 this test is very sensitive

to hippocampal dysfunction.

Motor coordination was assessed by the balance beam task

described previously22 and the gait analysis test was adapted from

the protocol used by Kloefkorn et al25 and Dunnett.26 Briefly, the gait

analysis consisted of a runway apparatus made of acrylic (30 cm long

× 5 cm wide × 25 cm high) coated with absorbent filter paper. A dark

box was placed at the end of the apparatus that allowed the mouse to

enter spontaneously and avoid the open area. Mice had their

hindlimbs marked with red paint, the right forelimb with black ink and

the left forelimb with blue ink. Furthermore, they were placed to walk

on the runway, allowing the marking of the footprints. The test was

performed over 2 days; the first day focused on the training of mice,

and the second day focused on evaluating their performance. On both

days, only one attempt was allowed. Spatial variables, including the

stride length, step length and step width, were evaluated for the hind-

and forelimbs (Figure 1). The stride length symmetry (step length

divided by stride length) was calculated as previously described.27

TST and FST have been used to assess the sensory and motor

function of mutant mice.22,28 The TST was performed as described

previously.22,29 Briefly, tape attached to a hook was used to suspend

mice by the tail in a single 6-minute trial shot with a camera in front

of the apparatus. Immobility time was defined as the mouse not

struggling.

The FST was performed as described previously.30 Briefly, each

mouse was individually placed into a vertical glass cylinder with a

28 cm in diameter that contained water at a depth of 25 cm and a

temperature of 23�C to 25�C. After 6 minutes in the cylinder, the

animals were removed, dried and moved to a warm cage; the latency

YAMAMOTO ET AL. 3 of 12

to immobility and total immobility time was recorded. Additionally,

the angle during immobility was measured as described previously.31

Briefly, after 2 minutes elapsed from the test start, one frame was

taken out from each video when the mouse was floating and the

angle formed by the body axis relative to the water surface was

measured.

2.5 | Statistical analysis

Two-way analysis of variance (ANOVA) followed by Bonferroni's

post-hoc test was employed to evaluate the strain and sex differences

in the behavior tests. Fisher's exact test was used to analyze the irrita-

bility parameter. Statistical analysis was completed with the GraphPad

TABLE 1 Parameters related to the general activity, sensory nervous system and psychomotor system

Parameter Description Scores

Surface-righting reflex Mouse response when placed in a supine position and latency to

return to its original position

1—does not move (absent)2—turns slowly with difficulty3—turns slowly4—turns faster5—turns immediately (baseline)

Grasp strength Mouse strength to hold onto an inverted down wire grid 1—does not hold the grid (absent)2—holds the grid but immediately released it3—holds the grid for up to 15 seconds but release it4—holds the grid for more than 15 seconds but

release it

5—grabs tightly and does not release it (baseline)

Auricular reflex Position of the ears after snap your fingers next to the mouse

several times in a row

1—ears perpendicular to the head and directedforward (absent)

2—ears rotate outwards and/or back (baseline)3—mouse pulls ears back4—ears slightly placed against the head5—ears laid flat against the head

Corneal reflex Mouse response when a forceps is slowly approached to your

eyes, but not touching

1—eyes remain open (absent)3—only blinks5—eyes completely closed (baseline)

Response to touch Mouse response when touched with forceps for more than

15 seconds

1—does not move (absent)2—moves but stays in the same place3—mouse takes a few steps4—walks with difficulty5—walks or runs with agility (baseline)

Tail squeeze Mouse response when the tip of the tail is pressed by forceps 1—no response (absent)2—moves slowly3—moves quickly4—moves quickly and jump (baseline)5—move quickly, jump and run

Irritability If the mouse shows a response to being touched and/or blown Absent

Present

Adapted from Manes et al.22

F IGURE 1 Representative scheme used in the gait analyze test. Spatial gait parameters (stride length, step length and step width) are shownfor the BALB/cJ mouse hindlimbs (footprints with red ink). The right forelimb prints are black, and the left forelimb prints are blue


Prism 6 software (GraphPad Software, Inc., La Jolla, California). The

data were expressed as the mean ± SEM or as the medians, and

results were considered significant at P < 0.05.

