Personalized Diagnosis and

Management of Congenital Cataract by

Next-Generation Sequencing
1

Rachel L. Gillespie, MSc, James OSullivan, BSc, Jane Ashworth, FRCOphth,

3

1,2

1,2

Sanjeev Bhaskar, MSc,

Simon Williams, PhD, Susmito Biswas, FRCOphth, Elias Kehdi, FRANZCO, Simon C. Ramsden, FRCPath,
1,3
1,3
1,2
Jill Clayton-Smith, FRCP, Graeme C. Black, DPhil, FRCOphth, I. Christopher Lloyd, FRCOphth

1,3

Purpose: To assess the utility of integrating genomic data from next-generation sequencing and phenotypic
data to enhance the diagnosis of bilateral congenital cataract (CC).
Design: Evaluation of diagnostic technology.
Participants: Thirty-six individuals diagnosed with nonsyndromic or syndromic bilateral congenital cataract
were selected for investigation through a single ophthalmic genetics clinic.
Methods: Participants underwent a detailed ophthalmic examination, accompanied by dysmorphology
assessment where appropriate. Lenticular, ocular, and systemic phenotypes were recorded. Mutations were
detected using a custom-designed target enrichment that permitted parallel analysis of 115 genes associated
with CC by high-throughput, next-generation DNA sequencing (NGS). Thirty-six patients and a known positive
control were tested. Suspected pathogenic variants were confirmed by bidirectional Sanger sequencing in
relevant probands and other affected family members.
Main Outcome Measures: Molecular genetic results and details of clinical phenotypes were identified.
Results: Next-generation DNA sequencing technologies are able to determine the precise genetic cause of
CC in 75% of individuals, and 85% patients with nonsyndromic CC were found to have likely pathogenic
mutations, all of which occurred in highly conserved domains known to be vital for normal protein function. The
pick-up rate in patients with syndromic CC also was high, with 63% having potential disease-causing mutations.
Conclusions: This analysis demonstrates the clinical utility of this test, providing examples where it altered
clinical management, directed care pathways, and enabled more accurate genetic counseling. This comprehensive screen will extend access to genetic testing and lead to improved diagnostic and management
outcomes through a stratified medicine approach. Establishing more robust genotypeephenotype correlations
will advance knowledge of cataract-forming mechanisms. Ophthalmology 2014;121:2124-2137 2014 by the
American Academy of Ophthalmology.
Supplemental material is available at www.aaojournal.org.

Congenital cataracts (CCs) affect 2.5 to 3.5 per 10 000

children aged younger than 15 years of age in the United
1
Kingdom. They have been estimated to cause lifelong
visual loss in approximately 200 000 children worldwide
2
each year. Approximately 50% of CC cases have a genetic
3
basis resulting from disturbances in the packaging of
proteins within lens fibers, protein folding and solubility,
4e6
and the organization of lens fibers themselves.
Congenital cataract is frequently inherited as an autosomal
7
dominant trait, although autosomal recessive inheritance is
increasingly recognized, in particular in populations in
8
whom parental consanguinity is prevalent. The number of
cases with a genetic basis could be underestimated because
a proportion of children with sporadic CC represent de
9
novo dominant or recessively inherited mutations. In as
many as 50% of bilateral and the majority of unilateral CC
10,11
cases, the exact cause currently remains undetermined.

2124

2014 by the American Academy of Ophthalmology

Published by Elsevier Inc.

12

Inherited CC can present as an isolated anomaly with or

without other ocular malformations, such as microphthalmia
or anterior segment dysgenesis, but in the absence of other
13
systemic problems (nonsyndromic CC) or as a manifestation
14
of a systemic condition (syndromic CC). Approximately one
third of nonsyndromic bilateral CC cases are estimated to be
1
caused by genetic mutations, with approximately 50% of
mutations occurring in crystallin genes, a further 25% in
connexins, and the remainder in mainly structural proteins and
15
transcription factors. To date, mutations in approximately 25
genes have been associated with nonsyndromic CC, with more
16
yet to be identified. Congenital cataract is often one of the
first presenting features of many syndromic and metabolic
disorders, which collectively are thought to account for
9,15
approximately 15% of all CC cases.
Examples include
oculocerebrorenal (Lowe) syndrome, in which CC presents
http://dx.doi.org/10.1016/j.ophtha.2014.06.006
ISSN 0161-6420/14

Gillespie et al NGS for the Diagnosis of Congenital Cataract

alongside hypotonia, intellectual disability, and renal abnor17
malities ; Nance Horan syndrome, which features
18,19
characteristic dental anomalies together with CC
; and
Cockayne syndrome, a premature aging syndrome in which
cutaneous photosensitivity and sensorineural hearing loss
20
manifest in conjunction with CC.

Determining the precise genetic cause of CC has significant clinical relevance, defining clinical diagnosis and
clarifying inheritance patterns, thereby guiding genetic
counseling. Establishing a robust genotypeephenotype correlation permits correlations to be made between disease
progression and surgical outcomes, thereby increasing
prognostic accuracy. For patients with syndromic CC, ge-netic
testing enables prompt diagnosis, more appropriate
monitoring, and implementation of early treatment strategies.

Next-generation DNA sequencing (NGS) is increasingly

powerful as a diagnostic tool that offers speed, precision,
and cost-effectiveness that alternative methods of diagnosis
21
do not. More than 110 genes have been associated with
CC, making this an ideal area for parallel interrogation of
22
these genomic targets using NGS. This has proven
successful in determining the cause of other genetically
heterogeneous conditions, such as congenital muscular
dystrophy, hereditary hearing loss, dilated cardiomyopathy,
23e30
and inherited eye diseases.
This technology recently
31
has been translated into clinical practice, extending
access to genetic testing. The aim of this study was to
assess the clinical utility and validity of NGS in the
diagnosis of CC, and we show that such an approach allows
efficient identification of the genetic cause of CC in the
majority of cases, improves diagnosis and management,
and allows definition of specific care pathways.

Methods

Recruitment and Selection of Patients

with Congenital Cataract
Patients with CC were diagnosed through the National Health Service
(NHS) ophthalmic genetics clinic at Saint Marys Hospital
(Manchester). A 3-generation family history and a full medical history
were obtained from all patients, and all patients underwent a detailed
ophthalmic examination. Where additional problems or dysmorphic
features were present, a full systemic and dysmorphic assessment was
undertaken by a clinical geneticist (Table 1, available at
www.aaojournal.org). Informed consent was obtained as an essential
prerequisite for inclusion in the study. Thirty-six patients were
selected for NGS screening, along with a single positive control who
had previously undergone conventional diagnostic testing by
bidirectional Sanger sequencing at a National
Health Service facility and was known to carry a homozygous
pathogenic mutation in RAB3GAP1 (c.2801delC). Ethics commit-

tee approval was obtained from the NW Research Ethics Committee (11/NW/0421).

