Duplications involving the long range HMX1 enhancer are associated with human isolated bilateral concha-type microtia

Abstract

Background:
Microtia is a congenital anomaly of ear that ranges in severity from mild structural abnormalities to complete absence of the outer ears. Concha-type microtia is considered to be a mild form. The H6 family homeobox 1 transcription factor gene (HMX1) plays an important role in craniofacial structures development. Copy number variations (CNVs) of a downstream evolutionarily conserved enhancer region (ECR) of Hmx1 associated with ear and eye abnormalities have been reported in different animals, but not yet in human. To date, no genetic defects responsible for isolated human microtia has been reported except for mutations in HOXA2. Here we recruited five Chinese families with isolated bilateral concha-type microtia, and attempt to identify the underlying genetic causes.

Methods:
Single Nucleotide polymorphism (SNP) array was performed to map the disease locus and detect CNVs on a genome scale primarily in the largest family (F1). Whole genome sequencing was performed to screen all SNVs and CNVs in the candidate disease locus. Array comparative genomic hybridization (aCGH) was then performed to detect CNVs in the other four families, F2-F5. Quantitative real-time polymerase chain reaction (qPCR) was used to validate and determine the extent of identified CNVs containing HMX1-ECR region. Precise breakpoints in F1 and F2 were identified by gap-PCR and sanger sequencing. Dual-luciferase assays were used to detect the enhancer function. qPCR assays were also used to detect HMX1-ECR CNVs in 61 patients with other types mictrotia.

Results:
Linkage and haplotype analysis in F1 mapped the disease locus to a 1.9 Mb interval on 4p16.1 containing HMX1 and its downstream ECR region. Whole genome sequencing detected no potential pathogenic SNVs in coding regions of HMX1 or other genes within the candidate disease locus, but it detected a 94.6 Kb duplication in an intergenic region between HMX1 and CPZ. aCGH and qPCRs also revealed co-segregated duplications in intergenic region downstream of HMX1 in the other four families. The 21.8 Kb minimal overlapping region encompassing the core sequences consensus with mouse ECR of Hmx1. Luciferase assays confirmed the enhancer function in human sequences, and proved that HOXA2 could increase its enhancer activity. No CNVs were detected in HMX1-ECR regions in 61 patients with other type of microtia.

Conclusion:
Duplications involving long range HMX1 enhancers are associated with human isolated bilateral concha-type microtia. We add to evidences in human that copy number variations in HMX1-ECR associates with ear malformations, as in other species. This study also provides an additional example of functional conserved non-coding elements (CNEs) in humans.

Full text

Sietal. J Transl Med (2020) 18:244
https://doi.org/10.1186/s12967-020-02409-6
RESEARCH
Duplications involving thelong range HMX1
enhancer are associated withhuman isolated
bilateral concha-type microtia
Nuo Si1,2† , Xiaolu Meng2†, Xiaosheng Lu3, Zhe Liu4, Zhan Qi2, Lianqing Wang2, Chuan Li1, Meirong Yang1,
Ye Zhang1, Changchen Wang1, Peipei Guo1, Lingdong Zhu5, Lei Liu6, Zhengyong Li7, Zhenyu Zhang7,
Zhen Cai8, Bo Pan1*, Haiyue Jiang1* and Xue Zhang2
Abstract
Background: Microtia is a congenital anomaly of ear that ranges in severity from mild structural abnormalities to
complete absence of the outer ears. Concha-type microtia is considered to be a mild form. The H6 family homeobox 1
transcription factor gene (HMX1) plays an important role in craniofacial structures development. Copy number vari-
ations (CNVs) of a downstream evolutionarily conserved enhancer region (ECR) of Hmx1 associated with ear and eye
abnormalities have been reported in different animals, but not yet in human. To date, no genetic defects responsible
for isolated human microtia has been reported except for mutations in HOXA2. Here we recruited five Chinese families
with isolated bilateral concha-type microtia, and attempt to identify the underlying genetic causes.
Methods: Single Nucleotide polymorphism (SNP) array was performed to map the disease locus and detect CNVs
on a genome scale primarily in the largest family (F1). Whole genome sequencing was performed to screen all SNVs
and CNVs in the candidate disease locus. Array comparative genomic hybridization (aCGH) was then performed to
detect CNVs in the other four families, F2-F5. Quantitative real-time polymerase chain reaction (qPCR) was used to
validate and determine the extent of identified CNVs containing HMX1-ECR region. Precise breakpoints in F1 and F2
were identified by gap-PCR and sanger sequencing. Dual-luciferase assays were used to detect the enhancer function.
qPCR assays were also used to detect HMX1-ECR CNVs in 61 patients with other types mictrotia.