3 | RESULTS

3.1 | Genetic mapping, exome enrichment andsequencing

The genome scan using microsatellite markers located the bapa muta-

tion in an interval of 17.02 cM on mouse chromosome 15 between

D15Mit100 (19.26 cM) and D15Mit68 (36.28 cM). We used this

mapped region to select SNPs that were only found between these

markers.

On average, approximately 92% of the reads produced by exome

sequencing from all the samples were aligned to the mouse reference

genome. Reads that were mapped to exonic target regions represen-

ted an average of 80.17% and only 4.2% of target regions were not

covered. At least 90.74% of target bases were covered at least 5× by

all the sequenced samples. The mean base coverage obtained was

94.7× and the total number of raw SNPs called for in bapa mutant,

BALB/cJ and C57BL/6J mice was 77 075, 78 786 and 2900 SNPs,

respectively (Supporting Information, Table S2).

The SNV filtering strategy for locating the candidates for causal

mutations was based on the following assumptions: the genetic back-

ground of the mutant (BALB/cJ); the region previously mapped by

microsatellites—Chr. 15: 19-37cM; the type of phenotype inheritance—

for bapa mutant is recessive (homozygous); and the uniqueness of the

mutation in relation to the dbSNP polymorphism bank (Table S3).

Finally, a causative variant implies a nonsynonymous exchange or must

be located in splicing sites (up to two nucleotides from the exon bor-

der). To ensure a better SNV identification, we aimed not to use a mini-

mum coverage filter, even if this implies false positives. This strategy

was very efficient at filtering candidate SNVs (Figure 2A). We found

only one candidate in the bapa mutant using these variant filtering

steps, a NM_001033276:c.A3865G:p.Thr1289Ala mutation with 40× cov-

erage in the Kmt2d gene. The SNV found implies a shift in the N-

terminal region of the protein prior to the start of the second group of

plant homeodomain (PHD) domains due to the nonsynonymous

exchange of a threonine (Thr) residue to an alanine residue (Ala). This

mutation was validated by the Sanger method and was not found in

any control; thus, it was exclusive to the bapa mutant (Figure 2B).

(+/+)

(-/-)

Kmt2d

exon13:c.A3865G:p.T1289A

Post-translationalmodification

PHD HMG-box SET

Filter SNPs

Raw SNPs 77075

Mapped region 1668Homozygous 1525WT controls 87dbSNP 18Exonic/splice site 3Unrelated mutants 1

(A)

(C)

(B)F IGURE 2 Comparison andfiltering steps were performed to selectonly exclusive homozygousnonsynonymous or splice site variantsin the bapa mutant compared withinbred strains as well as those in theMouse Genomes Project database(REL-1211) (A); Validation of thecandidate SNV by Sanger sequencingof genomic DNA samples from bapamutant (−/−) and C57BL/6J, BALB/cJ,A/J mice as well as an unrelated ENU-mutant controls (+/+) (B); SNVcandidate found in exon 13 of Kmt2dgene, which creates a nonsynonym

exchange of a threonine residue to analanine residue in KMT2D protein (C)


A multiple alignment of the sequences likely to be homologous to

mammalian, avian and amphibian KMT2D was performed and the

mutated Thr residue is conserved in all analyzed sequences

(Figure 2C). Threonine residues can be serine-threonine or even gly-

cosylation phosphorylated, so we used the in-silico phosphorylation

prediction tools Net-O-Glyc, iGPS and NetPhos to evaluate the possi-

bility of phosphorylation or even glycosylation of the Thr residue, indi-

cating that a possible posttranslational modification may occur at the

residue, which would be prevented by the presence of the mutant

allele. This region is also consistent with the phosphorylation consen-

sus sequence RxRxxS */T * in human PI3K/AKT (RSK, mTOR), and

the target residue in KMT2D was found to be phosphorylated in

phosphoprotection experiments in human cells.32 Inhibition of FLT3

(tyrosine kinase) affects the phosphorylation of KMT2D in humans33;

thus, posttranslational modifications may be important in the regula-

tion of KMT2D function, localization or protein-protein interactions.