Enrichment Design, Genomic Capture, and

Resequencing
To investigate whether NGS was an effective method of identi-fying
disease-causing variants in patients with bilateral CC, an Agilent
SureSelect (Agilent Technologies, Santa Clara, CA) target enrichment
was designed to capture all exons and 50 base pair (bp)

of the flanking intronic sequence of 115 genes known to cause

nonsyndromic and syndromic forms of CC. Library preparations
were sequenced across 3 different platforms, SOLiD 5500 (Life
Technologies, Grand Island, NY), MiSeq (Life Technologies), and
HiSeq 2500 (Life Technologies), to compare sequencing quality
and target coverage.
The Agilent SureDesign system eArray (available at https://
earray.chem.agilent.com/suredesign/) was used to design a custom
target enrichment for the capture of all coding exons and 50 bp of the
flanking intronic sequence of 115 genes known to be associated with
autosomal dominant (adCC) autosomal recessive (arCC), and Xlinked forms of nonsyndromic and syndromic CC. The Cat-Map
32
database (available at http://cat-map.wustl.edu/) and the literature
were searched for relevant genes. A list of all genes used in the design
can be found in the Supplementary Data (available at
www.aaojournal.org). Genomic DNA was extracted from peripheral
blood or mouthwash samples by the Genome Diagnostics Laboratory
at Manchester Centre for Genomic Medicine using the Chemagic
Separation Module I and polyvinyl-alcohol magnetic bead technology
(PerkinElmer, Wal-tham, MA) according to the manufacturers
protocols. The Agilent SureSelect Target Enrichment Kit (Agilent
Technologies) was used for the solution-based hybridization and
capture of genomic tar-gets. This methodology was adopted because it
31
had been validated for diagnostic use within our laboratory. Library
preparations were conducted on 3 mg of high-quality DNA,
according to the manufacturers instructions. Thirty-seven DNA
libraries were prepared and sequenced. This included those made
from the 36 DNA samples from patients randomly selected from the
study cohort and the positive control sample. Eight library
preparations underwent amplification by emulsion polymerase chain
reaction for subsequent sequencing on the SOLiD 5500 platform. The
remaining 29 libraries underwent cluster generation by bridge
amplification, 12 on board the MiSeq platform and 17 on the c-Bot
(Illumina Inc.), in preparation for sequencing on the HiSeq 2500
platform. For the SOLiD 5500 and MiSeq runs, libraries were
sequenced in indexed batches of 4. The preparations that under-went
sequencing on the HiSeq 2500 were indexed in a batch of 17 and
sequenced across a single lane of a FlowCell (Illumina Inc.)

Bioinformatics, In Silico Analysis, and Variant

Prioritization
Because the samples were run on a variety of sequencing plat-forms, a
combination of different bioinformatic tools were used for mapping
the reads to the human reference sequence hg19 (build GRCh37) and
subsequent variant calling. Sequenced reads from the SOLiD5500 and
MiSeq were aligned to the human reference sequence with LifeScope
software version 2.5.1 (Life Technolo-gies). Variant calling was
conducted with LifeScope using diBayes and small.indel modules.
Sequenced reads from the HiSeq were aligned to the human reference
sequence (Hg19) with the Burrows-Wheeler aligner version 0.6.2
33
(https://ww.msi.umn.edu/sw/bwa) The Genome Analysis Toolkit
34
Lite version 2.0.39 (https:// www.broadinstitute.org/gatk) was used
for base quality score recalibration and insertion/deletion realignment
before variant calling using the UnifiedGenotyper.

Single nucleotide polymorphisms (SNPs) with 5 coverage and

insertions/deletions were annotated to genes using Ensembl
version 68, and the functional consequences were then defined
against an in-house list of Refseq transcripts (http://www.
ncbi.nlm.nih.gov/refseq). To ensure high-quality variants, SNPs
were filtered on novel allele depth and mean quality value at
thresholds established during an in-house validation process. 31
The minimum thresholds were set at 18 for novel allele depth and
18 mqV for the SOLiD and MiSeq processed variants and 50 /45

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Ophthalmology Volume 121, Number 11, November 2014

Table 2. Mutation Classification: Details of the Inclusion Criteria and Mutation Classification Scores Assigned to Each of the
Variants Reported in this Study
Mutation
Classification

Description

Evidence

1
2

Clearly not pathogenic

Unlikely to be pathogenic

Variant of unknown
significance

Likely pathogenic

Clearly pathogenic

Present on EVS or dbSNP at >1% frequency therefore is a common non-disease-causing polymorphism.

May be present at low frequency on EVS or dbSNP, no in silico evidence that the change is pathogenic, no
other reports of variant in the literature, thus not confirmed molecularly.
Not present on EVS or dbSNP, conflicting in silico evidence (i.e., 2 programs predict pathogenic with
moderate/low confidence scores or 1 program predicts highly pathogenic but others predict change to be
benign; if a splice-site change <3 programs predict splicing is altered), affected amino acid is poorly/
moderately conserved across paralogs or orthologs, co-segregation information not available, no other
reports in the literature.
Note: Variants that appear to have arisen as de novo events that cannot be confirmed as absent in parental
samples are assigned variant of unknown significance status regardless of in silico predictions and previous
literature reports until ruled out as an incidental finding.
Not present on EVS or dbSNP or present at very low frequency (<1%), strong in silico evidence of
pathogenicity (i.e., 2 programs predict change is pathogenic with moderate/high confidence scores; if a
splice site change, 3 programs predict splicing is affected with moderate/high confidence values),
affected amino acid is moderately conserved, mutation co-segregates with disease and fits predicted mode
of inheritance or, where co-segregation data are not available, the zygosity status of the mutation fits the
inheritance pattern. May have been previously reported in the literature but without functional
evidence.
Not present on EVS or dbSNP or present at very low frequency (<1%), strong in silico evidence of
pathogenicity (i.e., 2 programs predict change is pathogenic with moderate/high confidence scores; if a
splice site change, 3 programs predict splicing is affected with moderate/high confidence values),
affected amino acid is highly conserved, mutation co-segregates with disease and fits predicted mode of
inheritance or, where co-segregation data are not available, the zygosity status of the
mutation fits the inheritance pattern, mutation is previously reported in the literature,
preferably >1 time or with functional evidence.
Note: Nonsense and insertion or deletion mutations that disrupt the reading frame of the gene and
cause premature termination or elongation of the encoded protein are automatically assigned a
mutation classification of 5 where presenting in the zygosity previously reported for the gene
concerned, i.e., the mutation is homozygous where the gene has been reported, to demonstrate a
recessive mechanism of pathogenicity. For in-frame insertion or deletion mutations, an additional
requirement is that the mutation has been reported in the literature as pathogenic more than once.

EVS Exome Variant Server; dbSNP Single Nucleotide Polymorphism Database.

mqV for HiSeq variants. Additional annotation was provided from

Online Mendelian Inheritance in Man and Genomic Evolutionary
Rate Profiling 35 species alignment), as well as population
frequencies from the 1000 Genomes Project (phase 1 release) and
the National Heart, Lung, and Blood Institute Exome Sequencing
Project (v6500). PolyPhen-2 and Sorting Intolerant from Tolerant
(SIFT) predictions were included to help determine pathogenicity.

being established as genuine calls via confirmation by

bidirectional Sanger sequencing. Suspected pathogenic variants
were assigned a pathogenicity classification score based on the
Association for Clinical Genetic Science best practice guidelines
for the evaluation of pathogenicity and reporting of sequence
variants
(available
at
http://www.acgs.uk.com/qualitycommittee/best-practice-guide lines/). The inclusion criteria and
mutation classification scoring system are detailed in Table 2.

To find the likely pathogenic change within the data, an in-house

hierarchy system of functional consequence was used to prioritize
variants with the most detrimental consequence. Variants reported in
the Human Gene Mutation Database were investigated further to
confirm or refute their disease-causing status. In the first instance, the
Exome Variant Server and Single Nucleotide Poly-morphism
Database were searched for the presence of the variant. Where
present, coverage of the variant in the relevant control study was
investigated. If covered enough to verify that the base call was
accurate and the allele frequency was >1% in the control popu-lation,
it was deemed a benign polymorphism. When investigating the
frequency of a variant in control populations, ethnicities were
matched where possible. Novel missense and splice-site changes, that
is, those not reported in the Single Nucleotide Polymorphism
Database, Exome Variant Server, or Human Gene Mutation Database,
were investigated further using the in silico analysis tools
35
36
37
38
AlignGVGD, SIFT, PolyPhen2, Mutation Taster, and splicing
39
40
41
effect tools (MaxEntScan, NNSplice, Gene Splicer, Human
42
Splicing Finder ) via Alamut (Interactive Biosoftware, LLC) to
assess their predicted disease-causing potential before

Sanger Sequencing Confirmation

2126

All suspected pathogenic variants were confirmed by Sanger

sequencing. Co-segregation analysis of other affected family
members was carried out where samples were available. Primers
were designed to flank the variant plus a minimum of 200 bp of
surrounding sequence using Primer3 software (version 0.4.0;
available
at
http://frodo.wi.mit.edu/primer3/).
Standard
polymerase chain reaction was used to amplify targets before the
products underwent sequencing on the ABI3730XL sequencer
(Applied Biosystems, Carlsbad, CA).