Results: Linkage and haplotype analysis in F1 mapped the disease locus to a 1.9 Mb interval on 4p16.1 containing
HMX1 and its downstream ECR region. Whole genome sequencing detected no potential pathogenic SNVs in cod-
ing regions of HMX1 or other genes within the candidate disease locus, but it detected a 94.6 Kb duplication in an
intergenic region between HMX1 and CPZ. aCGH and qPCRs also revealed co-segregated duplications in intergenic
region downstream of HMX1 in the other four families. The 21.8 Kb minimal overlapping region encompassing the
core sequences consensus with mouse ECR of Hmx1. Luciferase assays confirmed the enhancer function in human
sequences, and proved that HOXA2 could increase its enhancer activity. No CNVs were detected in HMX1-ECR regions
in 61 patients with other type of microtia.
Conclusion: Duplications involving long range HMX1 enhancers are associated with human isolated bilateral
concha-type microtia. We add to evidences in human that copy number variations in HMX1-ECR associates with ear
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Open Access
Journal of
Translational Medicine
*Correspondence: 13810855912@163.com; haiyuejiang2019@163.com
†Nuo Si and Xiaolu Meng contributed equally to this work
1 Plastic Surgery Hospital, Chinese Academy of Medical Sciences
and Peking Union Medical College, Beijing, China
Full list of author information is available at the end of the article
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Background
Dumbo, the famous Disney cartoon character, is an
elephant with oversized ears that enable it to fly. Some
real-life animals with abnormal external ears are also
named “dumbo” such as the dumbo mouse and dumbo
rat [1, 2]. Almost all mammals have outer ears (pinna)
of variable sizes. e main function of the pinna is to
collect sound waves and direct them into the ear. In
some species, pinna also serve functions such as dis-
sipating heat and signaling mood. In humans, different
congenital pinna malformations are observed. Microtia
(OMIM 600674) is an external ear developmental mal-
formation characterized by a small, abnormally shaped
pinna [3]. It ranges from mild structural abnormali-
ties to complete absence of the ear, affects one or both
ears, and occurs as isolated or syndromic birth defects.
e concha-type microtia is considered a mild form
of microtia, with remnant ear lobule, concha, acoustic
meatus, tragus, and incisura intertragica [4]. Because of
the variety of severity and forms, it is hard to estimate
microtia prevalence—reported data varies from 0.83 to
17.4 in 10,000 live births worldwide [3]. e causes of
microtia among most patients are unknown, although
some risk factors have been reported, such as gesta-
tional exposure to teratogens, maternal diabetes, and
higher maternal parity [3].
Genetic studies have made great progress in under-
standing ear development and function by identifying
underlying genetic defects of certain diseases, espe-
cially in hearing loss, but less is known about genetic
control of external ear morphogenesis. To date, only
HOXA2 mutations have been reported as responsible
for isolated bilateral microtia with or without hearing
loss in humans [5–7]. Single-gene defects and chromo-
somal aberrations have also been reported in different
microtia-associated syndromes [3, 8]. Nevertheless,
efforts in finding coding region mutations in genes
responsible for microtia-associated syndromes have
failed for isolated microtia. Among these, the H6 fam-
ily homeobox 1 transcription factor gene (HMX1) in
4p16.1 deserves special attention. It plays an important
role downstream of embryonic patterning genes in lat-
eral facial mesenchyme differentiation [1]. In human,
recessive loss of function mutations in HMX1 have
been associated with oculoauricular syndrome (OAS,
OMIM 142992) characterized by malformation of the
external ear and eyes [9, 10]. A linkage locus of 10-Mb
encompassing 4p16 has been reported in a five-gener-
ation Chinese family with isolated bilateral microtia
[11].
In the human genome, 98% of sequences are non-
coding but harbor many regulatory elements that direct
the precise spatial and temporal expression of coding
genes. Comparing genomic sequences from diverse
vertebrate species has revealed numerous highly con-
served non-coding regions near developmental regula-
tory genes, particularly transcription factors and these
regions are considered to have potential regulatory
functions [12]. ese functional regions are collectively
referred to conserved non-coding elements (CNEs).
