Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
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Simon Williams, PhD, Susmito Biswas, FRCOphth, Elias Kehdi, FRANZCO, Simon C. Ramsden, FRCPath,
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Jill Clayton-Smith, FRCP, Graeme C. Black, DPhil, FRCOphth, I. Christopher Lloyd, FRCOphth
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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.
2124
12
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.
Methods
tee approval was obtained from the NW Research Ethics Committee (11/NW/0421).
2125
Description
Evidence
1
2
Variant of unknown
significance
Likely pathogenic
Clearly pathogenic
2126
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
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.
2127
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.
2128
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
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
4
5
Deleterious
Class 0 NA/yes
(0.02)
Deleterious (0) Class 65 NA/yes
d
d
Yes/yes
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.
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1
2
3
4
Frequency
y
(MAF%)
Greek key motif d
Cytoplasmic loop d
Greek key motif d
Transmembrane d
Patient
In Silico Predictions
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In Silico Predictions
Patient
18
20
21
EVS/dbSNP
Mutation
c.453T>G
p.Tyr151Ter
CRYGD
Het
NM_006891
c.71T>A
p.Leu24Gln
RAB18
Hom
NM_021252
Hom
NM_004431
Probably
Deleterious Class 65
damaging
(0)
(1)
d
d
d
c.213C>T
Hom
NM_005208
Het
NM_005208
p.Gly71Gly
Gene
CRYBA1
Transcript
Protein Domain
Frequency
NA/yes
NA/yes
Yes/yes
CYP51A1
Het
NM_000786
CYP51A1
Het
NM_000786
FOXE3
Het
NM_012186
Het
NM_021954
Extracellular loop
Hom
NM_198270
Het
NM_006918
26
27
p.Phe193Ser GJA3
Positive control
Het
Pathogenicity
Score
AlignGVGD
Yes/yes
23
25
SIFT
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
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
Patient 27 has phenotypic features of bilateral CC, neurodevelopmental delay, learning difficulties, and microcephaly. This
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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.
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68
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.
44,69
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
References
1. Rahi JS, Dezateux C; British Congenital Cataract Interest G.
Measuring and interpreting the incidence of congenital ocular
anomalies: lessons from a national study of congenital cataract in
the UK. Invest Ophthalmol Vis Sci 2001;42:14448.
2. Foster A, Gilbert C, Rahi J. Epidemiology of cataract in
2135
27.
28.
29.
30.
31.
32.
33.
2136
55.
56.
57.
58.
59.
60.
61.
62.
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.
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.
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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
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
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
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
No
12
13
14
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
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
27
28
Male
Male
Posterior opacities
Nuclear, lamellar
d
ASD, amblyopia, deep-set eyes, divergent
squint
29
Male
30
32
33
34
(?PFV)
Male
Nuclear, sutural
Female R: nuclear; L: lamellar
31
35
36
d
Posterior lenticonus, long axial length,
Cardiomyopathy
No
Yes (daughter)
No
No
Yes
No
No
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
No
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.
22
Global/Systemic Features