Results
Patient Profiles
The systemic and ophthalmic features of the 36 patients selected
for screening are listed in Table 1 (available at
www.aaojournal.org), and a selection of cataract morphologies

Gillespie et al NGS for the Diagnosis of Congenital Cataract

Figure 2. Average coverage of target exons at a minimum read depth of 50

and 20 . Bar chart shows the average percentage of target regions covered
at each read depth for the 40 library preparations sequenced on each
platform and the average coverage across all platforms. Actual figures for
each are displayed above each bar. For the ABI SOLiD 5500 (Applied
Biosystems, Carlsbad, CA), 95.1% of genomic targets were covered at a
depth of 50 and 96.6% at a depth of 20 . For the Illumina MiSeq platform
(Illumina Inc., San Diego, CA), on average 99.0% of targets were covered at
a depth of 50 and 99.1% at a depth of 20 . Slightly better coverage was seen
for results from the HiSeq 2500 platform (Illumina Inc.), with 99.5% covered
at 50 and 99.7% covered at 20 .

Figure 1. Congenital cataract morphologies. The range of cataract morphologies identified during this study is shown. A, Left eye of patient 3
with coralliform cataract. B, Right eye of patient 4 displays a total
lenticular opacity. C, Right eye of patient 5 shows Y-sutural and lamellar
opacities. D, The opacity in the left eye of patient 9 displays an
interesting spoke-like morphology with a nuclear component. E, The left
eye of patient 34 dis-plays lamellar opacities and ectopia lentis. F, The
right eye of patient 18 displays nuclear and sutural lenticular opacities.
G. The right eye of patient 20 shows a nuclear and cortical cataract. H,
Lamellar opacities in the right eye of patient 31.

identified during this study are displayed in Figure 1. Analysis of

the patient information revealed that 20 patients with
nonsyndromic CC and 16 patients with syndromic CC had been
selected for testing. It is also noteworthy that 10 of the probands
were of consanguineous parentage.

Next-Generation DNA Sequencing Yields Excellent

Coverage of Genes Implicated in Human
Cataractogenesis
Results from the sequencing of CC-targeted enrichment libraries
across different NGS platforms showed a marginal percentage
difference in coverage and are detailed in Figure 2. Targets that

consistently receive zero or poor (<18 ) coverage can be found in

Table 3.
Differences in the number of high-quality SNP calls were seen
across the range of platforms used (Fig 3). This difference is most
likely attributable to the combinations of platforms and variant
calling software used in this study. Sequencing coverage and base
quality scores assigned by bioinformatics software packages play
an important part in variant calling and are an important factor for
consideration when validating targeted NGS assays for clinical
diagnostic use. The number of novel or rare variants typically
identified in each patient was lower (between 4 and 8, depending
on the platform) (Fig 3), thereby reducing the number of variants
for investigation as potential pathogenic changes. There was also
a difference in the average number of insertion and deletion
mutations detected on each platform, although many
insertion/deletion variants appeared in several patients and thus
likely represent sequencing or bioinformatics artefacts, such as
mismapped sequencing reads or alignment errors caused by
homopolymeric tracts.43

Targeted Next-Generation Sequencing

Demonstrates High Mutation Pick-up Rate
in Patients with Congenital Cataract
The CC target enrichment achieved an overall mutation pick-up
rate of 75%; 27 of the 36 individuals tested were found to har-bor
putative pathogenic variants within the genes included on the CCtargeted panel. Of these suspected disease-causing variants, 48%
were assigned a mutation classification of 5dclearly path-ogenic,
41% were assigned a classification of 4dlikely patho-genic, and
11% were assigned a classification of 3dvariant of unknown
significance because of the unavailability of parental samples for
testing. All suspected causative mutations were

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Ophthalmology Volume 121, Number 11, November 2014

Table 3. Target Regions that Consistently Receive Poor (<18 ) or Zero Coverage on Next-Generation Sequencing
Gene

Chromosome

Exon

Genomic Coordinates

13
21
1
11
16
X
8

1
1
1
1 (intron)
1
1
2

31774171-31774341
46825095-46825206
47881937-47882997
68080132-68080323
79632631-79633849
17393830-17394495
145742747-145743218

21
1
11
16

1
1
1
1

46825095-46825206
47881988-47882947
68,080,183-68,080,273
79632678-79633799

13
13
1
8
8

1
54
17
1
14

31,774,222-31,774,291
110,959,291-110,959,374
16,482,343-16,482,427
145,742,986-145,743,168
145,738,601-145,738,864

SOLiD 5500
Platform* (Life Technologies, Grand Island, NY)
B3GALTL
COL18A1
FOXE3
LRP5
MAF
NHS
RECQL4
MiSeq Platform (Illumina Inc., San Diego, CA)
COL18A1
FOXE3
LRP5
MAF
HiSeq 2500 Platform (Illumina Inc.)
B3GALTL
COL4A1
EPHA2
RECQL4
RECQL4

*Genes listed for the SOLiD platform are in addition to those listed for MiSeq platform.

segregated as expected in other affected family members, where

samples were available.

Nonsyndromic CC. Of the 20 patients with nonsyndromic

CC, 17 were found to have suspected pathogenic mutations (Table 4).
A variety of changes were detected, including 9 missense, 3 nonsense,
3 splice site changes, and 2 deletion mutations. This equates to a pickup rate of 85% in this sub-group. Some 35% of the suspected
pathogenic variants occurred in crystallin genes, and 35% occurred in
connexin genes. The ma-jority (76%) of these mutations appear to be
dominant; however, recessive changes (24%) also were detected.

Several patients had cataract in association with complex

ocular abnormalities: Patient 19, an individual with bilateral CC,
micro-phthalmia, microcornea, and nystagmus, was found to
harbor a homozygous c.71T>A; p.(Leu24Gln) missense change in
the RAS-associated protein RAB18. This mutation, previously reported as a functionally null founder mutation in the Pakistani
population,44 is a finding that will radically alter the clinical
management of this patient.
A homozygous c.1059-1060dupCA mutation was detected in exon
5 of EPHA2 in patient 20, an individual with bilateral CC and

microcornea. This mutation causes a frame shift that is predicted

to create a stop codon 39 amino acids downstream of the
duplication site that may generate a truncated protein that is
targeted for nonsense-mediated decay.45 Loss of functional
EPHA2 could be accountable for both the bilateral CC and
microcornea. EPHA2 has recently been found to be expressed
extensively within the layers of the cornea 46 and the cells of the
ocular lens,47 where it is thought to play an important role in
establishing and maintaining corneal architecture 46 and directing
lens fiber cell migration during development. 46e48
Patient 24, who presented with bilateral CC and suspected
vitreoretinal dysplasia, was found to have a heterozygous

Figure 3. Average number of variants detected across each sequencing

platform. Bar chart displays the average number of variants detected in
8 patients sequenced on each platform after bioinformatic filtering of
sequencing data, including all single nucleotide polymorphism (SNPs),
novel (Nov)/rare variants, and insertion and deletions (In/Dels). For the
SOLiD 5500 and MiSeq platforms, an average of 60 to 70 high-quality
SNPs were identified for each patient following bioinformatics filtering.

