Genetics

Mutations in the ABCA3 Gene Are Associated With CataractMicrocornea Syndrome

Peng Chen,1 Yunhai Dai,1 Xiaoming Wu,1 Ye Wang,1 Shiying Sun,1 Jingjing Xiao,2
Qingyan Zhang,3 Liping Guan,2 Xiaowen Zhao,1 Xiaodan Hao,1 Renhua Wu,2 and Lixin Xie1
1

State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong
Academy of Medical Sciences, Qingdao, Shandong Province, China
2BGI-Shenzhen, Shenzhen, China
3
BGI Tianjin Corporation, Tianjin, China

Correspondence: Lixin Xie, State

Key Laboratory Cultivation Base,
Shandong Provincial Key Laboratory
of Ophthalmology, Shandong Eye
Institute, Shandong Academy of
Medical Sciences, Qingdao, Shandong Province 266071, China;
Lixinxie@public.qd.sd.cn.
PC and YD contributed equally to the
work presented here and should
therefore be regarded as equivalent
authors.
Submitted: February 13, 2014
Accepted: October 27, 2014
Chen P, Dai Y, Wu X, et al. Mutations
in the ABCA3 gene are associated with
cataract-microcornea syndrome. Invest Ophthalmol Vis Sci.
2014;55:80318043. DOI:10.1167/
iovs.14-14098

PURPOSE. Cataract-microcornea syndrome (CCMC) is an autosomal dominant inherited disease

characterized by the association of congenital cataract and microcornea without any other
systemic anomaly or dysmorphism. Although mutations of several genes have been shown to
cause dominant CCMC, in many patients the causative gene has not yet been identified. Our
aim was to identify the disease-associated gene in Chinese patients with CCMC.
METHODS. The CCMC patients from two unrelated Chinese families and 26 sporadic patients
were enrolled. All the patients were screened by Sanger sequencing with no identified
mutations. Genetic variations were screened by whole-exome sequencing and then validated
using Sanger sequencing.
RESULTS. By sequencing the whole exome of three patients in a Chinese four-generation
dominant CCMC family (Family A), three heterozygous missense mutation (c.115C>G,
c.277G>A, and c.4393G>A) were identified in ATP-binding cassette protein A3 (ABCA3). At
highly conserved positions, changes (c.115C>G and c.4393G>A) were predicted to have
functional impacts and completely cosegregated with the phenotype. We further confirmed
our finding by identifying another heterozygous missense mutation, c.2408C>T, in ABCA3 in
an additional dominant CCMC family (Family B), which also cosegregated with the
phenotype. Moreover, four heterozygous mutations, two missense mutations (c.4253A>T,
c.2069A>T) and two splice site mutations (c.40532T>C, c.2765-1G>T) were identified
from the sporadic patients. The ABCA3 protein was expressed in human lens capsule,
choroid-retinal pigment epithelium and retinal pigment epithelial cells.
CONCLUSIONS. Mutations in the human ABCA3 gene were associated with lethal respiratory
distress. Our study showed, for the first time to our knowledge, that mutations in ABCA3
were associated with CCMC, warranting further investigations on the pathogenesis of this
disorder.
Keywords: cataract-microcornea syndrome, exome sequencing, ABCA3, heterozygous
mutation

ongenital cataract is a leading cause of childhood blindness,

accounting for approximately 10% to 38%,1 with a
prevalence of approximately 0.006% to 0.06% in live births.2,3
It may occur alone or in association with other ocular or
systemic abnormalities. Microcornea, one of the most frequent
abnormalities associated with congenital cataract, results from
secondary damage of the lens maldevelopment or from
mutations in some growth or transcription factors.4
The combination of congenital cataract and microcornea,
cataract-microcornea syndrome (CCMC; OMIM 116200), appears as a distinct phenotype affecting 12% to 18% of heritable
congenital cataract patients.5 Genetically, CCMC is a heterogeneous condition. To date, approximately 200 genes and loci
have been associated with cataracts.4,6 Of these genes,
mutations in at least nine genes were reported to be responsible
for congenital cataract associated with microcornea, including
genes encoding crystallins (crystallin alpha-A [CRYAA], OMIM
123580; crystallin beta-A4 [CRYBA4], OMIM 123631; crystallin

beta-B1 [CRYBB1], OMIM 600929; crystallin beta-B2 [CRYBB2],

OMIM 123620; crystalline gamma-C [CRYGC], OMIM 123680;
and crystallin gamma-D [CRYGD], OMIM 123690),5,714 gap
junction protein alpha 8 (GJA8, OMIM 600897),5,15 v-maf avian
musculoaponeurotic fibrosarcoma oncogene homolog (MAF,
OMIM 177075),16,17 and solute carrier family 16 member 12
(SLC16A12, OMIM 611910).18 Only the transcription factor,
MAF, has exclusively been associated with CCMC, whereas
mutations in the other genes were reported in congenital
cataract without additional malformations.5
Whether the CCMC phenotype is related to specific cataract
gene alleles or closely linked modifiers remains to be clarified.
Fortunately, the technique of exome sequencing has come to
the aid, enabling the identification of disease-associated
mutations by sequencing the whole exome of a small number
of affected individuals.1921 In the present study, diseaseassociated mutations were identified in a Chinese family with
dominant CCMC using the exome sequencing technique.

Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.
www.iovs.org j ISSN: 1552-5783

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ABCA3 Gene and Cataract Microcornea Syndrome

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FIGURE 1. Pedigrees of the two Chinese families with dominant cataract-microcornea syndrome. (A) Pedigree of Family A. (B) Pedigree of Family B.
Affected men and women are indicated by filled squares and circles, respectively. Normal individuals are shown as empty symbols. Deceased
individuals are indicated with slashes (/).