e general features of CNEs were noticed, including
their non-random distribution in line with key devel-
opmental regulatory target genes across genomes, the
distinguished sequence features with AT-rich and runs
of identical nucleotides, the overlapping with tran-
scription factor binding sites and known function as
developmental enhancers in many cases [12].Human
diseases and phenotypic changes have been associated
with alterations in CNEs [13–18]. One of the well-char-
acterized example is the SHH ZRS enhancer, in which
point mutations and copy number variations could
result in limb malformation in both human and other
species [19–21]. In wild populations of animals, a CNE
proximal to the Hmx1 was also noticed and proved to
be associated with external ear development [22].Struc-
tural variants (SVs) such as deletions, duplications,
insertions and inversions can disrupt or rearrange
functional genomic elements [23, 24]. e genetic eti-
ology of many diseases such as limb malformation and
autism has been proven to relate to rare inherited SVs
in coding gene cis-regulatory elements [25–27]. For
ear development, evidence in mice implicated an evo-
lutionarily conserved enhancer region (ECR) down-
stream of Hmx1 as an important regulatory element
driving ear development. Hoxa2, Meis and Pbx can
act cooperatively on a 32bp core sequence within the
ECR to regulate Hmx1 expression [28]. Mutations in
Hmx1 coding region and SVs involving the Hmx1-ECR
region have been found in animals with dysmorphic
external ears, including ‘dumbo’ or ‘misplaced ears’ in
mice, ‘dumbo’ in rats, ‘crop ear’ in highland cattle, and
‘short ear’ in Altay sheep (Table1) [1, 2, 10, 29, 30]. In
human genome, ~ 600bp conserved sequence homolo-
gous to mouse Hmx1-ECR was also observed. However,
malformations, as in other species. This study also provides an additional example of functional conserved non-cod-
ing elements (CNEs) in humans.
Keywords: Microtia, Duplication, HMX1, Long range enhancer, Conserved non-coding elements
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Sietal. J Transl Med (2020) 18:244
whether genetic changes affecting this region are asso-
ciated with human ear malformations is unknown.
In the present study, we show that duplications
involving HMX1-ECR are associated with human
isolated bilateral concha-type microtia. A ~ 600 bp
human ECR sequence may function as a tissue-specific
enhancer regulating HMX1 expression and response to
HOXA2 in the lateral facial mesenchyme that contrib-
utes to outer ear development.
Materials andmethods
Subjects
Five Han Chinese families with isolated bilateral microtia
were included in the present study (Fig.1). All patients
were clinically evaluated, and digital photographs were
taken to document ear phenotypes in affected individu-
als. Family 1 (F1) consisted of 25 individuals including
10 affected individuals with microtia in four generations.
Family 2 (F2) and Family 3 (F3) each had six affected
individuals in four generations. Family 4 (F4) and
Table 1 Genomic changes anddysmorphic outer ear phenotypes acrossspecies
Phenotype description Phenotype/disease entry Species Genomic changes Inheritance References
Enlarged ear pinnae with a
distinctive ventrolateral shift,
microphthalmic anomalies
Dumbo (dmbo) Mouse Nonsense mutation in Hmx1
exon1 Recessive Munroe et al. [1]
Laterally-protruding ears and
microphthalmic anomalies Misplaced ears (mpe) Mouse 8 bp deletion in Hmx1 exon2 Recessive Munroe et al. [1]
Congenital malformations of
the pinna and modest reduc-
tion in ocular size
Dumbo (dmbo) Rat 5777 bp deletion encompass-
ing Hmx1-ECR Recessive Quina et al. [2]
Moderately to severely trun-
cated ear Crop ear Highland cattle 76 bp Hmx1-ECR duplication Dominant Koch et al. [29]
Shorter and thicker ear Short ear Altay sheep 76 bp Hmx1-ECR duplication Dominant He et al. [30]
Ophthalmic anomalies and
external ear abnormalities Oculoauricular syndrome (OAS) Human 26 bp deletion in HMX1 coding
region Recessive Schorder et al. [10]
Bilateral external ear malforma-
tion/cup ear Concha type microtia Human Duplications involving HMX1-
ECR Dominant This study
Fig. 1 Five families with isolated bilateral Concha-Type Microtia. a Pedigree of five families (F1-F5). Individuals with available blood samples are
indicated with an asterisk. b–q Identical pinna phenotypes in five families. All patients have identical bilateral concha-type microtia phenotype, and
representative individuals from each family are shown. IV-4 (b), III-8 (c), IV-6 (d), IV-5 (e) in F1; III-4 (f, g), III-6 (h), IV-7 (i) in F2; IV-1 (j, k), III-1 (l), IV-2 (m)
in F3; II-1 in F4 (n, o), II-1 in F5 (p, q)
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Sietal. J Transl Med (2020) 18:244
Family 5 (F5) are nuclear families with an affected child
and affected mother. e ear malformations are consist-
ent within the five families (Fig.1b–q). We also recruited
53 patients with oculoauricular syndrome, six patients
with severe bilateral isolated microtia and two patients
with bilateral syndromic microtia. Blood samples from
all available family members were collected following
informed consent. e study was reviewed and approved
by the institutional review board of the Chinese Academy
of Medical Sciences.