2128

c.289A>G; p.(Ile97Val) missense mutation in the Forkhead box E3

(FOXE3) gene. This amino acid resides within the second helix
(H2) of the helix-turn-helix motif of the forkhead (DNA-binding)
domain and is predicted to be pathogenic by in silico algorithms
(SIFT: deleterious [0]; Polyphen2: probably damaging [0.912]). The
affected amino acid is 100% conserved across 26 model species and
throughout 24 other forkhead box proteins. This very high degree of
orthologous and paralogous conservation implies that this amino acid
plays an important role in normal forkhead box

function. Investigations into the 3-dimensional structure of the

FOXE3 protein when complexed with DNA show that H2 is not
located on the side of the molecule that establishes direct contact

Table 4. Mutations Identified in Patients with Nonsyndromic Bilateral Congenital Cataract

EVS/dbSNP

c.402C>G
c.1273C>T
c.455T>G
c.293A>G

p.Tyr134Ter
p.Arg425Ter
p.Val152Gly
p.His98Arg

CRYGC
GJA8
CRYBB2
GJA8

Het
Hom
Het
Het

NM_020989
NM_005267
NM_000496
NM_005267

5
6
7
8

c.148T>C
c.596A>C
c.727T>C
c.3945-1G>C

p.Ser50Pro
p.Glu199Ala
p.Cys243Arg
d

GJA3
GJA3
GALK1
FYCO1

Het
Het
Hom
Hom

NM_021954
NM_021954
NM_000154
NM_024513

Extracellular loop
Extracellular loop
d
GOLD domain

d
d
d
d

9*

Het

NM_003571.2

10
11

c.697_
p.Glu233del BFSP2
699delAAG
c.134T>C
p.Leu45Pro CRYGC
c.3670C>T
p.Arg1224Ter FYCO1

Het
Hom

NM_020989
NM_024513

Greek key motif

FYVE-type

12

c.103C>T

Het

13*
14*

c.176C>T
p.Pro59Leu
c.269_
p.Gly91del
271delGAG
c.2151G>A d

15*

Mutation

Protein
Change

Gene

Status

Transcript

Protein Domain

17

c.184G>A

PolyPhen

3
5
4
4

Yes/yes
Yes/yes
Yes/yes
NA/yes

4
4
4
5

Yes/yes

Deleterious (0) Class 0 Yes/yes

d
d
NA/yes

4
5

Deleterious
Class 0 NA/yes
(0.02)
Deleterious (0) Class 65 NA/yes
d
d
Yes/yes

Predicted to abolish donor splice site

Yes/yes
(SSF: 73.4, MES: 8.4, NNS: 0.8, GS: 4.1,
HSF: 83)
Predicted to abolish donor splice site
Yes/yes
(SSF: 73.4, MES: 8.4, NNS: 0.8, GS: 4.1,
HSF: 83)
Probably damaging
Deleterious (0) Class 55 NA/yes
(0.957)

CRYBA1 Het

NM_005208

CRYBA1 Het

NM_005208

p.Glu62Lys

GJA3

NM_021954

Extracellular
domain

HSF4

Het

GVGD

Co-segregation/
Consistent with
Inheritance
Mutation
Pattern
Classification
NA/?yes
NA/yes
Yes/yes
Yes/yes

GJA3
Het
CRYBA1 Het

p.His35Tyr

SIFT

d
d
d
d
d
d
Probably damaging (1) Deleterious (0) Class 65
Probably
Deleterious (0) Class 25
damaging (1)
Probably damaging (1) Deleterious (0) Class 65
Probably damaging (1) Deleterious (0) Class 65
Probably damaging (1) Deleterious (0) Class 65
Predicted to abolish acceptor splice site
(SSF: 85.9, MES: 7.2, NNS: 0.6, GS: 5.7,
HSF: 86.1)
d
d
d

d
Probably damaging (1)
rs372348872 d
AG 1/6502;
AA 0/6502
(MAF%
0.0077)
NM_001040667 DNA binding
d
Probably damaging
domain
(0.992)
NM_021954
Extracellular loop d
Probably damaging (1)
NM_005208
Greek Key motif d
d

c.2151G>A
16*

Align

5
5

5
4

dbSNP Single Nucleotide Polymorphism Database; EVS Exome Variant Server; GS Gene Splicer (max score 15); Het heterozygous; Hom homozygous; HSF Human
Splicing Finder (max score 100); MAF% minor allele frequency percentage; MES MaxEntScan (max score 16); NNS NNSplice (max score 1); SIFT Sorting Intolerant
from Tolerant; SSF splice site finder (max confidence score 100).
Table displays the range of mutations identified in patients with nonsyndromic congenital cataract tested by the target enrichment, including the details of the complementary DNA sequence change and the
protein change (using Human Genome Variation Society nomenclature), the gene the mutation is occurring in (named according to HUGO Gene Nomenclature Committee), mutation status, the protein domain
affected (as listed in UniProt), frequency of variant as listed EVS and dbSNP (d indicates not listed), in silico predictions and scores (note that predictions cannot be made for insertion, deletion, or nonsense
mutations, as indicated by d), information on whether the mutation co-segregates with the phenotype within the family (NA indicates samples not available for testing), and whether the transmission of the
mutation follow the suspected inheritance pattern (?yes denotes that the mutation could have arisen as a sporadic event). Pathogenicity scores (classified as recommended by the Association for Clinical Genetic
Science): 1 clearly not pathogenic, 2 unlikely to be pathogenic, 3 variant of unknown significance, 4 likely to be pathogenic, 5 clearly pathogenic.

2129

*Previously reported mutation.

y
Frequencies listed are heterozygous and homozygous observed allele counts for all populations (MAF% for all populations).

Gillespie et al NGS for the Diagnosis of Congenital Cataract

1
2
3
4

Frequency
y
(MAF%)
Greek key motif d
Cytoplasmic loop d
Greek key motif d
Transmembrane d

Patient

In Silico Predictions

2130

Table 5. Mutations Identified in Patients with Syndromic Congenital Cataract

In Silico Predictions
Patient
18

20

21

EVS/dbSNP

Mutation

Bilateral CC, microphthalmia, upslanted palpebral fissures, mild

microcephaly and small tapering
fingers
Bilateral cortical rim opacities,
microphthalmia,
microcornea, nystagmus
Bilateral CC, microcornea,
phacodonesis,
neurodevelopmental
delay, mild dysmorphic features
Bilateral CC and learning
difficulties

c.453T>G

p.Tyr151Ter

CRYGD

Het

NM_006891

Greek key motif

c.71T>A

p.Leu24Gln

RAB18

Hom

NM_021252

c.1059_1060 p.Ser354Metfs EPHA2

dupCA
Ter40

Hom

NM_004431

Fibronectin type III

Probably
Deleterious Class 65
damaging
(0)
(1)
d
d
d

c.213C>T

Hom

NM_005208

Greek key motif

Het

NM_005208

p.Gly71Gly

Gene

CRYBA1

Transcript

Protein Domain

Frequency

NA/yes

Predicted to activate cryptic donor splice

site: SSF, 73.3; MES, 8.4; NNS, 0.8; GS,
4.1; HSF, 83
Greek key motif
d
Probably
Deleterious Class 15
damaging
(0)
(0.99)
Lanosterol 14-alpha
d
Benign
Deleterious Class 65
demethylase
(0.381)
(0.0)
Lanosterol 14-alpha
rs141654764 d
d
d
demethylase
TC 1/6502;
TT 0/6502
(MAF%
0.0077)
Forkhead (DNA binding) d
Probably
Deleterious Class 25
domain
damaging
(0)
(0.912)

NA/yes

Yes/yes

CYP51A1

Het

NM_000786

CYP51A1

Het

NM_000786

Bilateral CC, vitreoretinal

c.289A>G
dysplasia,
neurodevelopmental delay, joint
laxity
Bilateral CC and macrocephaly c.578T>C

FOXE3

Het

NM_012186

Het

NM_021954

Extracellular loop

Hom

NM_198270

WAVE homology domain d

Het

NM_006918

26
27

p.Phe193Ser GJA3

Bilateral CC, severe mental

c.3019C>T p.Gln1007Ter NHS
retardation
Bilateral CC, neurodevelopmental c.479C>G p.Pro160Arg SC5D
delay, microcephaly
c.630C>A* p.Asp210Glu SC5D