3.2 | Phenotypic characterization

The OFT test results including the general activity and parameters

relating to the psychomotor, autonomous and sensory nervous sys-

tems in bapa and BALB/cJ mice are shown in Figure 3.

A main effect of strain was showed on the distance traveled

(F1,55 = 15.20, P = 0.0003), average speed (F1,55 = 17.26, P = 0.0001),

F IGURE 3 General activity in the open field: distance traveled (A); average speed (B); rearing (C); grooming (D) and parameters related to thepsychomotor and autonomous nervous systems (E-H) and sensory nervous system (I-N) in bapa mutant (n = 15 males and 15 females) andBALB/cJ (n = 15 males and 15 females) mice. Data are presented as the means ± SEM or medians and the interquartile range. Two-way ANOVAfollowed by Bonferroni's post-hoc test was employed to evaluate strain and sex differences. Fisher's exact test was used to analyze the irritabilityparameter. * P < 0.05; ** P < 0.01; *** P < 0.001


rearing frequency (F1,56 = 5.85, P = 0.0189) and grooming

(F1,56 = 5.59, P = 0.0216), in which male and female bapa mice

showed enhanced parameters compared with the BALB/cJ controls

(Figure 3A-D).

Analysis of the psychomotor system showed a reduction in the

surface righting reflex in male and female mutants (F1,56 = 141.13,

P < 0.0001) (Figure 3E) and an increased hindquarter fall in male bapa

mice (F1,56 = 6.86, P = 0.0113) (Figure 3G) compared with the

BALB/cJ controls. Additionally, we observed difference in the hind-

quarter fall between the sexes (F1,56 = 11.34, P = 0.0014) and the

interaction between strain and sex (F1,56 = 6.86, P = 0.0113)

(Figure 3G). The grasp strength (Figure 3F) did not show differences

between the strains and sexes.

With respect to the autonomous nervous system parameters,

males showed increased micturition (F1,56 = 15.51, P = 0.0002) com-

pared with the female group. No differences were observed between

strains (Figure 3H).

A main effect of strain (F1,56 = 4.69, P = 0.0345) and strain by sex

interactions were showed on auricular reflex (F1,56 = 4.69,

P = 0.0345) (Figure 3I). A main effect of sex was also significant in

females that presented reduced auricular reflex compared with males

(F1,56 = 13.04, P = 0.0007) (Figure 3I). The sex by strain interaction

was significant in female bapa mice that presented diminished tail

squeeze compared with the female BALB/cJ group (F1,56 = 9.77,

P = 0.0028), while there was no strain difference in male mice

(Figure 3L). Additionally, a significant sex difference was found for tail

squeeze, as females presented higher scores than males

(F1,56 = 12.21, P = 0.0009) (Figure 3L). Other parameters involved in

the sensorial system, such as corneal reflex, response to touch and

irritability, did not show differences between the strains (Figures 3J,K,

M,N).

In the T maze alternation task, the tendency of the mouse is

always to explore the opposite side of the labyrinth arm to the one

previously chosen.24 Bapa mice did not present different behaviors

from BALB/cJ mice (data not shown).

The latency of the first immobility in the TST (F1,50 = 30.45,

P < 0.0001) and FST (F1,52 = 7.06, P = 0.0104) was lower in females

compared with males (Figures 4A,C). The total immobility time in the

FST was higher in the bapa male and female groups compared with

the controls (F1,52 = 4.46, P = 0.0395) (Figure 4D). Additionally, the

total immobility time in the TST was higher in the bapa male group

compared with the BALB/cJ male group (F1,48 = 8.25, P = 0.0060)

(Figure 4B). Moreover, a significant sex difference was found in the

TST, as males presented reduced immobility times compared with

females (F1,48 = 10.80, P = 0.0019) (Figure 4B). Regarding the floating

posture in FST, which was quantified by measuring the angles during

the immobility time, there were no significant differences between

the groups (data not shown).