Genotyping, whole genome linkage andhaplotype
analysis
Affymetrix Genome-Wide Human SNP array 5.0 was
used to perform whole genome linkage analysis in the
four-generation F1 family. Genomic DNA samples from
10 individuals were genotyped following the manufac-
turer’s instructions. Genotype calling and quality control
were performed with the Affymetrix Genotyping Con-
sole 2.1 package. Parametric multipoint linkage analysis
was performed using MERLIN v.1.1.2 under the assump-
tions of autosomal-dominant inheritance with 99% pen-
etrance, a disease allele frequency of 0.1%, and equal
SNP allele frequency (50%). Genotyping and data analy-
sis were accomplished at the CapitalBio Corporation
(Beijing, China). Selected polymorphic micro-satellite
markers within candidate disease loci were genotyped.
Polymorphic micro-satellite markers and amplification
primers are summarized in Additional file1: TableS1.
Whole genome sequencing
Ten individuals from F1 (III2, III3, III4, III5, III8, III9,
IV1, IV4, IV6, IV7) underwent whole genome sequencing
(WGS) using the NEBNext Ultra II DNA Library Prep kit
for Illumina (New England Biolabs, Ipswich, MA, USA)
and a HiSeq X Ten sequencer (Illumina, San Diego, CA,
USA). Reads were aligned to the GRCh37/hg19 human
reference sequence using the Burrows-Wheeler Aligner
(BWA, v.0.7.8-r455) and variant calling was performed
with SAMtools (v.1.0) and annotated using ANNO-
VAR (v.2015Dec14). Picard (v.1.111) was used to merge
BAM files of the same sample and filter out duplicate
reads marked. SNP/Indel, CNV, and SV variants were
called and classified by SAMtools (v.1.0), Control-FREEC
(v.V7.0), and CREST (v.V0.0.1), respectively. WGS was
performed and bioinformatic analysis accomplished at
the Novogene Corporation (Beijing, China).
Microarray analysis
Genomic copy number changes at locus 4p16.1 in F2-5
were further tested by the Laboratory of Clinical Genetics
of Peking Union Medical College Hospital using a High-
Resolution Array CGH analysis (SurePrint G3 Human
1x1M; Agilent Technologies, Santa Clara, CA, USA). One
patient from each family was selected to undergo micro-
array analysis. e experiment and data analysis were
performed according to the manufacturer’s instructions.
In brief, patient and control DNA were labeled and com-
bined to hybridize to the 60mer oligonucleotide-based
microarray. e resulting fluorescent signals were auto-
matically scanned by the Agilent SureScan Microarray
Scanner. Agilent CytoGenomics software was then used
to extract and translate the signal into log ratios for fur-
ther analysis of copy number changes.
Real‑time quantitative PCR (qPCR) andGap‑PCR
We performed qPCR to confirm the HMX1-ECR dupli-
cation and determine the extent of duplications in dif-
ferent families. qPCR primer sequences and amplicon
positions are given in Additional file2: TableS2. qPCR
assays were performed using SYBR premix Ex Taq
(TaKara Bio., Dalian, China), and reactions were run in
a Rotor-gene 6000 real-time rotary analyzer (Qiagen,
Hilden, Germany) as previously reported [19]. Data
were analyzed by Rotor Gene Q series software (Qia-
gen, Hilden, Germany). e relative copy number (RCN)
of the target sequence was determined by the compara-
tive ΔΔCt method where ΔCt = (mean CtTarget) − (mean
CtReference) and ΔΔCt = ΔCtpatient − ΔCtcontrol. An RCN of
~ 1.5 indicated a heterozygous duplication. For F1 and F2,
Gap-PCR was designed according to the extent of dupli-
cation implicated by qPCR assays. q20 forward and q3
reverse primers were used for Gap-PCR in F1, while q35
forward and q8 reverse primers were used for Gap-PCR
in F2 (Additional file2: TableS2). Breakpoint junctions
were detected by direct Sanger sequencing of Gap-PCR
products.
Dual‑luciferase activity assay
hECR and mECR fragments were PCR amplified from
genomic DNA and inserted into the pGL4.23 firefly lucif-
erase vector (Promega, Madison, WI, USA) using either
a restriction digest strategy or the In-Fusion cloning kit
(TaKaRa Bio, Beijing, China). e human HOXA2 cDNA
sequence was inserted into the multiple cloning site of the
pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA)
using HindIII and BamHI. All plasmids were sequenced
to confirm correct fragment insertion. Primers for
plasmid construction are summarized in Additional
file 3: Table S3. COS-1 cells were plated into 24-well
plates1day before transfection and grown until 70–90%
confluent. For each well, 500ng luciferase reporter vec-
tor was transfected into the cells using Lipofectamin™
3000 Reagent (Invitrogen, Carlsbad, CA, USA) with or
without the pcDNA3.1 expression vector, with 25ng of
the pRL-TK Renilla luciferase vector used as an internal
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Sietal. J Transl Med (2020) 18:244
control to normalize transfection efficiency. 24h post-
transfection, cells were harvested and lysed with 100μl
passive lysis buffer (Promega, Madison, WI, USA). e
firefly and renilla luciferase activities for each 20μl cell
lysate were measured by the Microplate Luminometer
CentroLB 960 (Berthold, Germany). Relative luciferase
activity was calculated by the ratio of firefly luciferase
activities/renilla luciferase activities as fold change com-
pared to pGL4.23. Assays were conducted as indicated
in the dual luciferase reporter assay system manual (Pro-
mega, Madison, WI, USA). Normalized luciferase activ-
ity fold change (mean ± SD) of three experiments with six
duplicates each is reported.