Positive control

Het

c.2818delC p.Pro941Leu RAB3GAP1 Hom

fsTer87

NM_001172435 Rab3 GTPase-activating d

protein catalytic
subunit

Pathogenicity
Score

Bilateral CC, cryptogenic neonatal c.935T>C p.Ile312Thr

liver cirrhosis,
spastic diplegia
c.1263G>A p.Tyr421Ter

AlignGVGD

Yes/yes

23

25

SIFT

Bilateral CC and cardiomyopathy c.626C>G

p.Ile97Val

Polyphen2

NA/?yes

22

24

p.Ser209Trp CRYBA1

Status

Phenotype

NA/?yes

Probably
Deleterious Class 65
damaging
(0)
(1)
d
d
d

Yes/yes

Yes/yes

Probably
Deleterious Class 65
damaging
(0)
(0.99)
Probably
Deleterious Class 35
damaging
(0)
(0.99)
d
d
d

NA/yes

NA/Yes

Ophthalmology Volume 121, Number 11, November 2014

19*

Protein
Change

Cosegregation/
Consistent
with
Inheritance
Pattern

Frequencies listed are heterozygous and homozygous observed allele counts for all populations (minor allele frequency percentage for all populations).

pro ling.fi
Con rmed biochemically by sterolfi

CCcongenitalc ataract;dbS NP SingleNucleotidePolymorphis mDatabase;EV SE xomeVariantS erver;GSGeneS plicer(maxscore15);Hetheterozygous;Homhomozygous;HS FHumanS plicingFinder(maxscore100);MES MaxE ntS can(maxscore16);NA notavailable;N NS NNSplice(maxscore1);S IFTS ortingIntolerantfromTolerant;S S Fsplicesitender(maxcondencescore100);WA VE WA SP-familyverprolin-homologousprotein.fifiTabledisplaystherangeofmutationsidentiedinpatientswithsyndromicCCtestedbythetargetenric hment,includingthedetailsofthepatientphenotype,thecomplementaryDNAsequencechange,thefiproteinchange(usingHumanGenomeV ariationSocietynomenclature),thegenethemutationis occurringin(namedaccordingtoHUGOGeneNomenclatureCommittee),mutationstatus,theproteindomainaffected(aslis tedinUniP rot),frequencyofvariantaslistedE VS anddbSNP (indicatesnotlis ted),insili copredictionsandscores(notethatpredictionscannotbemadeforinsertion,deletion,ord

transm issionofthemutationfollowthesuspectedinheritancepattern(?yesdenotesthatthemutationcouldhavearisenasasporadicevent).Pathogenicityscores (c lassi ed as rec ommended by thefiAssociationforClinicalGeneticScience):1clearlynotpathogenic ,2unlikelytobepathogenic ,3variantofunknownsignicance,4likelytobepathogenic ,5clearlypathogenic.fi*Previouslyreportedmutation.y

nonsensemutations,asindicatedby),informationonwhetherthemutationco-segregateswiththephenotypewithinthefamily(NAindicatessamplesnot available for testing), and whether thed

Gillespie et al NGS for the Diagnosis of Congenital Cataract

with the DNA helix,49 suggesting that the mutation identified in
patient 24 would not directly inhibit DNA binding. Ormestad et
al50 identified a heterozygous mutation, p.Arg90Leu, in a patient
with Peters anomaly, eccentric corneal opacities, and glaucoma.
Functional analysis of the mutation revealed that DNA binding
was not affected by this nonconservative change, and this led
them to speculate that the mutation affected interaction with
proteins responsible for nuclear transport.50 A similar mechanism
of pathogenicity could be responsible for the phenotype identified
in patient 24. However, in vivo analysis of localization of the
mutant protein would be required to confirm this. Although
mutations in FOXE3 have not specifically been associated with
vitreoretinal dysplasia, there have been reports of mutations in
FOXE3 in association with Peters anomaly, for which
vitreoretinal dysplasia can be mistaken. 51

Syndromic CC. Sixteen patients with syndromic CC were

tested by the CC-targeted NGS panel. Nine patients, excluding the
positive control, were found to have potential disease-causing
variants; there was a calculated pick-up rate of 63% in this subset of
patients (Table 5). Similar to the findings in the patients with
nonsyndromic CC, the majority (60%) of suspected pathogenic
mutations were nonsynonymous missense changes, whereas a further
30% were nonsense mutations. The CC-targeted array also
successfully and accurately detected a single bp deletion in the
RAB3 GTPase activating protein subunit 1 (RAB3GAP1) gene
within the positive control sample from a patient with known Warburg
Micro syndrome. Unlike the nonsyndromic CC findings, the
proportion of dominant versus recessive changes was more equivalent
(55% dominant, 45% recessive), as might be expected given that
several of the families with syndromic cataract were consanguineous.
Unexpectedly, 40% of the mutations in syndromic patients occurred in
genes associated with nonsyndromic CC.
A number of patient phenotypes are consistent with the molec-ular
findings: Patient 23 presented with a phenotype of bilateral CC,
cryptogenic neonatal liver cirrhosis, and spastic diplegia. The CCtargeted array detected a compound heterozygous mutation in the
cytochrome P450, family 51, subfamily a, polypeptide 1 (CYP51A1)
gene. CYP51A1 is responsible for the removal of the 14-alpha52

methyl group of lanosterol during the biosynthesis of cholesterol. A

homozygous mutation in this gene was recently reported in a family
53
with arCC from Saudi Arabia. Unfortunately, any details of systemic
features additional to the bilateral CC in this family were not
disclosed in the report. However, the liver has been found to express
52
high levels of CYP51 activity, and reports of other disorders of
sterol metabolism indicate cataracts and liver disease as
manifestations, such as cerebrotendinous xanthomatosis and
lathosterolosis. Subsequent sterol profiling in patient 23 identified
significantly elevated lanosterol and mildly elevated 7-

dehydrocholesterol levels, providing biochemical confirmation of

the pathogenic effect of the CYP51A1 mutations.
Patient 26 was a young male with bilateral CC and severe mental
retardation. The CC-targeted array detected a homozygous c.3019C>T; p.
(Gln193Ser) nonsense mutation within the Nance Horan syndrome (NHS)
gene. This mutation creates a premature stop codon that is predicted to
truncate the protein by 623 amino acids, potentially making it a target for
45

nonsense-mediated decay. This mechanism of pathogenicity would

require confirmation by functional analyses but could possibly account for
the severity of this childs phenotype.

Patient 27 has phenotypic features of bilateral CC, neurodevelopmental delay, learning difficulties, and microcephaly. This

2131

Ophthalmology Volume 121, Number 11, November 2014

individual was found to be compound heterozygous for mutations in
the sterol C5-denaturase-like (SC5DL) gene. Recessive mutations in
SC5DL have been reported to cause lathosterolosis, another cholesterol biosynthesis disorder, this time due to sterol-C5-desaturase
deficiency. There have been reports of a pair of siblings with lath-

osterolosis who were compound heterozygous for mutations in

SCDL5; however, their phenotype was also more severe than that
seen in patient 27, with features including microcephaly, cataract,
postaxial hexadactyly, bilateral clubbed feet, and intrahepatic
cholestasis with liver fibrosis.54,55 The diagnosis of lathosterolosis
in patient 27 awaits biochemical confirmation.
A number of patients with presumed syndromic forms of cataract
were found to have mutations in genes underlying nonsyndromic CC,
suggesting the presence of dual pathology. For example, a
heterozygous c.435T>G, p.(Tyr151Ter) nonsense mutation in
crystallin-gD (CRYGD) was detected in patient 18. This change is
likely accountable for her CC and microphthalmia but unlikely to be
the cause of her other dysmorphic features. Likewise, the putative

pathogenic c.213C>T, p.(Gly71Gly) mutation in crystallin- bA1

(CRYBA1) identified in patient 21 is probably causative of the
bilateral CC but not the learning difficulties. Of note, this potential
CC-causing variant was a synonymous change. Although the majority of synonymous variants would not be considered disease
causing, this particular variant is predicted to create a cryptic donor
splice site by 4 splice prediction programs with moderately high
confidence and so is the likely CC-causing variant. However, the
cause of the accompanying phenotype also reported in this same
individual remains undetermined because learning difficulties have
never been associated with mutations in CRYBA1. Patient 22 pre-

sented with bilateral CC and cardiomyopathy. On clinical assessment, it was predicted that this individual may have a variant in
the CRYAB gene because mutations in association with a
myopathy and cataract phenotype have been reported. 56 However,
testing with the CC-targeted array did not identify any variants in
this gene. Instead, we identified a dominant c.626C>G, p.
(Ser209Pro) missense variant in the CRYBA1 gene, suggesting
that the cardiomyopathy is occurring as an unrelated trait.