METHODS
Ethics Statement
The study was performed in accordance with the Declaration
of Helsinki and approved by the Ethics Committee of Shandong
Eye Institute (Qingdao, China). Written informed consent was
obtained from all participants (or guardians).

Study Population
Two Han Chinese families (designated as Family A and Family
B) with dominant CCMC, 26 sporadic patients with CCMC and
their parents, and 200 matched, normal controls (27.20 6 7.13
years old, 117 males) were included. The 26 sporadic patients
and their parents, and the 200 controls of Han Chinese
ethnicity were nonrelated Qingdao locals recruited at the
Qingdao eye hospital of Shandong Eye Institute (Qingdao,
China). All participants underwent an extensive, standardized
examination by ophthalmologists. The diagnosis was confirmed with ophthalmic examinations, including visual acuity,
slit-lamp microscopy, tonometry, keratometry, specular microscopy, ultrasonic A/B scan, and a history of cataract extraction.
Ocular photographs were taken by slit-lamp photography.
There was no family history of other systemic abnormalities in
Family A, Family B, and the 26 sporadic patients.

Mutation Screening in Reported Causative Genes

of CCMC
All exons and exon/intron border regions were directionally
sequenced and aligned to the GenBank reference sequences.
The following genes were included in the DNA sequencing
analysis: CRYAA, NM_000394; CRYBA1, NM_005208; CRYBB1,
NM_001887; CRYGC, NM_020989; CRYGD, NM_006891;
GJA8, NM_005267; MAF, NM_005360. Primers used to amplify

the coding exons and adjacent intronic regions of the seven

genes were determined according to previous reports.16,22
Individual exon was amplified by PCR and analyzed on an
ABI 3730XL Genetic Analyzer (Applied Biosystems, Foster City,
CA, USA). Sequencing data were compared pair-wisely with the
Human Genome database.

Targeted Capture and Exome Sequencing

The exome sequencing approach was used to identify the
disease-causing genetic variant for the dominant CCMC Family
A. Exome sequencing was performed on three patients (II2,
III1, and III4) at the BGI, Inc., Shenzhen, China. Venous blood
(5 mL) was collected from the participants, and total human
genomic DNA was isolated with the DNA isolation kit for
mammalian blood (Tiangen, Beijing, China). Venous blood and
genomic DNA samples were stored at 808C before use.
NimbleGen (44 Mb) target enrichment system (Roche NimbleGen, Inc., Madison, WI, USA) was used to collect the
protein coding regions of human genome DNA.
The array was able to capture 18,283 (99.6%) of the 18,357
genes. The gene sequences for this array were available in the
Consensus Coding Sequence Region (CCDS) database (available in the public domain at http://www.ncbi.nlm.nih.gov/
projects/CCDS/). The exon-enriched DNA libraries then were
subjected to a second library construction in preparation for
Illumina GA sequencing and were sequenced using the
Illumina Genome Analyzer II platform, following the manufacturers instructions (Illumina, San Diego, CA, USA).

Variant Analysis
The sequencing reads were aligned to the human reference
genome (NCBI Build 36.3). Alignment of the sequences from
the three affected individuals was performed using SOAPaligner after the duplicated reads were removed,23 and single

ABCA3 Gene and Cataract Microcornea Syndrome

IOVS j December 2014 j Vol. 55 j No. 12 j 8033

FIGURE 3. B-scan ultrasonagraphic photographs of the affected

individuals in Family A (AD) and Family B (EL). The phenotypes
are described and summarized in Table 1.
FIGURE 2. Slit-lamp photographs of the affected individuals in Family A
(AD) and Family B (EK). The phenotypes are described and
summarized in Table 1. OD, right eye; OS, left eye.

nucleotide polymorphisms (SNPs) were called using SOAPsnp

set with the default parameters.24 Indels affecting coding
sequence or splicing sites were identified as described
previously.25
Data were provided as lists of sequence variants (SNPs and
short indels) relative to the reference genome. Identified
variants were filtered against the Single Nucleotide Polymorphism database (dbSNP 129, available in the public domain at
http://www.ncbi.nlm.nih.gov/projects/SNP/snp_summary.cgi/),
1000 genome project (February 28, 2011 releases for SNPs, and
February 16, 2011 releases for indels, available in the public
domain at http://www. 1000genome.org/), HapMap 8 (available
in the public domain at http://hapmap.ncbi.nlm.nih.gov/)
database, and YH database.26

Verification of Variants
Sanger sequencing was used to determine whether any of the
remaining variants cosegregated with the disease phenotype in
Family A. Primers flanking the candidate loci were designed

based on genomic sequences of Human Genome (hg18/

build36.3) and synthesized at the BGI, Inc. All shared variants
of the three affected individuals after filtering then were
confirmed by PCR and analyzed on an ABI 3730XL Genetic
Analyzer. Sequencing data were compared pair-wisely with the
Human Genome database.
Afterwards, we sequenced all the exons and flanking
introns of the ABCA3 gene (NM_001089) in patients of Family
B and 26 sporadic patients to detect other mutation sites using
the Sanger sequencing method. As an additional step, the
detected variants were sequenced in 200 normal control
subjects.