Results
Mapping ofasusceptibility locus on4p16.1
Genome-wide linkage analysis in F1 suggested three can-
didate loci: a 20Mb interval on 4p16.1, a 25Mb interval
on 4q, and a 2Mb interval on 5q. Genotyping of selected
polymorphic microsatellite markers within candidate
regions of 4q and 5q showed no co-segregation status
in F1. However, one polymorphic microsatellite marker
(CHLC.GATA151E03) on 4p16.1 co-segregated with
phenotype in F1. Fine mapping using Affymetrix SNP
5.0 microarray probe-sets refined the critical region to
1.9Mb between rs4696668 to rs16891285 (chr4:8061832–
9954880, hg19) with a HLOD score of 1.8. e interval
includes 13 protein-coding genes including HMX1, yet
we found no potential coding region mutations in these
genes by sanger sequencing.
Identication oftheHMX1‑ECR duplication inve families
withisolated bilateral concha‑type microtia
We further performed WGS in 10 members of F1. Con-
sistent with the previous sanger sequencing result, no
potential mutations were identified in the coding region.
However, WGS implicated a ~ 95 Kb duplication in
the critical interval in six patients, but not in two unaf-
fected members or two unrelated members in the family
(Fig. 2a). e duplication encompasses a partial intra-
genic region between CPZ and HMX1, and involves
the ~ 600 bp evolutionarily conserved region down-
stream of HMX1 (HMX1-ECR). qPCR assays designed
within a 600bp critical region confirmed the duplication
and detected full-segregation status in F1 (Fig.2c). is
finding prompted us to detect copy number changes in
other families with isolated bilateral concha-type micro-
tia. ere are limited probes within the identified dupli-
cated region designed in commercial array CGH systems,
decreasing our accuracy and efficiency in CNV detection.
Nevertheless, SurePrint G3 Human 1x1M microarray
implicated increased copy number in a 46.2Kb intergenic
region (chr4: 8677567–8723767, hg19) between CPZ and
HMX1 in four additional families with the identical phe-
notype same as F1 (Fig.2b). Duplications were confirmed
by qPCR assay in the HMX1-ECR region in all four addi-
tional families (Fig.2c).
Determination oftheduplication extent andcritical region
To determine the size of the duplications in different
families, multiple qPCR assays were designed to cover a
region of 253Kb (chr4:8617326–8871246, hg19) encom-
passing CPZ, HMX1, and their intergenic region (Addi-
tional file2: TableS2). Extent and overlapping regions of
duplications in five families were detected (Fig. 3). We
performed qPCR assays on one affected individual per
family to determine the extent of duplication in each
family and identified duplications of 94.6 Kb, 147 Kb,
185–213Kb, 49.8–55.9 Kb, and 67.4–104Kb in F1, F2,
F3, F4 and F5, respectively (Fig. 3a). We detected the
precise duplicated segment and breakpoints by gap-PCR
and sanger sequencing in F1 (chr4:8638135–8732725,
hg19) and F2 (chr4: 8677560–8,824,629, hg19) (Fig.3b).
In F3, F4 and F5, multiple qPCR assays detected the
boundary regions harboring the breakpoints (Fig.3c). All
identified duplications contained a 21.8Kb overlapping
region (chr4:8,684,896–8,706,719, hg19) harboring the
HMX1-ECR.
A 600bp sequence withintheduplicated region shows
enhancer activity increased byHOXA2
A 594bp Hmx1-ECR region has been demonstrated to
be a specific enhancer determining endogenous Hmx1
lateral facial expression patterns in mouse [28]. us, a
600bp human sequence (hECR) in the identified dupli-
cated region homologous to the 594bp mouse sequence
(mECR) was tested for enhancer function by dual lucif-
erase assay (Fig. 4). As a result, constructs containing
hECR showed increased luciferase activity compared to
the empty group (replicate = 3, p < 0.0001), suggesting
hECR enhancer activity (Fig.4a). However, the induced
luciferase activity was significantly lower in the hECR
than in the mECR group, which is regulated by the Hox-
Pbx-Meis complex (Fig. 4a). HOXA2 mutations were
reported in patients with isolated bilateral microtia with-
out hearing loss. In the luciferase assay, co-transfection
with human HOXA2 expression vectors led to an 8.14-
fold increase in enhancer activation, indicating that the
hECR is responsive to HOXA2 (Fig.4b).