The Majority of Putative Congenital

CataracteCausing Mutations Occur in Highly
Conserved Protein Domains
During this study, 81% of the suspected pathogenic mutations
identified in the cohort were found to occur in highly conserved
domains thought to be important for the proper function of the
respective protein (Tables 3 and 4). However, in the absence of
functional investigations, it is difficult to comment on the possible
mechanisms that lead to the development of opacities in each of
these cases. Nine mutations were found in 4 different crystallin
genes, accounting for >30% of all mutations found during this
study. Three of these mutations were heterozygous changes
occurring in genes from the g-crystallin family: CRYGC (patient
1, c.402C>G; patient 10, c.134T>C) and CRYGD (patient 19,
c.453T>G). The remaining 6 mutations occurred in genes from
the b-crystallin family. Five of these changes were detected in
CRYBA1, 4 dominant (patient 14, c.269_271delGAG; patients 15

and 16, c.2151G>A; patient 22, c.626C>G) and 1 recessive

(patient 13, c.213C>T), and a dominant change in CRYBB2 (patient 4, c.455T>G). All were predicted to be pathogenic by in silico
analysis tools, and each occurred in Greek key motifs that are

2132

known to be important for the highly symmetric structure of these

exceedingly transparent proteins that are abundant within lens
fiber cells.57 Mutations in the Greek key motifs of genes from the
bg-crystallin family have been found to damage the b-sheet
structure that these motifs are known to form, which in turn
affects the tertiary structure of the protein that then has a
detrimental effect on its symmetry and interactions with other
crystallins, causing them to become insoluble within lens cells and
leading to the development of an opacity.57 Likewise, a significant
number of mutations were identified in the functional domains of
connexin proteins, accounting for 41% of mutations identified in
this cohort: 5 mutations were found in extracellular loop domains,
1 mutation in a cytoplasmic domain, and 1 mutation in a
transmembrane domain. It is difficult to speculate on the true
effect of these changes in the absence of functional data, but other
studies have suggested that mutations in connexin genes act in a
dominant negative manner and that the presence of a single
mutant subunit is enough to completely abolish the entire function
of the connexin.58

Next-Generation DNA Sequencing Genetic Diagnosis

of Patients with Congenital Cataract Alters Clinical
Management and Genetic Counseling
This research has shown that ascertaining the precise genetic
cause of the manifesting CC is of clinical utility and alters the
manage-ment and counseling of the patient. This is illustrated
with 3 brief case examples in Figure 4.

Case 1: Next-Generation DNA Sequencing Findings

Alter Clinical Hypothesis and Define Recurrence Risk.
Patient 1 was assumed to have a recessive syndromic form of CC
on the basis of her family history and consanguineous parentage.
Testing revealed the presence of a (likely de novo) dominant
nonsense mutation in the crystallin- gC gene (CRYGC), dramatically altering the counseling of her parents and the patient herself.
All previous reports of mutations in this gene have been
dominant, and this is highly likely to be the cause of her CC. 59e67

Case 2: Next-Generation DNA Sequencing Reveals

an Unsuspected Metabolic Condition. The second example is
that of patient 7, who was recruited to this study with suspected nonsyndromic arCC because of her consanguineous parentage and lack of
other discernible features. Testing by the CC-targeted NGS panel revealed
a homozygous missense mutation c.727T>C in exon 5 of the
galactokinase 1 (GALK1) gene that resulted in the amino acid change p.
(Cys243Arg). In silico analysis predicted this change to be patho-genic.
The affected amino acid is highly conserved throughout 11 model species,
and there is a large physiochemical difference between the polar,
hydrophilic cysteine residue and the basic, positively charged arginine
residue that it is substituted for. Recessive mutations in GALK1 cause
galactokinase deficiency and CCs in approximately 75% of cases,
presumed to be osmotically induced as the result of the accumulation of
galactitol in the lens. Avoiding galactose intake by strict dietary
management may reduce and sometimes even reverse lenticular
opacities.

68

However, diagnosis requires biochemical

measurement of galactokinase enzyme activity in erythrocytes,

which may then be confirmed by genetic screening of GALK1.

Case 3: Next-Generation DNA Sequencing Directs

Clinical Management. Patient 19, who was referred for
ophthalmic assessment of his CC before surgery, is the third example.
He was noted to have nuclear cataracts with cortical rim opacities,

Gillespie et al NGS for the Diagnosis of Congenital Cataract

Figure 4. Examples of genetic diagnosis altering the clinical care of patients with congenital cataract (CC). Examples from 3 different patients in whom establishing the
precise cause of the presenting CC defined diagnosis and altered the clinical management of the respective patient and the counseling of the family.

microphthalmia, and microcornea, with nystagmus. A provisional

diagnosis of arCC was made. Next-generation DNA sequencing
identified a homozygous p.Leu24Gln missense change in the Rasrelated protein Ras18 (RAB18). This mutation has been reported as a
founder mutation in the Pakistani population and was identified in 4
44
Warburg Micro families by Bem et al. Functional work conducted
by this group also highlighted the serious pathogenic effect of the
change on the normal function of the encoded RAB18 protein and
demonstrated the possible role of this mutation in the development of
44
the disease. Warburg Micro syndrome is a severe progressive
neurodevelopmental disorder in which early phenotypic signs are
subtle. Children may appear normal at birth apart from their CC but
fail to reach developmental milestones and develop little if any
speech. They develop progressive limb spasticity, severe epilepsy,

44,69

and become wheelchair bound by late childhood, if they survive.

It is
unlikely that patient 19 would have received a diagnosis of War-burg
Micro syndrome at such a young age without the NGS approach because
the presentation of the condition in infancy is subtle. The delineation of
the underlying genetic cause of CC in this individual is likely to alter
decisions about his clinical management and the genetic counseling that he
and his family receive. It has also provided the option for prenatal
diagnosis for the family in a future pregnancy if wished and the
opportunity for carrier testing for other family members.

Discussion
In this study, we used a custom-designed target enrichment
for the capture of all coding exons and flanking intronic

Figure 5. Next-generation DNA sequencing (NGS) alters care pathways. Flow diagrams depict the current traditional care pathway for
patients with congenital cataract (left) and our proposal for a new pathway in which NGS is central to diagnosis (right).