Human Ocular Tissues and Cell Culture

Expression of the ABCA3 gene in the human cornea, sclera,
conjunctiva, iris-ciliary body (ICB), retina, choroid-RPE, lens
capsule, and human retinal pigment epithelial cell line (ARPE19, catalog No.CRL-2302; ATCC, Manassas, VA, USA) were
evaluated.
Human donor ocular tissues were provided by the Eye Bank
of Shandong Eye Institute. The whole globes, enucleated
within 10 hours of death from human donors, were

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ABCA3 Gene and Cataract Microcornea Syndrome

TABLE 1. The Clinical Features of Patients With Cataract-Microcornea Syndrome in the Two Chinese Families
Patient

Age,
y/Sex

Eye

Visual
Acuity

Family A:II2

59/F

OD

FC

FC

OS

FC

OD

Family A:III1

Family A:III4

Family B:III2

Family B:III3

Family B:III7

Family B:IV2

Family B:IV5

35/F

22/F

68/F

65/M

49/M

48/F

44/M

Best Corrected
Visual Acuity

Nystagmus

IOP,
mm Hg

Axial Length,
mm

Aphakia

Yes

23

25.38

FC

Aphakia

Yes

22

26.13

FC

FC

Cataract

Yes

16

22.95

OS

HM

HM

Cataract

Yes

18

23.26

OD

FC

FC

Aphakia

Yes

20

23.35

OS

20/200

20/200

Aphakia

Yes

19

23.5

OD

10/200

10/200

Aphakia, posterior subcapsular

cataract

Yes

18

32.44

OS

HM

HM

Cataract

Yes

22

32.37

OD

FC

FC

Aphakia, posterior subcapsular

cataract

Yes

16

26.97

OS

10/200

10/200

Aphakia, posterior subcapsular

cataract

Yes

18

26.98

OD

0.02

0.05

Aphakia

Yes

16

22.22

OS

0.05

0.05

Aphakia

Yes

17

21.45

OD

0.02

0.02

Cataract

Yes

14

28.44

OS

0.05

0.2

IOL, posterior subcapsular

cataract

Yes

15

25.38

OD

0.3

0.5

Aphakia

Yes

14

30.78

OS

0.05

0.05

IOL

Yes

14

30.53

Lens

FC, finger counting; F, female; IOL, intraocular lens; M, male; NA, not available; OD, right eye; OS, left eye.

immediately dissected, and the cornea, sclera, conjunctiva,

ICB, retina, choroid-RPE, lens capsule, and RPE were collected.
The ARPE-19 cells were cultured in 1:1 of Dulbeccos
modified Eagle medium/nutrient mixture F12 (DMEM/F12;
Invitrogen, Carlsbad, CA, USA), containing 10% fetal bovine
serum (FBS; Invitrogen). The cells were incubated at 378C in a
humidified atmosphere of 5% CO2 and 95% air. The culture
medium was changed every 3 days.

(TaKaRa, Dalian, China). Gene-specific cDNA fragments were

amplified with DNA polymerase (Tiangen). The expression of
genes was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primer sequences used for ABCA3 were
forward-5 0 -GAGGAGAGCCTCACTTCTGG-3 0 , reverse-5 0 -CGTAC
ATGACCAGCATCTCC-3 0 and for GAPDH were forward-5 0 ACCACAGTCCATGCCATCAC-3 0 , reverse-5 0 -TCCACCACCCTGT
TGCTGTA-3 0 . The PCR amplification products were analyzed
by agarose gel electrophoresis.

Reverse-Transcription PCR
Total RNA was prepared from venous blood (0.2 mL) of the
family members, using the RNA isolation kit for mammalian
blood (Tiangen). Total RNA was prepared from each human
ocular tissue, using the NucleospinRNA kit (BD Biosciences,
Palo Alto, CA, USA), and reversely transcribed into first-strand
cDNA, using Primescript First-Strand cDNA Synthesis kit

Western Blotting and Antibodies

Total protein was prepared from each tissue using radioimmunoprecipitation assay (RIPA) buffer (50 mmol/L Tris, 150
mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1%
SDS, sodium orthovanadate, and sodium fluoride, pH 7.4;
Galen, Beijing, China) and quantified. Protein (50 lg in 15 lL