Detection ofHMX1‑ECR CNVs inpatients withother types
ofmicrotia
To determine whether HMX1-ECR CNVs associate with
other ear malformations, we performed qPCR assays in
the 600 bp HMX1-ECR in 53 patients with unilateral
lobule-type microtia, six patients with isolated bilateral
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Fig. 2 Detected duplications involving the long range HMX1 Enhancer in five families. a Whole genome sequencing indicated duplications in F1.
Red bar shows the duplicated region. b Duplications detected by array-CGH in F2-F5. Blue arrows show where the probes detected 3 copies. c qPCR
assays in the HMX1-ECR region confirm the duplication and co-segregation status with phenotype in five families
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lobule-type microtia, one patient with bilateral concha-
type microtia with preauricular sinus, and one patient
with bilateral concha-type microtia with atrial septal
defect. No duplications or deletions were detected in
these microtia cases. In health population, some duplica-
tions involving the HMX1-ECR region were documented
in the database of genomic variants (DGV) [31], but they
were relatively large in size and involved other nearby
Fig. 3 Extent and overlapping regions of duplications in five families. A. Schematic diagram showing detected duplications, 600 bp core hECR
sequences, and CNVs in the database of genomic variants. The blue line highlights the ECR region. B. Chromatogram of the breakpoint junctions in
F1 and F2. C. A series of qPCR assays detected the extent of duplications in F3, F4 and F5
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Sietal. J Transl Med (2020) 18:244
genes (at least CPZ) at the same time. ere were also
some duplications only involving the CPZ and HMX1
intergenic region, but they did not contain the HMX1-
ECR. While deletions only involving the intragenic region
between CPZ and HMX1, and containing HMX1-ECR
were also documented in the DGV database (Fig.3a).
Discussion
Microtia is phenotypically and etiologically heteroge-
neous. Little is known about the genetic background
underlying microtia. Among candidate loci for microtia,
Chromosome 4p16 deserves special attention. A par-
tial deletion from the short arm of chromosome 4 (4p
deletion) results in Wolf-Hirschhorn syndrome (WHS,
OMIM#194190) featuring a distinct craniofacial pheno-
type and intellectual disability [32]. WHS patients with
pure and translocated forms of monosomy 4p16.1 → pter
(M4p16.1) have different types of external ear malforma-
tion such as poorly rolled descending helix edge, short
ear lobes, or deep or long concha [33]. By studying 72
oculoauriculovertebral spectrum (OAVS) patients with
highly heterogeneous phenotypes involving ears, eyes,
face, neck and other organs, Bragagnolo etal. observed
recurrent chromosomal imbalances predominantly
in chromosome 4 in four patients [34]. Balikova et al.
reported on a large family with autosomal-dominantly
inherited microtia, eye coloboma, and imperforation of
the nasolacrimal duct, and found the phenotype linked
to a cytogenetically visible alteration at 4p16 consisting of
five copies of a copy-number-variable region [35]. Li etal.
reported a 10Mb susceptibility locus for isolated bilateral
microtia on 4p15.32–4p16.2 in a 5-generation Chinese
family [11].
HMX1 harbored in 4p16.1, also known as NKX5-3, is
an important transcription factor in craniofacial struc-
ture development, especially in eye and ear. Expression of
Hmx1 was observed in the external ear, lens, and retina
of mice as early as E13.5. In humans, HMX1 expression
was observed in the optic vesicle in the 5–6-week embry-
onic period and in the developing pinna and auricular
mesenchymatous cells at the 20-week fetus period [10].
e different expression patterns of HMX1 in ear and
eye development suggest that there may be different
regulatory elements determining strict spatial–tempo-
ral expression. Meanwhile, homozygous mutation in the
human HMX1 gene leads to abrogation of gene func-
tion causing oculoauricular syndrome (OAS, OMIM
#612109) affecting both the eye and external ear [9, 10,
36]. us, isolated microtia and syndromic microtia
without eye affects are unlikely to be caused by mutations
in the HMX1 coding region. Accordingly, we found no
potential HMX1 coding region mutations in 120 OAVS
patients by whole exome sequencing (unpublished data).
Conserved non-coding elements (CNEs) are sequences
outside of protein coding regions highly conserved across
diverse vertebrate species [37]. ey may act as cis-regu-
latory modules (CRMs) that interact with nearby genes
to determine tissue-specific gene expression, and they are
enriched near transcription factor genes expressed dur-
ing embryogenesis, suggesting a possible role in regulat-
ing the expression of essential developmental genes [38,
39]. CNEs are required for normal development, and
mutations in CNEs have been established as causal for
human diseases and subtle phenotypic changes that likely
lead to decreased fitness over evolutionary time [12].