2133

Ophthalmology Volume 121, Number 11, November 2014

sequences of genes associated with CC for subsequent
massively parallel sequencing. We tested 36 patients and
have shown a high mutation detection rate, proving the efficacy of this method in determining the precise genetic
cause of CC and providing further evidence of the utility of
NGS in the diagnosis of a genetically heterogeneous condition. The absence of previously available Sanger
sequencing data precludes a retrospective analysis of
sensitivity or concordance.
The majority of genetic studies of CC have involved
sequencing of candidate genes indicated by the inheritance
pattern and cataract morphology in single families. There are
surprisingly few studies investigating significant numbers of
patients across a large number of genes. Next-generation
sequencing technologies will thus transform the scale of CC
research. Gaining a better understanding of lens proteins and
how specific mutations affect their function will lead to greater
comprehension of cataract-forming pathways and ultimately
could lead to insights into the pathogenesis of the more
common age-related cataract. Establishing robust genotypeephenotype correlations from large-scale studies will
facilitate delineation of the clinical phenotypes and the provision of more accurate prognoses. It may also lead to the
development of novel gene therapies, in particular for syndromic forms of CC, as seen in other inherited eye dis70,71
eases.
The detection of likely pathogenic variants in 27 of
36 patients, the accurate detection of the positive control
mutation, and the finding of 5 mutations previously reported as
pathogenic demonstrate considerable potential diagnostic
value. The wide range of mutation types (homozygous and
heterozygous changes, point mutations, a splice site change,
and deletions) detected in patients with nonsyndromic CC and
patients with syndromic CC of varying ethnicities provides
further validity of the use of NGS technologies in establishing
the molecular genetic basis of inherited CC. The high number
of suspected pathogenic variants found in crystallin and
connexin genesdcollectively accounting for 59% of all
mutations identified and 70% of mutations in patients with
nonsyndromic CCdcorrelates with the literature findings.
Conversely, the identification of rare recessive mutations in
72
genes only recently or rarely associated with CC (FYCO1,
44
54
47
RAB18, SC5DL, CYP51A1 ) endorses the importance

of the inclusion of less frequently mutated CC genes in

such a target enrichment.

Nonsense, splice site, and deletion mutations identified

during this study can relatively clearly be assigned as disease
causing. However, in the absence of functional studies,
determining the true effect of novel or rare missense changes
is more difficult and relies on relatively crude assessment of
function, including evidence of conservation and amino acid
type. It is notable that 78% of the suspected pathogenic mutations identified occurred in highly conserved protein domains that are integral to normal protein function, and it is
likely that functional assessment can be targeted in a protein
domain-specific manner. High coverage of virtually all
genomic targets was achieved in the samples tested. This
credits the quality of the library preparations and the efficacy
of the SureSelect technology (Agilent) used in the current
31
study and previously validated for diagnostic use. Coverage
was, on average, slightly better for the Illumina platforms by
2134

approximately 4% at 50 read depth and 2.5% at 20 read depth

compared with the ABI SOLiD 5500 (Applied Biosystems).
Because library preparation is largely the same for each
sequencing platform, the difference in coverage could be due
to bias during the final amplification of the libraries because
73
this section of the methodology is platform specific. Better
coverage also could be due to the longer sequencing reads
used by the Illumina MiSeq and HiSeq platforms, allowing the
successful mapping of more repetitive regions during
73,74
bioinformatic analysis.
Shared regions of poor coverage
across the 2 platforms are likely due to high GC or AT content
of the target sequence and could harbor CC-causing mutations
in some patients that would potentially be missed. The results
also highlight the presence of potentially false small
insertion/deletion variants within the data sets. This is
recognized to complicate the determination of real diseasecausing variants and exposes a limitation of current
75e78
bioinformatics analysis.
Both insertions and
deletions have been reported in genes associated with CC
66,79e81
; 4 were identified in the relatively small cohort of this
study. Although our CC-targeted array demonstrated accurate
detection of 3 small deletions (including the control sample)
and 1 small duplication, standard bioinformatics analysis of
NGS sequencing data would fail to detect larger copy number
variants, such as those involving whole genes or entire exons.
Although there have been advancements in the detection of
copy number variants and small structural rearrangements
from NGS data, they are not widely used and suffer their own
76
limitations. At present, alternative strategies for the detection
of such abnormalities should be pursued. A robust means of
detecting this type of variant will be important for the future
improvement for diagnostic applications.
This study has demonstrated that determining the precise
genetic cause of CC can dramatically alter the initial working
diagnosis hypothesized at clinical assessment and lead to more
appropriate management and counseling of the patient and
family. Accurate diagnosis of CC requires a multidisciplinary
pathway with close communication be-tween different
departments and specialties. A typical clin-ical assessment of a
child with CC involves taking a careful family history to
explore the presence of ocular phenotypes and other
conditions and to gather the details of any ante-natal, perinatal,
and postnatal problems. In particular, this needs to include
enquiry about maternal illness and expo-sure to drugs and
82
other potential teratogens during preg-nancy. Ascertainment
of when the cataracts or any associated visual symptoms were
first noticed is important not only to enable assessment of the
likely amblyogenic effect of the cataracts and subsequent
optimal timing of intervention but also to provide important
83
diagnostic clues ; for example, cataracts may be present at
birth in some conditions, such as Cockayne syndrome, but
develop after birth in some metabolic disorders. A feeding and
developmental history also should be taken. A thorough
ophthalmic examination and age-appropriate acu-ity
assessment are performed to clarify cataract morphology and
identify any coexistent ocular structural abnormalities. Retinal
examination may be difficult or impossible because of the
cataract but should be attempted. A B-scan ultrasound
examination can be used to look for any significant posterior

Gillespie et al NGS for the Diagnosis of Congenital Cataract

segment abnormality, such as evidence of a mass or retinal
detachment. In addition, parents and family members should
be examined to look for signs of lens or ocular abnormality.
All children with bilateral cataracts without a family history
of nonsyndromic dominant cataract should be referred for a
pediatric and genetic assessment to look for evidence of
congenital infection, neurodevelopmental problems, metabolic
disorders, and chromosomal abnor-mality. Investigations
performed will typically include chromosomal analysis and a
TORCH screen to look for evidence of intrauterine infection
with toxoplasmosis, rubella, cytomegalovirus, and herpes. A
number of biochemical tests also are usually ordered. These
include urinary assays of amino acid profile, oligosaccharides,
organic acids, and reducing substances together with blood
tests for plasma amino acid profile, liver function tests, renal
profile, and assay of Gal-1-P-uridyl transferase. The cost of
these investigations is significant and cumulative. Additional
neuroimaging and other radiologic investigations, some of
which involve general anesthetics or are invasive, also may be
indicated in helping to reach a diagnosis in the dysmor-phic or
developmentally delayed infant.
This traditional diagnostic approach to children with CC,
based on clinical assessment, is reactive, expensive for health
services, and often unsuccessful in identifying an underlying
diagnosis. It is frequently unsuccessful in assisting with genetic counseling and identifying individuals in whom prevention or metabolic intervention may play a role.
In conclusion, it is evident from our study that a single targeted
gene screen undertaken early in the diagnostic algo-rithm would
be swift and effective, and when placed against the large numbers
of investigations run in series, it is also likely to be more costefficient. By eradicating the need for many of the routine
investigations, this can potentially provide the oppor-tunity for the
development of an individualized care pathway (Fig 5). We
successfully developed an NGS target enrichment for 115 genes
associated with CC. We believe that the use of this assay as a
frontline diagnostic tool will establish a more linear, efficient, and
cost-effective clinical care algorithm for this patient group.
Adoption of this strategy will lead to better diagnostic outcomes
attributable to the implementation of personalized genomic
medicine. We also hope that the use of high-throughput
sequencing technologies for genetic diagnosis will increase the
known mutational basis of the condition and lead to a more
thorough understanding of the epidemiology of CC. Ascertaining
more robust genotypeephenotype correla-tions will in time also
lead to greater comprehension of the mechanisms leading to
cataract formation.

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Footnotes and Financial Disclosures

Originally received: March 4, 2014.
Final revision: May 2, 2014.
Accepted: June 4, 2014.
Available online: August 19, 2014.

Manuscript no. 2014-344.

Funded by Fight for Sight (grant no. 1831) and supported by the Manchester Academic Health Science Centre and the Manchester National
Institute for Health Research Biomedical Research Centre. The funding
organization had no role on the design or conduct of this research.

Manchester Centre for Genomic Medicine, Institute of Human Development, Faculty of Medical and Human Sciences, University of Manchester,
Manchester Academic Health Science Centre, Saint Marys Hospital,
Manchester, United Kingdom.