IOVS j December 2014 j Vol. 55 j No. 12 j 8035

ABCA3 Gene and Cataract Microcornea Syndrome

TABLE 1. Extended
Corneal Curvature,
D

Corneal Diameter,
mm

Endothelial Cells
Density, mm

Min:53.1

Transverse:8.0 Vertical:8.5

2197

Aphakia, Vitreous bodies opaque,

posterior scleral staphyloma

Phacoemusification

MAX:53.25
Min:52.73

Transverse:8.0 Vertical:8.0

2341

Aphakia, Vitreous bodies opaque,

posterior scleral staphyloma

Phacoemusification

Transverse:8.0 Vertical:8.2

2419

NA

No

Transverse:8.0 Vertical:8.0

2395

NA

No

Transverse:9.5 Vertical:9.0

2572

Aphakia

Phacoemusification

Transverse:9.0 Vertical:9.5

2216

Aphakia

Phacoemusification

Transverse:9.8 Vertical:9.5

2637

Aphakia, Vitreous bodies opaque,

posterior scleral staphyloma

Phacoemusification

MAX:48.4
Min:50.5

Transverse:9.6 Vertical:9.5

2919

Vitreous bodies opaque, posterior

scleral staphyloma,cataract

No

MAX:52.6
Min:40.90

Transverse:9.0 Vertical:9.0

2176

Vitreous bodies opaque, posterior

scleral staphyloma

No

MAX:42.48
Min:41.10

Transverse:9.0 Vertical:9.5

2305

Vitreous bodies opaque, posterior

scleral staphyloma

No

MAX:42.04
Min:44.12

Transverse:8.0 Vertical:8.0

2693

Aphakia, Vitreous bodies opaque,

posterior scleral staphyloma

Phacoemusification

Transverse:8.0 Vertical:8.5

2512

Aphakia, Vitreous bodies opaque

Phacoemusification

Transverse:9.5 Vertical:9.8

2692

Vitreous bodies opaque, posterior

scleral staphyloma

No

MAX:45.38
Min:43.96

Transverse:9.6 Vertical:9.8

2247

Vitreous bodies opaque, posterior

scleral staphyloma

PhacoemusificationIOL

MAX:46.27
Min:45.10

Transverse:9.8 Vertical:9.5

2350

Aphakia, Vitreous bodies opaque,

posterior scleral staphyloma

Phacoemusification

MAX:45.60
Min:45.10

Transverse:9.5 Vertical:9.5

2587

Vitreous bodies opaque, posterior

scleral staphyloma

PhacoemusificationIOL

MAX:53.12
Min:45.6
MAX:47.5
Min:47.8
MAX:49.3
Min:47.5
MAX:51.9
Min:45.9
MAX:50.75
Min:47.03

MAX:47.82
Min:45.91
MAX:47.15
Min:45.25

B-Scanning

Surgery and Trauma

History

MAX:46.00

loading buffer) was resolved in 10% SDS-PAGE gel before

transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The blots were blocked in 5% nonfat
dry milk dissolved in Tris-buffered saline Tween-20 (TBST; 20
mmol/L Tris, pH 7.5, 0.5 mmol/L NaCl, 0.05% Tween-20) for 1
hour and then incubated with the primary antibody in TBST for
1 hour, followed by incubation with horseradish peroxidaseconjugated secondary antibody (Amersham Biosciences, Uppsala, Sweden) for 1 hour. All incubations were conducted at
258C, and three washes with 10 mL TBST were performed
between each step. The membranes then were developed with
SuperSignal West Femto Maximum Sensitivity substrate (Pierce
Biotechnology, Rockford, IL, USA) and exposed to X-ray film
(Kodak, Rochester, NY, USA). The immunoreactive bands were
quantified using National Institutes of Health (NIH) Image 1.62
software (NIH, Bethesda, MD, USA). All the experiments
reported in this study were performed three times, and the
results were reproducible. For each sample, the levels of

proteins of interest were normalized to that of GAPDH.

Primary antibodies included anti-ABCA3 antibody (Abcam,
Cambridge, MA, USA) and anti-GAPDH antibody (Kangchen,
Shanghai, China).

Immunocytochemical Staining
Cells were plated in a glass culture dish (Biousing Biotech Co,
Wuxi, China) at a density of approximately 4.0 3 104 cells/35mm dish in complete medium and grown to subconfluence.
After the medium was removed, the cells were fixed in 4%
paraformaldehyde in PBS for 15 minutes, followed by three PBS
washes. The fixed cells were incubated with 0.2% Triton X-100
in PBS for 5 minutes, blocked in 5% BSA, and incubated with
anti-ABCA3 antibody (Abcam) for 30 minutes, before incubation with the fluorescence-conjugated secondary antibody for
1 hour. Images were obtained using an Eclipse TE2000-U
confocal laser scanning microscope (Nikon, Tokyo, Japan).

0.996
1.0

PROBABLY DAMAGING
PROBABLY DAMAGING

DAMAGING
DAMAGING

N1418I
E690V

AAT4253ATT
GAG2069GTG

Novel
Novel
Novel
Novel
chr16/2331134/ABCA3
chr16/2347524/ABCA3
chr16/2347541/ABCA3
chr16/2333185/ABCA3

Missense
Missense
Splice site
Splice site

0.801
POSSIBLY DAMAGING
DAMAGING
T803M
ACG2408ATG
Novel
chr16/2345597/ABCA3

Missense

0.006
BENIGN
TOLERATED
V93I
GTC277ATC
rs199840288

Missense

0.857
POSSIBLY DAMAGING
TOLERATED
L39V
CTC115GTC
Missense

Prediction
From SIFT
Substitution
Codons
Mutation
Type

PROBABLY DAMAGING

chr16/2376053/ABCA3

To identify the causative gene of CCMC, exome sequencing

was performed on DNA samples obtained from three affected
members of Family A (II2, III1, and III4). At the depth of
coverage, approximately 94.4% of each targeted exome was
sufficiently covered to pass our thresholds for variant calling.
After identification of variants, we focused only on nonsynonymous (NS) variants, splicing site (SS) mutations, and
short, frame-shift coding insertions or deletions (indel) that
were more likely to be pathogenic mutations than other
variants. The total NS/SS/indel variants in patients II2, III1, and
III4 of Family A are shown in Table 2.
Because CCMC is a rare disorder, but has a clear phenotype,
there was a very low likelihood of the causal mutation in these
patients being shared with a wider healthy population. We
compared the shared variants in these patients with the dbSNP
129, 1000 genome project, HapMap 8 database, and YH

rs200090198

Exome Sequencing Identified ABCA3 as the

Associated Gene

chr16/2376215/ABCA3

Direct sequencing of the CRYAA, CRYBA1, CRYBB1, CRYGC,

CRYGD, GJA8, and MAF exons showed no pathogenic
mutations in any of the affected individuals in Family A and
Family B.

dbSNP rs#
Cluster ID

Mutation Screening in Reported Causative Genes

of CCMC

Chromosome/
Position/Gene Name

Herein we described two Han Chinese families from Qingdao

that had monogenic CCMC with a dominant inheritance model
(Fig. 1A). By ophthalmic examinations, three of 24 members in
Family A, a four-generation family, were identified to be affected
with CCMC (Figs. 1A, 2AD). Another patient in this family was
deceased. Five affected individuals (Figs. 1B, 2EK) were found
among the 48 examined family members in the five-generation
Family B, which also had three deceased patients.
The two families in our study had some common features,
such as congenital cataract and nystagmus. The corneal
diameters ranged from 8.0 to 9.8 mm (8.89 6 0.76 mm in
the transverse meridian and 8.99 6 0.68 mm in the vertical
meridian). The axial length ranged from 21.45 to 32.44 mm
(26.38 6 3.62 mm). The B-scan ultrasonagraphy showed
posterior scleral staphyloma in patient II:2 of Family A (Figs.
3A, 3B) and patients III:2, III:7, IV:2, and IV:5 of Family B (Figs.
3EL). There was no family history of other systemic
abnormalities. Detailed clinical characteristics of the patients
from the two families are summarized in Table 1.