In the balance beam test, male mice presented higher scores than

females when crossing the bar (F1,42 = 8.95, P = 0.0046) (Figure 5A).

For the crossing time, control mice moved faster than mutants of both

sexes (F1,40 = 16.23, P = 0.0002). Additionally, a main effect of sex

was observed, with females walking faster than males (F1,40 = 16.54,

P = 0.0002) (Figure 5B).

Figure 6 shows the parameters (stride length, step length, step

width and gait symmetry) used in the gait analysis. Male bapa mice

showed shorter stride lengths for their right and left fore-

(F1,24 = 8.34, P = 0.0081; F1,24 = 10.33, P = 0.0037) and hind-

(F1,24 = 7.98, P = 0.0094; F1,24 = 7.76, P = 0.0103) limbs compared

with BALB/cJ controls (Figures 6A,B,E,F). The strain by sex interaction

was significant in bapa males' left fore- (F1,24 = 10.12, P = 0.0040)

and hind- (F1,24 = 7.76, P = 0.0103) limbs that were shorter than the

F IGURE 4 Latency to immobility(A) and total immobility time (B) in theTST and latency to immobility (C) andthe total immobility time (D) in the FSTwith bapa mutant (n = 15 males and15 females) and BALB/cJ (n = 15 malesand 15 females) mice. Data arepresented as the means ± SEM ormedians and the interquartile range.Two-way ANOVA followed byBonferroni's post-hoc test wasemployed to evaluate strain and sexdifferences. * P < 0.05; ** P < 0.01;*** P < 0.001


controls (Figures 6B,F). Additionally, mutant males showed shorter

hindlimb step lengths (F1,24 = 4.70, P = 0.0403) (Figure 6G), and strain

by sex interactions were found for the fore- (F1,24 = 5.27, P = 0.0308)

and hind- (F1,24 = 4.64, P = 0.0415) limbs (Figures 6C,G). Regarding

the step width, there were no significant differences between the

groups (Figures 6D and H). The forelimb spatial symmetry was signifi-

cantly different and greater than 0.5 for bapa males compared with

BALB/cJ controls (F1,52 = 6.77, P = 0.0120) (Figure 6I). A main effect

of sex was observed in the fore- (F1,52 = 12.60, P = 0.0008) and hind-

(F1,52 = 9.18, P = 0.0038) limb symmetry (Figure 6I,J).

4 | DISCUSSION

The bapa mutant is maintained in a co-isogenic BALB/cJ background

and exhibits phenotypic changes of recessive inheritance character-

ized by the repetitive movement of the hindlimbs when suspended by

the tail. They are fertile and have a normal life span.

Sequencing analysis of the mutant exome indicated a single candi-

date SNV located on the Kmt2d gene, formerly known as Mll2 or

Mll4.34 The Kmt2d gene is a specific lysine (K) methyltransferase

whose primary function is the methylation (mono, di or mainly

trimethylation) of the K4 residue in histone H3, also known as H3K4

methylation. This type of methylation, especially H3K4 trimethylation

by KMT2D, is associated with histone modification in the 50-regions

and the consequent increase in gene transcription levels of virtually

all-active genes.34 The main domain responsible for methyltransferase

action is the SET domain (family proteins originally identified in Dro-

sophila suppressor of variegation [Su(var)3-9], enhancer of zeste [E(z)]