Abbreviations and Acronyms:

arCC autosomal recessive congenital cataract; bp base pair; CC
congenital cataract; NGS next-generation DNA sequencing; SIFT
Sorting Intolerant from Tolerant; SNP single nucleotide polymorphism.

Manchester Royal Eye Hospital, Manchester Academic Health Science

Centre, The University of Manchester, Central Manchester Foundation
Trust, Manchester, United Kingdom.
3

Manchester Centre for Genomic Medicine, Central Manchester University

Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre,
Saint Marys Hospital, Manchester, United Kingdom.

Correspondence:
Professor Graeme Black, The University of Manchester, Manchester
Centre for Genomic Medicine, Institute of Human Development, 6th
Floor, Saint Marys Hospital, Oxford Road, Manchester, Lancs M139WL,
United Kingdom. E-mail: graeme.black@manchester.ac.uk.

Financial Disclosure(s):
The author(s) have no proprietary or commercial interest in any materials
discussed in this article.

2137

2137.e1

Table 1. Patient Profiles: Details of Cataract Morphology, Extraocular Features, and Systemic Phenotypic Features in the Study Patients
Patient
1
2

Sex

Cataract Morphology

Male

6
7
8
9

Male
Female
Female
Male

10

Male

11

Male

Family History of CC?

Consanguineous (Affected Family Members)

d
d

Yes
Yes

No
No

d
Amblyopia, convergent squint, all other
measurements within normal range
Subcapsular, lamellar, y-sutural d

d
d

No
No

No

Nuclear and y-sutural

Nuclear, lamellar
R: nuclear; L: subcapsular
Spoke-like, nuclear component,
subcapsular, lamellar
Nuclear, lamellar

d
Intermittent divergent squint
Iris slightly atrophic (more in R than L)
d

d
d
d
d

No
Yes
Yes
No

Yes (mother)
Yes (mother and maternal
grandfather)
Yes (mother, grandmother,
great grandmother, 3
maternal aunts)
Yes (mother)
Yes (father)
No
Yes (father, paternal cousin)

No

Posterior lenticonus, amblyopia and

secondary glaucoma
Horizontal manifest latent nystagmus
d
d

Yes

d
d
d

No
No
No

d
d
d
Mild microcephaly, developmental delay,
hypotonia, up-slanted and long palpebral fissures,
small tapered fingers
Mild microcephaly and LD

No
No
Yes
Yes

Yes (mother)
Yes (brother)
Yes (mother, maternal
grandfather, maternal
cousins)
Yes (mother and brother)
Yes (father and sister)
Yes (2 affected children)
No

Yes

No

Hypotonia, small size, poor weight gain due to

No
feeding problems, developmental delay,
communication and comprehension limited (no
speech at 6 yrs, uses symbols to communicate),
hand flapping, unusual head shape, fine hair with
unusual hairline and chaotic patterning, single
palmar crease, unusual low columnar nose, downslanted palpebral fissures, thin upper lip, contact
dermatitis, diffuse cerebral and cerebella atrophy,
and reduced white matter on MRI
Delayed speech and motor movements,
Yes
developmental delay

No

12
13
14

R: total cataract, L: central and

posterior
Female Total
Female ND
Male
Lamellar

15
16
17
18

Female
Female
Female
Female

19

Male

20

Male

21

Male

Microcornea
d

Global/Systemic Features

Lamellar, sutural
Nuclear
ND
R: nuclear, sutural, posterior
plaque

d
d
d
R: PFV, microphthalmia, horizontal jerk
nystagmus, right divergent squint,
strabismus
Nuclear
Bilateral microphthalmia, convergent squint,
low-frequency multiplanar nystagmus, R
scalloped pupil, roving eye movements,
pupils difficult to dilate, no recordable
VEP response
R: lamellar; L: minimal changes Vitreous abnormality, optic nerve hypoplasia
(worse in R), R divergent squint, R
exotropia, very pale fundi, hypermetropia,
bilateral abnormal hypoplastic discs,
abnormal trafficking of vessels, lens
subluxation, phacodonesis

Nuclear, cortical, lamellar

Watering eyes since birth

Yes (sister, mother, maternal

grandfather)
Yes (sister)

Yes (paternal great uncle)

(Continued)

Ophthalmology Volume 121, Number 11, November 2014

3
4

Female Nuclear
Male
Nuclear, lamellar, posterior
capsule opacity
Female Coralliform
Female Total cataract

Extraocular Features

Table 1. (Continued)
Family History of CC?
Patient

Sex

Cataract Morphology

Extraocular Features

Male

ND

23

Male

Nuclear, lamellar

24
25
26

Female ND
Male
ND
Male
L: dense nuclear; R: nuclear,
lamellar, posterior polar

Vitreal strands and clumps

d
L: hypoplastic iris, nystagmus

27
28

Male
Male

Posterior opacities
Nuclear, lamellar

d
ASD, amblyopia, deep-set eyes, divergent
squint

29

Male

Posterior capsule opacity

Astigmatism, nystagmus, hypermetropic

30

Congenital nystagmus, strabismus, alternate

large-angle squint
d

32

Female Anterior polar (white dot

Mild astigmatism
opacities)
Male
R: lamellar; L: minimal changes Vitreous abnormality, optic nerve hypoplasia
(worse in R), R divergent squint, R
exotropia, very pale fundi, hypermetropia,
bilateral abnormal hypoplastic discs,
abnormal trafficking of vessels, lens
subluxation, phacodonesis
Male
Posterior pigmented plaques Hypermetropia

33
34

(?PFV)
Male
Nuclear, sutural
Female R: nuclear; L: lamellar

31

35
36

d
Posterior lenticonus, long axial length,

myopia, ectopia lentis

Female Lamellar, sutural, small nuclear Nystagmus, roving eye movements
component
Male
Mild posterior plaques
Hypermetropia

Consanguineous (Affected Family Members)

Cardiomyopathy

No

Yes (daughter)

Developmental delay, spastic diplegia, cryptogenic

neonatal liver cirrhosis
Neurologic developmental delay, joint laxity
Macrocephaly
Severe mental retardation

No

No

Yes
No
No

Neurologic developmental delay, microcephaly

LD, developmental delay, low-set ears, skeletal
dysplasia, broad, flat nose, short stature,
microcephaly, deep-set eyes, small teeth, short
distal phalange, septal defect, epilepsy, recurrent
chest infections, fragile-looking skin
LD, night incontinence, short attention span,
asthma, horseshoe kidney, ?neuromuscular/
connective tissue disorder, prone to infections,
asthma, involuntary head jerking, unilateral
gynecomastia, markedly elevated FSH, small
testes
d
Hypotonia, small size, poor weight gain due to

No
Yes

No
Yes (daughter)
Yes (maternal great
grandmother, maternal
cousins)
No
Yes (brother)

No

No

No
No

No
No

d
No
Learning difficulties, sensorineural hearing loss, No

No
No

microcephaly
Delayed dental eruption and dental abnormalities No
IUGR, vitamin D deficiency, developmental delay, No

No
No

feeding problems, developmental delay,

communication and comprehension limited (no
speech at 6 yrs, uses symbols to communicate),
hand flapping, unusual head shape, fine hair with
unusual hairline and chaotic patterning, single
palmar crease, unusual low columnar nose, downslanted palpebral fissures, thin upper lip, contact
dermatitis, diffuse cerebral and cerebella atrophy
and reduced white matter on MRI

poor growth, mild high frequency sensorineural

hearing loss
d

No

Yes (father, paternal

grandmother, paternal
great aunt)

2137.e2

? possible; ASD anterior segment dysgenesis; CC congenital cataract; FSH follicle-stimulating hormone; IUGR intrauterine growth retardation; L left (eye); LD learning
difficulty; MRI magnetic resonance imaging; ND not disclosed; PFV persistent fetal vasculature; R right (eye); VEP visual evoked potential.

Gillespie et al NGS for the Diagnosis of Congenital Cataract

22

Global/Systemic Features