Patient

Clinical Assessment and Findings

TABLE 3. Genetic Variants Identified in ABCA3 in the Two Chinese Families and the 5 Sporadic Patients With Cataract-Microcornea Syndrome

RESULTS

DAMAGING

22588
22693
22104
254

D1465N

7095
7088
6992
105

GAT4393AAT

2612
2638
2574
33

Missense

12881
12967
12538
116

Total

rs201955122

II2
III1
III4
II2III1III4Not in
1000 Genomes
Project, the
dbSNP129, HapMap
8, YH database

Nonsynonymous Splicing
SNP
Site
Indel

Prediction From
PolyPhen2

Filter

chr16/2329098/ABCA3

Genetic Variants

II2, III1, III4 (family

A)
II2, III1, III4 (family
A)
II2, III1, III4 (family
A)
III2, III3, III7, IV2,
IV5 (family B)
Sporadic 4
Sporadic 13
Sporadic13
Sporadic12, 15, 17

Score of Prediction
From PolyPhen2

TABLE 2. Genetic Variants Identified in the Three Patients (II2, III1,

and III4) of Family A Through Exome Resequencing

0.994

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ABCA3 Gene and Cataract Microcornea Syndrome

ABCA3 Gene and Cataract Microcornea Syndrome

IOVS j December 2014 j Vol. 55 j No. 12 j 8037

FIGURE 4. Sequence chromatograms of all ABCA3 mutations identified in the study. The left chromatogram represents the sequences of the affected
individuals. The right chromatogram represents the sequence of a normal family member. The arrow indicates the location of the mutations.

database. This left a total of 254 variants that were shared

among these three patients. Of these, 116 nonsynonymous
SNPs, 33 SSs, and 105 indels were predicted to potentially have
a functional impact on the gene (Table 2).
Segregation then was analyzed by Sanger sequencing on the
116 nonsynonymous SNPs, 33 SSs, and 105 indels, with the 24
members of the Family A. Three variants in ABCA3 gene
cosegregated with the disease phenotype in Family A:
c.115C>G resulting in an L39V amino acid change,
c.277G>A resulting in an V93I amino acid change, and
c.4393G>A resulting in an D1465N amino acid change (Table
3, Fig. 4).

Mutations in ABCA3 Gene

To confirm ABCA3 as the associated gene of CCMC, we used
Sanger sequencing to screen all members of Family B. All five
clinically affected subjects, but none of those who were
unaffected in Family B, carried the heterozygous c.2408C>T
mutation resulting in a T803M amino acid change (Table 3; Fig.
4). Thus, the heterozygous missense mutation T803M in

ABCA3 completely cosegregated with the dominant CCMC

phenotype within Family B.
We further carried out direct Sanger sequencing of the
ABCA3 exons in the 26 unrelated (based on their self-identified
geographical ancestry), sporadic patients with CCMC. A total
of 43 eyes of 26 patients (12.6 6 17.41 years old, 10 males)
had congenital cataract and nystagmus. The corneal diameter
of the 43 eyes ranged from 5.0 to 9.5 mm (7.84 6 1.39 mm in
the transverse meridian and 7.90 6 1.34 mm in the vertical
meridian).
In the 26 sporadic patients, we identified a total of 4
variants when we sequenced the ABCA3 exons, and all the 4
variants (present in 5 unrelated individuals) were filtered
against the 1000 Genomes Project, the dbSNP129 database,
HapMap 8, YH database, and 200 controls. Detailed clinical
characteristics of the five patients are summarized in Table 4
and Figure 5. There were no ocular abnormalities and no family
history of other systemic abnormalities in the parents of the 26
sporadic patients.
These four heterozygous mutations identified from the
sporadic patients were c.4253A>T resulting in an N1418I

IOVS j December 2014 j Vol. 55 j No. 12 j 8038

ABCA3 Gene and Cataract Microcornea Syndrome

TABLE 4. The Clinical Features of the 5 Patients With ABCA3 Gene Mutation in the 26 Sporadic Patients With Cataract-Microcornea Syndrome

Lens

Nystagmus

IOP,
mm
Hg

FC

Cataract

Yes

14

21.37

Min:43.90
MAX:44.90

Transverse:8.0
Vertical:8.0

HM

HM

Cataract

Yes

14

22.18

Min:43.60
MAX:44.70

Transverse:8.0
Vertical:8.0

OD

0.5

0.8

Normal

No

14

21.03

OS

HM

HM

Cataract

Yes

15

18.33

OD

HM

HM

Cataract

Yes

16

25.8

Min:44.05
MAX:45.25
Min:43.50
MAX:46.50
NA

Transverse:10.8
Vertical:10.5
Transverse:8.5
Vertical:8.5
Transverse:5.0
Vertical:5.0