and trithorax, which is also present in the group of Drosophila melano-

gaster homologous trithorax genes (Su(var) enhancer-of-Zestes and

trithorax), important for embryonic development and involved in regu-

lating the hox gene pattern.35 The SET domain in mammals,

SUV39H1, appears to be related to methylation of lysine-9 in the his-

tone H3 N terminus.36,37

The function of H3K4 methyltransferases in mice does not appear

to be redundant and may be related to the formation of different pro-

tein complexes. Those complexes are similar to the structuring of the

COMPASS complex in Saccharomyces cerevisiae, which is related to

the balance between different types of methylation and histone acet-

ylation as well as to the different localization of the expression of

these genes.38 The major members of this complex include the

KDM6A protein, Menin, UTX and the CTD portion of RNA polymer-

ase II.38 The KMT2D gene also appears to be involved with the devel-

opment of cancer and the regulation embryonic cell differentiation

into cardiac tissue.39

Recently, with the advent of large next-generation sequencing

(NGS) studies, there was a correlation between the presence of muta-

tions in the KMT2D gene in humans as the primary cause of KS.40 KS

is a rare childhood congenital disease that affects approximately 1 in

32 000 births and is characterized by a broad spectrum of symptoms,

including craniofacial anomalies and intellectual disability; KS is often

confused with autistic spectrum disorder.41 Most mutations found in

patients result in the loss of KMT2D gene function and are located in

the C-terminal portion of the SET domain region.40,41 The mutation

location also seems to directly influence the type of sym-

ptom/anomaly found in patients, highlighting an interesting genotype-

phenotypic association that may be related to the molecular function

of the gene product.14

Bjornsson et al13 characterized a Kmt2d+/βGeo murine model with

a loss-of-function of the Kmt2d gene in heterozygosis. The homozy-

gous is embryonic lethal, which indicates that Kmt2d is an essential

gene and that the bapa mutation is hypomorphic, allowing phenotype

studies on homozygous mice. The main behavior phenotype of

Kmt2d+/βGeo mouse was learning and memory impairment, which

could be explained by the failing of the dentate gyrus granule cell

layer in the hippocampus. Cognitive deficiencies were related, at

least in part, with hippocampal dysfunction in KS patients. Additionally,

the authors showed a decrease in H3K4me3 in the dentate gyrus

granule of Kmt2d+/βGeo mice. The results also showed that H3K4

methylation is not correctly balanced in the brain and spleen of bapa

mutants, presenting more H3 monomethylated histones in K4 residues

using an anti-H3K4me1 antibody specific for histone H3 lysine-4

residues that are monomethylated (data not shown).

The Kmt2d mouse gene has 55 exons, the primary transcript is

39 kb and the 5588 amino acid residues is located on chromosome

15 (GRCm38/mm10). The mutation detected in bapa mice is a T > C

exchange in the transcript (c.A3865G: p.T1289A) located in exon 13 out-

side of the SET domain. The SNV was detected with 40× local cover-

age and validated by Sanger sequencing compared with samples

extracted from isogenic lines from an unrelated mutant and from

another mutant bapa individual, corroborating the exclusivity of the

F IGURE 5 Scores (A) and timespent crossing the bar (B) in thebalance beam test for bapa mutant(n = 15 males and 15 females) andBALB/cJ (n = 15 males and15 females) mice. Data are presentedas the means ± SEM or medians andthe interquartile range. Two-wayANOVA followed by Bonferroni'spost-hoc test was employed toevaluate strain and sex differences.** P < 0.01; *** P< 0.001


exchange and the strong evidence of the presence of the homozygous

allele in the mutant population.

Different neurological diseases cause significant motor impair-

ments, and they can induce the same changes in a particular behav-

ioral measurement; the use a battery of behavioral tests can help to

deal with this problem and to estimate the differences in the mutant

and wild-type phenotypes.42 All together, the sequence of behavioral

tests could assess the phenotypic behavior when investigating the dif-

ferent outcomes from the animal model.43 Thus, the phenotypic

screening aimed to characterize the motor, sensory and nervous

F IGURE 6 Spatial parameters inthe gait analyze test: right (A) and left(B) forelimb stride length, forelimbstep length (C) and step width (D),right (E) and left (F) hindlimb stridelength, forelimb step length (G) andstep width (H), forelimb (I) andhindlimb (J) gait symmetry with thebapa mutant (n = 7 males and7 females) and BALB/cJ (n = 7 malesand 7 females) mice. The dotted lineindicates spatial symmetry value of0.5. Data are presented as the means± SEM or medians and theinterquartile range. Two-way ANOVAfollowed by Bonferroni's post-hoc testwas employed to evaluate strain andsex differences. * P < 0.05; ** P < 0.01;*** P < 0.001


system aspects in mutant bapa mice compared with BALB/cJ

control mice.