Dickel etal. created knock out mice with individual or
Fig. 4 Human ECR within the duplicated region shows an enhancer activity increased by HOXA2. a hECR showed increased luciferase activity. b
hECR is responsive to HOXA2. ****p < 0.0001
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Sietal. J Transl Med (2020) 18:244
pairwise deletion of four CNEs near ARX, the essential
neuronal transcription factor [40]. ese knockout mice
showed substantial alterations of neuron populations and
structural brain defects that potentially detrimental in
the wild, although they were viable and fertile in labora-
tory conditions. Rosin etal. showed that Hmx1 has such
a CNE that functions as a strong and highly dynamic
lateral facial enhancer [28]. e CNE is a ~ 600bp evo-
lutionarily conserved region (ECR) with a 32 bp core
sequence containing consensus binding sites for Hoxa2,
Pbx, and Meis, and it has tissue-specific enhancer func-
tion in the craniofacial mesenchyme which contributes to
the pinna. Genomic structural variations disrupting the
ECR enhancer role associate with loss of Hmx1 expres-
sion specifically in the first and second branchial arch
(BA1 and BA2) mesenchyme, leading to dysmorphic
outer ears across species (Table 1). Genomic findings
in human patients with isolated bilateral concha-type
microtia reinforce the enhancer role of HMX1-ECR
in conserved pinna developmental processes. We also
noticed that hECR has weaker enhancer activity com-
pared to mECR via luciferase assay. However, it remains
unclear whether the difference in the relative size of the
pinna between human and mice is related to the level of
enhancer activity.
e core sequence of hECR is highly homologous to
mECR including the consensus binding sites of HOXA2,
PBX and MEIS [22, 28]. In dual luciferase assays, co-
transfection of HOXA2 and hECR resulted in increased
expression level, suggesting that the hECR may also be
regulated by the HOX-PBX-MEIS complex. HOX, PBX
and MEIS are all homeobox proteins involved in tran-
scriptional regulation by forming heterodimers and are
essential contributors to developmental programs. Genes
encoding this homeoprotein complex associate with con-
genital anomalies with craniofacial phenotypes. HOXA2
is the only reported gene responsible for isolated microtia
to date. Patients with homozygous mutations in HOXA2
display more severe microtia than hECR duplicated car-
riers, presenting middle ear deformities and hearing loss
[7]. PBX1 mutations lead to congenital kidney and uri-
nary tract anomalies with or without hearing loss, abnor-
mal ears, or developmental delay [41]. MEIS2 mutations
associate with cleft palate, cardiac defects, and mental
retardation [42, 43]. ese findings suggest that HOXA2,
PBX1, and MEIS2 act early in patterning of the branchial
arch region and transactivate HMX1 by binding to hECR.
erefore, we speculate that any genetic changes affect-
ing hECR regulation by the HOX-PBX-MEIS complex
may lead to developmental defects involving ears and
eyes.
Regulatory elements and their target gene clusters
often exist in the same local chromatin interaction
regions, called topologically associated domains (TADs),
to ensure that the regulatory elements are specific to
their target genes rather than other nearby genes [44].
Boundaries between TADs are required and provide
an insulator function to prohibit interference between
opposing activities of neighboring domains [44, 45]. We
used the 3D Genome Browser (http://promo ter.bx.psu.
edu/hi-c/) to visualize the chromatin interaction sur-
rounding HMX1. According to Hi-C profile data from
human embryonic stem cells, HMX1 and CPZ are in
two different TADs, while hECR sequences appear in the
same TAD with HMX1 but not with CPZ (Additional
file4: Figure S1). e detected duplications in isolated
bilateral concha-type microtia patients are in the same
TAD with HMX1, and do not interrupt the TAD bound-
ary. ey contain hECR but not the HMX1 gene. ere-
fore, these duplications may result in overexpression of
HMX1 by increasing the number of local enhancers but
not the coding gene. Meanwhile, the copy number varia-
tion (nsv1014219) in the DGV database detected in nor-
mal population involves the hECR, the CPZ gene, and
the boundary between two TADs. erefore, due to the
insulator effect of TAD boundaries, the increased hECR
could not interact with HMX1, thus it probably does not
change gene expression level.
Notably, the size of the hECR region (600 bp) is
small and its copy number changes could be missed by
chromosomal microarray analysis (CMA). Duplica-
tions detected in the present study range from ~ 50 to
~ 200Kb. Although they were implicated in the Agilent
SurePrint G3 Human CGH 1X1M microarray analysis,
they could not be automatically detected in standard
analysis process due to limited probes designed within
the region. e genomic findings in these patients indi-
cate the importance of checking the HMX1-ECR copy
number status and highlight the necessity for custom
designed microarrays with higher probe density covering
this region.