OS

0.4

0.8

Normal

No

17

25.05

Min:40.05
MAX:41.6

Transverse:11.2
Vertical:11.0

OD

0.08

0.08

Cataract

Yes

23

19.95

NA

Transverse:7.0
Vertical:7.0

OS

FC

FC

Cataract

Yes

16

13.3

NA

OD

NA

NA

Cataract

Yes

16

17.86

Min:43.60
MAX:46.60

Transverse:5.0
Vertical:5.0
Transverse:7.0
Vertical:7.0

OS

NA

NA

Normal

No

18

19.73

Min:45.36
MAX:46.70

Transverse:10.6
Vertical:11.0

Patient

Age,
y/Sex

Eye

Sporadic 4

33/M

OD

FC

OS

Sporadic 12

Sporadic 13

Sporadic 15

Sporadic 17

6/F

7/M

7/F

0.5/F

Visual
Acuity

Best
Corrected
Visual Acuity

amino acid change, c.2069A>T resulting in an E690V amino

acid change, 40352T>C, and 2765-1G>T (Table 3, Figs. 4IP).
In the parents of the 5 sporadic patients (sporadic 4, 12, 13, 15,
and 17), no variant in ABCA3 gene was detected when we
sequenced the ABCA3 exons.
The c.4393G>A generated a heterozygous missense mutation (p.Asp1465Asn). The c.2408C>T generated a heterozygous missense mutation (p.Thr803Met). The c.2069A>T
generated a heterozygous missense mutation (p.Gly690Val).
The c.4253A>T generated a heterozygous missense mutation
(p.Asn1418Ile). The p.Gly690Val mutation was located in the
first nucleotide binding domain. The p.Asn1418Ile and
p.Asp1465Asn mutations were located in the second nucleotide binding domain. The p.Thr803Met mutation was located
in the loop between the first nucleotide binding domain and
the seventh membrane-spanning domain (Fig. 6A).
Multiple alignments of Asp1465, Thr803, Gly690, and
Asn1418 of the human ABCA3 protein (Homo sapiens,
NP_001080) from different species revealed 100% identity,
which suggested that they were highly conserved during
evolution (Fig. 6B). The Sorting Intolerant From Tolerant (SIFT)
and Polymorphism Phenotype (PolyPhen) tool analysis revealed a score for each of the four mutations and predicted that
the replaced amino acid was damaging to protein function.
The c.115 C>G generated a missense mutation (p.Leu39Val). Multiple alignments of Leu39of the human ABCA3 protein

Axial
Length,
mm

Corneal
Curvature,
D

Corneal
Diameter,
mm

B-Scanning
Vitreous
bodies
opaque
Vitreous
bodies
opaque
Normal
Short axial
length
Vitreous
bodies
opaque,
posterior
scleral
staphyloma
Vitreous
bodies
opaque,
posterior
scleral
staphyloma
Short axial
length,
posterior
scleral
staphyloma
Short axial
length
Posterior
scleral
staphyloma
Normal

from different species revealed 100% identity, which suggested

that it was highly conserved during evolution (Fig. 6B). The
SIFT tool analysis predicted that the replaced amino acid was
tolerated to protein function. The PolyPhen-2 tool analysis
predicted that the replaced amino acid was possibly
damaging to protein function.

ABCA3 Expression
To get an insight on the expression of ABCA3 expression in eye
development, we performed an investigation of mouse embryo
in the Eurexpress database (available in the public domain at
http://www.eurexpress.org/ee/intro.html). The ABCA3 was
expressed in the eye of mouse embryo. To get an insight on
the expression of ABCA3 in human eye tissues, we performed
an extensive examination of human expressed sequences
located in the NCBI UniGene database (available in the public
domain at http://www.ncbi.nlm.nih.gov/unigene/) and in the
Eyebrowse site (available in the public domain at http://
eyebrowse.cit.nih.gov/), which displayed expressed sequence
tags (ESTs) obtained from complementary DNA clones from
eye tissues derived from NEIBank and other sources. We
identified a number of ESTs matching to the ABCA3 gene in
different parts of the eye, such as fetal eyes, lens, eye anterior
segment, optic nerve, retina, retinal fovea and macula, RPE,
and choroid (Table 5).

IOVS j December 2014 j Vol. 55 j No. 12 j 8039

ABCA3 Gene and Cataract Microcornea Syndrome

TABLE 5. Results of Nucleotide BLAST (blastn) Searches of ABCA3 Expressed Sequence Tags Located in the Unigene Database
GenBank EST
CK299203.1
CK301091.1
BE251915.1
BG471039.1
BG474383.1
BM465915.1
BM711374.1
BM716567.1
BM717900.1
BQ186032.1
BQ187006.1
BQ189474.1
BU153899.1
BU176836.1
BU184378.1
BU738127.1
CA393439.1

Sequence Length, bp
679
493
618
664
906
1009
317
726
506
689
590
741
870
907
894
645
484

Eye Tissue
Fetal eyes, lens,
Fetal eyes, lens,
Retinoblastoma
Retinoblastoma
Retinoblastoma
Retinoblastoma
Fetal eyes
Fetal eyes
Fetal eyes, lens,
Fetal eyes, lens,
Fetal eyes, lens,
Fetal eyes, lens,
Retinoblastoma
Retinoblastoma
Retinoblastoma
Fetal eyes
RPE/choroid

eye anterior segment, optic nerve, retina, retina foveal and macular, RPE and choroid
eye anterior segment, optic nerve, retina, retina foveal and macular, RPE and choroid

eye
eye
eye
eye

anterior
anterior
anterior
anterior

To make sure that the ABCA3 was expressed in the eye, we

examined ABCA3 expression in different human ocular tissues
using RT-PCR and Western blotting. The ABCA3 gene and
protein were expressed in the human lens capsule, choroid-