The general activity in the OFT assesses locomotor and behavior

activity levels of mice and can be correlated with the psychomotor

system function.43 OFT is useful for investigating motor impairment

in animal models of neuromuscular diseases. The test is also used to

assess anxiety-like and exploratory behaviors in mice.43,44 The data

showed an increased rearing frequency, distance traveled and average

speed of bapa mice compared with the control group, denoting hyper-

activity. By contrast, genetically modified Kmt2d+/ßGeo mice pres-

ented general open-field activity similar to their controls.13 According

to the literature, hyperactivity is one of the behavioral problems

observed in patients with KS.12,45

Data from sensory parameters showed a reduction in auricular

reflex in the mutant males compared with the controls. Therefore,

both groups of females showed less auricular reflex compared with

males; however, further investigation is required to clarify whether

the bapa mutation may cause hearing impairment. The auricular reflex

is fundamentally connected to cochlear neuron pathways and is often

used as a model of sensory integration.46 Intense acoustic stimulation

leads to this reflex, and lesions or traumas in this pathway are

reflected as a reduced response to stimulus,47 suggesting hearing

impairment. Hearing loss is one of the neurosensory abnormalities of

KS observed over 30% of the patients.48–50

As for the psychomotor system aspect, surface righting reflex

impairment and hindquarter fall was observed in the bapa mutant,

which potentially presupposes the presence of hypotonia in these ani-

mals. Hypotonia is often noticed from birth in patients with KS, affect-

ing approximately 70% of patients,48,51 and this clinical feature could

be linked to the presence of a KMT2D mutation.49

The TST was designed by Steru et al52 based on the principle of

FST.30,53 In both tests, mice are exposed to an inescapable situation

with a moderate level of stress in which immobility is interpreted as a

lack of escape-related behavior.30 These situations are common and

the best validated tests used to evaluate the efficacy of antidepressant

drugs but have also been applied to the characterization of genetic

mutations in mice.22,30 Our data showed no differences in latency to

immobility for TST and FST in bapa compared with the controls, but dif-

ferences between sexes were registered. In evaluating the despair-

related behaviors of female BALB/cJ,54 found a significant effect of the

estrous cycle in TST. Moreover, both tests can also determine balance

and motor information.28 Notably, the total immobility time in FST was

longer in bapa mice than in BALB/cJ mice of both sexes. Furthermore,

the mutant and controls presented similar body posture angles when

floating. The floating posture in mice depends on the buoyancy force,

and abnormal positions can indicate psychomotor impairment.31

Performance on the balance beam is a useful tool to assess fine

motor coordination and the balance of mutant mice.55 The bapa mice

had a longer crossing time compared with BALB/cJ controls. During

the crossing bar, bapa mice showed some difficulty in maintaining

their balance and used their tails as an aid; furthermore, they slipped

frequently, resulting in an increased wooden beam crossing time,

whereas BALB/cJ mice did not present difficulty crossing. On the

other hand, our findings are similar to the motor impairment described

in Kmt2d+/βGeo mice13 corroborating with the loss-of-function of

KMT2D gene.

Male and female mice were evaluated by phenotypic characteriza-

tion. Although the use of males alone is the most common approach,

it is frequent to find discussions about variability related to the

estrous cycle,42 and tests with females showed some interesting

results related to the mutant phenotype. Mutant males showed a

greater hindquarter fall and increased micturition frequency compared

with the female group. As expected, male mice urinate throughout the

cage as a territorial marking, whereas females limit micturition to

restricted zones.56 Furthermore, female mutant bapa mice presented

a diminished tail squeeze compared with the female BALB/cJ. By con-

trast, both female groups presented an increased tail squeeze and

diminished auricular reflex compared with the male groups. Consider-

ing the balance beam score and time, both groups of females had

worse performance than the males.