Conclusions
In this study, we found various genomic duplications
involving the HMX1-ECR long range enhancer in five
families with isolated bilateral concha-type Microtia.
e HMX1-ECR duplications were specifically associ-
ated with isolated bilateral concha-type microtia but
not with other ear malformations or syndromic micro-
tia. We add to evidence in humans that copy number
variations in HMX1-ECR, a conserved non-coding ele-
ments (CNEs), associates with ear malformations, as
in other species. We provide additional evidence that
the dosage sensitive effects of HMX1 may result in dif-
ferent types of ear malformations. Unveiling genetic
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Page 10 of 11
Sietal. J Transl Med (2020) 18:244
causes of isolated microtia provides an entry point into
understanding the regulatory network for common lat-
eral facial birth defects and complex syndromes involv-
ing external ear malformations. Meanwhile, the results
could be used for genetic counseling and screening for
isolated bilateral concha-type microtia.
Supplementary information
Supplementary information accompanies this paper at https ://doi.
org/10.1186/s1296 7-020-02409 -6.
Additional le1: TableS1. Polymorphic micro-satellite markers and
primers.
Additional le2: TableS2. qPCR primers in 4p16.1.
Additional le3: TableS3. Primers for plasmid construction.
Additional le4: Figure S1. Visualization of chromatin interaction sur-
rounding HMX1.
Abbreviations
CNVs: Copy number variations; ECR: Evolutionarily conserved enhancer region;
HMX1: The H6 family homeobox 1 transcription factor gene; SNP: Single
nucleotide polymorphism; aCGH: Array comparative genomic hybridization;
qPCR: Quantitative real-time polymerase chain reaction; OAS: Oculoauricular
syndrome; SVs: Structural variants; WGS: Whole genome sequencing; hECR:
Human sequence of evolutionarily conserved enhancer region; mECR: Mouse
sequence of evolutionarily conserved enhancer region; DGV: Database of
genomic variants; WHS: Wolf–Hirschhorn syndrome; OAVS: Oculoauriculover-
tebral spectrum; CNEs: Conserved non-coding elements; CRMs: Cis-regulatory
modules; BA1 and BA2: First and second branchial arch; TADs: Topologically
associated domains; CMA: Chromosomal microarray analysis.
Acknowledgements
We thank all participants in the study.
Authors’ contributions
NS, BP and conceived and designed the study. XL, CL, MY, YZ, CW, PG, LZ, LL,
ZL, ZZ, ZC, BP and HJ carried out the study of the clinical part. NS, XM, ZL, ZQ,
LW and BP performed the genetic analysis. NS, XM and BP wrote the manu-
script. NS and BP revised the manuscript. XZ, HJ contributed to supervision.
XZ, HJ, BP and ZL contributed to funding acquisition. All authors read and
approved the final manuscript.
Funding
This study was financially supported by the National Key Research and
Development Program of China (2016YFC0905100), the CAMS Innovation
Fund for Medical Sciences (CIFMS) (2016-I2M-1-002), the National Natural
Science Foundation of China (81571863, 81871574), the Key Laboratory of
craniofacial congenital malformation of Chinese Academy of Medical Sciences
(2018PT31051).
Availability of data and materials
All data generated or analyzed during this study were included in this pub-
lished article and its additional files.
Ethics approval and consent to participate
The study was approved by institutional review board of Chinese Academy of
Medical Sciences, and all participants signed written informed consent.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1 Plastic Surgery Hospital, Chinese Academy of Medical Sciences and Peking
Union Medical College, Beijing, China. 2 Institute of Basic Medical Sciences
Chinese Academy of Medical Sciences, School of Basic Medicine Peking, Union
Medical College, Beijing, China. 3 Department of Plastic Surgery, Affiliated
Hospital of Weifang Medical University, Beijing, China. 4 Laboratory of Clinical
Genetics, Peking Union Medical College Hospital, Chinese Academy of Medi-
cal Sciences & Peking Union Medical College, Beijing, China. 5 Department
of Plastic Surgery, Qilu Children’s Hospital of Shandong University, Jinan, China.
6 Department of Burns and Plastic Surgery, Second Hospital of Shandong
University, Beijing, China. 7 Department of Plastic and Burn Surgery, West
China School of Medicine, West China Hospital, Sichuan University, Sichuan,
China. 8 Department of Plastic Surgery, Sichuan Academy of Medical Sciences
& Sichuan Provincial People’s Hospital, Sichuan, China.
Received: 25 February 2020 Accepted: 5 June 2020
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