segment,
segment,
segment,
segment,

optic
optic
optic
optic

nerve,
nerve,
nerve,
nerve,

retina,
retina,
retina,
retina,

retina
retina
retina
retina

foveal
foveal
foveal
foveal

and
and
and
and

macular,
macular,
macular,
macular,

RPE
RPE
RPE
RPE

and
and
and
and

choroid
choroid
choroid
choroid

RPE, and ARPE-19 cells (Fig. 7). In ARPE-19, ABCA3 was

detected in the cytoplasm and on the plasma membrane by
immunofluorescence microscopy (Fig. 8).
To confirm the change of the ABCA3 expression in the
patients with CCMC and unaffected family members in
Families A and B, total RNA and protein were prepared from
venous blood. The ABCA3 mRNA level (normalized to GAPDH)
was approximately 55% lower in patients with CCMC than in
unaffected family members (*P < 0.01; Figs. 9A, 9C). The
ABCA3 protein level (normalized to GAPDH) was approximately 70% lower in patients with CCMC than in unaffected
family members (*P < 0.01; Figs. 9B, 9C).

DISCUSSION

FIGURE 5. Slit-lamp and B-scan ultrasonagraphic photographs of

sporadic patients with CCMC. (A, B) Slit-lamp photographs of sporadic
patient 13. (C, D) Slit-lamp photographs of sporadic patient 15. (E, F)
B-scan ultrasonagraphic photographs of sporadic patient 12. (G, H) Bscan ultrasonagraphic photographs of sporadic patient 15. (I, J) B-scan
ultrasonagraphic photographs of sporadic patient 17. The phenotypes
are described and summarized in Table 4.

Microcornea-cataract syndrome is an autosomal dominant

inherited disease characterized by the association of congenital
cataract and microcornea without any other systemic anomaly
or dysmorphism. Clinical findings include a corneal diameter
inferior to 10 mm in both meridians, and an inherited cataract.
Other rare ocular manifestations include myopia, iris coloboma, sclerocornea, and Peters anomaly. Transmission seems to
be autosomal dominant, sometimes with a high degree of
penetrance. Although mutations of several genes have been
shown to cause dominant CCMC, in many patients the
causative gene has not yet been identified.
In this study, we have identified a novel dominant CCMC
associated gene, ABCA3, which had different heterozygous
missense mutations in two autosomal dominant CCMC families
(c.115C>G, c.277G>A, c.4393G>A, and c.2408C>T). Another
four heterozygous mutations, 2 missense (c.4253A>T, N1418I;
c.2069A>T, E690V), and 2 splice site mutations (c.
40532T>C, c.2765-1G>T) were identified from the sporadic
patients. Parental clinical information of the 26 sporadic
patients had been gathered. There were no ocular abnormalities and no family history of other systemic abnormalities. In
the parents of the 5 sporadic patients (sporadic 4, 12, 13, 15,
and 17), no variants in ABCA3 gene were detected when we
sequenced the ABCA3 exons.
We provided several lines of evidence to support the
contention that the ABCA3 gene was the pathogenic causative
gene: (1) three heterozygous missense variant in ABCA3 gene
was identified in Family A with dominant CCMC via exome
sequencing, (2) a different heterozygous missense mutation
was identified in another family with dominant CCMC (Family

ABCA3 Gene and Cataract Microcornea Syndrome

IOVS j December 2014 j Vol. 55 j No. 12 j 8040

FIGURE 6. (A) Model of ABCA3 protein structure. The protein is embedded in the membrane, and has two similar domains, each consisting of six
membrane spanning domains (cylinders) and a nucleotide binding domain (NBD). The locations of the identified mutations associated with CCMC
are indicated. ECD1 and ECD2, extracellular domains; NBD1 and NBD2, nucleotide binding domains. Numbers refer to amino acid position. (B)
Alignment of sequences surrounding the L39V, V93I, E690V, T803M, N1418I, and D1465N mutation in human, chimpanzee, monkey, pig, rat, and
mouse. The five mutations (L39V, E690V, T803M, N1418I, and D1465N) in ABCA3 are highly conserved among different species. The various species
included Homo sapiens (NP_001080.2), Pan troglodytes (chimpanzee, XP_510744.2), Macaca mulatta (Rhesus monkey, XP_001085237.2), Sus
scrofa (pig, XP_003124787.2), Rattus norvegicus (Norway rat. XP_001054650.1), Mus musculus (house mouse, NP_001034670.1), and Danio
rerio (zebrafish, XP_002661144.3).

B), (3) these variants completely cosegregated with the disease

phenotype in the two families, (4) two heterozygous missense
mutations and two heterozygous mutations in SSs were
identified in 5 sporadic patients with CCMC, (5) five ABCA3
mutations (c.115C>G, c.4393G>A, c.2408C>T, c.4253A>T,
and c.2069 A>T) were present at a conserved site among
different vertebrates, (6) four missense mutations (c.4393G>A,
c.2408C>T, c.4253A>T, and c.2069 A>T) were predicted to be
functionally damaging by SIFT and PolyPhen-2 tools, (7) the
ABCA3 mRNA and protein levels were significantly lower in
patients with CCMC than in unaffected family members, (8)
neither mutation was seen in the 200 unaffected healthy
controls.
The ABC family of transporters is a large family of related
transmembrane proteins that bind and hydrolyze ATP to
translocate a wide variety of substrates across biological
membranes.27,28 These proteins share a common structure,
with half-transporters having 6 membrane spanning domains

and a cytoplasmic ATP-binding domain with conserved motifs

and full transporters containing 12 transmembrane regions and
2 nucleotide-binding domains. The ABCA3 protein is encoded
by a single gene, located on human chromosome 16 which
contains 33 exons, with the first 3 exons being untranslated,
and the gene is referred to as ABCA3.2935 The ABCA3 gene
spans over 80,000 nucleotide bases and is transcribed into an
approximately 6500base pair (bp) mRNA, which directs the
synthesis of a 1704amino acid protein.33 Although the cDNA
for ABCA3 was first isolated from thyroid tissue, it is most
highly expressed in lung tissue. Its expression is low in a wide
range of other human tissues, including heart, brain, and
kidney, and platelets.33,35
Recently, mutations in the human ABCA3 gene were
associated with lethal respiratory distress.36,37 Genetic variants
identified in ABCA3 in patients with surfactant metabolism
dysfunction-3 (SMDP3) were shown in Table 6.