In the gait analysis, spatial parameters such as the stride length,

step length and step width showed the geometric position of the foot

prints during mouse locomotion,27 and they are highly correlated to

walking speed.57 Additionally, it is known that the normal gait pattern

for rodents tends to be symmetric; thus, the step length is approxi-

mately 50% of the stride length for either the fore- or hindlimbs.27

The spatial gait pattern changes observed in male bapa mice showed a

shorter stride length for the fore- and hindlimbs, a shorter step length

for the hindlimbs, and a gait symmetry greater than 0.5. For a normal

rodent gait, a spatial symmetry value of approximately 0.5 indicates

that the right footprint is centered between the two left footprints.25

Gait abnormalities were described in mice models for Parkinson's

disease and Huntington's disease, as well as amyotrophic lateral scle-

rosis (ALS).58,59 Changes in the gait pattern would be related to mor-

phological and functional alterations in the cerebellum, including

those induced by damage to the nigrostriatal dopaminergic sys-

tem.58,60 Malformations in the cerebellum have been reported in

some KS patients61 and could be linked with central nervous system

symptoms, such as hypotonia and reduced fine motor coordination.

It is important to consider that the bapa mutants described here

presents a mutation in exon 13 outside of the SET domain. Although

most mutations responsible for KS in human are found in the C-

terminal portion, some mutations in the N-terminal region have also

been identified in humans.41

In conclusion, the mutation identified in the Kmt2d gene (c.

A3865G: p.T1289A) in bapa mice may be an exciting model to study the

function of this gene, as the mutation appears to affect the methyl-

transferase activity. Additionally, phenotypic characterization of the

bapa mouse highlighted a valuable animal model to study mechanisms

involved in psychomotor impairment, hypotonia and fine motor coor-

dination, as well as behavior disturbances as hyperactivity.

ACKNOWLEDGMENTS

We thank the Core Facility for Scientific Research—University of Sao

Paulo (CEFAP-USP/GENIAL) for NGS sequencing. This manuscript is


based upon work supported by the Fundação de Amparo à Pesquisa

do Estado de São Paulo (FAPESP) under grant numbers 2012/2538

7-2, 2016/23659-6 and 2017/21103-3; Conselho Nacional de Des-

envolvimento Científico e Tecnológico (CNPq) 14411/2017-1 and the

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—

Brasil (CAPES)—Finance Code 001.

ORCID

Claudia M. C. Mori https://orcid.org/0000-0002-9393-240X

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SUPPORTING INFORMATION

Additional supporting information may be found online in the

Supporting Information section at the end of this article.

How to cite this article: Yamamoto PK, de Souza TA,

Antiorio ATFB, et al. Genetic and behavioral characterization

of a Kmt2d mouse mutant, a new model for Kabuki Syndrome.

Genes, Brain and Behavior. 2019;e12568. https://doi.org/10.

1111/gbb.12568


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https://doi.org/10.1111/gbb.12568https://doi.org/10.1111/gbb.12568https://www.researchgate.net/publication/331901984

Genetic and behavioral characterization of a Kmt2d mouse mutant, a new model for Kabuki Syndrome1 INTRODUCTION2 MATERIALS AND METHODS2.1 Ethics statement2.2 Mice2.3 Genetic mapping, exome enrichment and sequencing2.4 Phenotypic characterization2.5 Statistical analysis

3 RESULTS3.1 Genetic mapping, exome enrichment and sequencing3.2 Phenotypic characterization

4 DISCUSSION4 ACKNOWLEDGMENTS REFERENCES

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Genetic and behavioral characterization of a Kmt2d mouse … · A study developed in Brazil by Massironi et al11 with the purpose of inducing new mutations in BALB/cJ mice allowed