ABCA3 Gene and Cataract Microcornea Syndrome

IOVS j December 2014 j Vol. 55 j No. 12 j 8041

FIGURE 7. Expression of ABCA3 in human ocular tissues and ARPE-19. (A) The mRNA expression of ABCA3 in the human cornea, conjunctiva, ICB,
sclera, retina, choroid-REP, lens capsule, and ARPE-19 cells. (B) Expression of ABCA3 in the human cornea, conjunctiva, ICB, retina, choroid-REP,
lens capsule, and ARPE-19 cells by Western blotting. ARPE-19, human retinal pigment epithelial cell; ICB, iris-ciliary body.

It may be that a specific interaction between an unknown

protein and ABCA3 during eye development is disrupted by the
specific mutations identified in congenital cataract patients;
these would rather not result from a decrease in the amount of
ABCA3 proteins but in conformational properties of the
mutant proteins. There were now some well documented
examples of severe bronchopulmonary dysplasia related to
heterozygous mutations in the ABCA3 gene.38,39 The existence
of an increasing number of instances where the heterozygous
state carries significant risk burdens is a compelling argument
for further study and clarification of the risk and the processes
by which these disorders are manifested.
All the mutations that we identified in the two dominant
CCMC families and 26 sporadic patients affected by CCMC
were heterozygous, and the mutations in Table 3 were entirely

FIGURE 8. Representative figures of immunolabeling of ABCA3 in

ARPE-19 cells. The arrowhead indicates the expression of ABCA3. The
asterisk indicates the location of nucleus. Magnification: 16003.

FIGURE 9. Confirmation of the change of ABCA3 expression in the

patients with CCMC and unaffected members of Family A and B. (A)
The ABCA3 mRNA expression in the patients with CCMC and
unaffected family members. (B) ABCA3 protein expression in the
patients with CCMC and unaffected family members. (C) Analysis of
ABCA3 expression as mean 6 SD. *P < 0.01, as compared with
unaffected family members.

IOVS j December 2014 j Vol. 55 j No. 12 j 8042

ABCA3 Gene and Cataract Microcornea Syndrome

TABLE 6. Genetic Variants Identified in ABCA3 in Patients With Surfactant Metabolism Dysfunction-3 (SMDP3)
dbSNP rs# Cluster ID
rs121909181
rs121909182
rs121909183
rs28936691
rs121909184

rs121909185

Codons

Substitution

Mutation Type

Mutation Mode

c.3426G>A
c.301T>C
c.4657T>C
c.4772A>C
c.1702G>A
c.49091G>A
c.977T>C

W1142X
L101P
L1553P
Q1591P
N568D

L326P

Missense
Missense
Missense
Missense
Missense
Splice site
Missense

Homozygosity
Homozygosity
Homozygosity
Heterozygosity
Heterozygosity
Homozygosity
Homozygosity

different from that in ABCA3 gene, which was associated with

lethal respiratory distress. The p.Gly690Val mutation was
located in the first nucleotide binding domain. The p.Asn1418Ile and p.Asp1465Asn mutations were located in the
second nucleotide binding domain. The p.Thr803Met mutation
was located in the loop between the first nucleotide binding
domain and the seventh membrane-spanning domain. Two
transmembrane domains consisting of multiple membranespanning a-helices (typically six a-helices per domain) form the
conduit through which substrate crosses the membrane. These
domains also contain a substrate-binding site (or sites), which
contributes to transport specificity. Two nucleotide binding
domains couple the energy of ATP hydrolysis for substrate
translocation. Multiple alignments of Asp1465, Thr803,
Gly690, and Asn1418 of the human ABCA3 protein were
highly conserved during evolution.
Given that ABCA3 is predicted to be a glycoprotein that
may hydrolyze ATP to provide energy for substrate transport
involved in eye development, a mutant ABCA3 protein may
impact the normal eye development. However, the exact
mechanism of ABCA3 action and its role in dominant CCMC
pathogenesis remain unclear, and future functional studies
will be important. To date, there have been no documented
studies on the ABCA3 gene in eye development, and the
data in our study indicating its involvement in a devastating
eye disease provided excellent motivation for future
investigation of the ABCA3 gene, which in turn should
enable dissection of its relationship with dominant CCMC
pathogenesis.

Acknowledgments
The authors thank the participating families and sporadic patients,
and the Single Nucleotide Polymorphism database, 1000 genome
project, HapMap 8 database, and YH database for the data set used
to filter variants.
Supported by the National Natural Science Foundation of China
(81070759, 81300742), and the Young and Middle-Aged Scientists
Research Awards Fund of Shangdong Province, China
(BS2013YY013). The authors alone are responsible for the content
and writing of the paper.
Disclosure: P. Chen, None; Y. Dai, None; X. Wu, None; Y. Wang,
None; S. Sun, None; J. Xiao, None; Q. Zhang, None; L. Guan,
None; X. Zhao, None; X. Hao, None; R. Wu, None; L. Xie, None

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