Identification of an HMGB3 Frameshift Mutation in a Family with an X-Linked Colobomatous Microphthalmia Syndrome Using Whole-Genome and X-Exome Sequencing

Research

Original Investigation Identification of an HMGB3 Frameshift Mutation in a Family With an X-linked Colobomatous Microphthalmia Syndrome Using Whole-Genome and X-Exome Sequencing

Alan F. Scott, PhD; David W. Mohr, BA; Laura M. Kasch, BS; Jill A. Barton, MS; Raquel Pittiglio, MS; Roxann Ingersoll, MHS; Brian Craig, MS; Beth A. Marosy, MS; Kimberly F. Doheny, PhD; † William C. Bromley, MD; Thomas H. Roderick, PhD ; Nicolas Chassaing, MD; Patrick Calvas, MD; Shreya S. Prabhu, MD; Ethylin Wang Jabs, MD

Supplemental content at IMPORTANCE Microphthalmias are rare disorders whose genetic bases are not fully jamaophthalmology.com understood. HMGB3 is a new candidate gene for X-linked forms of this disease.

OBJECTIVE To identify the causative gene in a pedigree with an X-linked colobomatous microphthalmos phenotype.

DESIGN, SETTING, AND PARTICIPANTS Whole-genome sequencing and chromosome X-exome–targeted sequencing were performed at the High Throughput Sequencing Laboratory of the Genetic Resources Core Facility at the Johns Hopkins University School of Medicine on the DNA of the male proband and informatically filtered to identify rare variants. Polymerase chain reaction and Sanger sequencing were used to confirm the variant in the proband and the carrier status of his mother. Thirteen unrelated male patients with a similar phenotype were also screened.

MAIN OUTCOMES AND MEASURES Whole-genome and X-exome sequencing to identify a frameshift variant in HMGB3.

RESULTS A 2–base pair frameshift insertion (c.477_478insTA, coding for p.Lys161Ilefs*54) in the HGMB3 gene was found in the proband and his carrier mother but not in the unrelated patients. The mutation, confirmed by 3 orthogonal methods, alters an evolutionarily conserved region of the HMGB3 protein from a negatively charged polyglutamic acid tract to a positively charged arginine-rich motif that is likely to interfere with normal protein function.

CONCLUSIONS AND RELEVANCE In this family, microphthalmia, microcephaly, intellectual disability, and short stature are associated with a mutation on the X chromosome in the

HMGB3 gene. HMGB3 should be considered when performing genetic studies of patients with Author Affiliations: McKusick- similar phenotypes. Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland (Scott, Mohr, Kasch, Barton, Pittiglio, Ingersoll, Craig, Marosy, Doheny, Jabs); The Jackson Laboratory, Center for Human Genetics, Bar Harbor, Maine (Bromley, Roderick); CHU Toulouse, Service de Génétique Médicale, Hôpital Purpan, Université Paul- Sabatier Toulouse III, Toulouse, France (Chassaing, Calvas); Icahn School of Medicine at Mount Sinai, New York, New York (Prabhu, Jabs). †Deceased. Corresponding Author: Alan F. Scott, PhD, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, 1034 Blalock Bldg, 600 JAMA Ophthalmol. 2014;132(10):1215-1220. doi:10.1001/jamaophthalmol.2014.1731 N Wolfe St, Baltimore, MD 21287 Published online July 3, 2014. ([email protected]).

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icrophthalmia can be classified into 2 major forms. X-exome sequencing was performed by the High Through- In simple microphthalmia, the eye is anatomically put Sequencing Laboratory of the Genetic Resources Core M normal except for decreased axial length. Complex Facility (http://grcf.jhmi.edu) at the Johns Hopkins Univer- microphthalmia is characterized by short axial length and sity School of Medicine. A total of 3 μg of DNA was used to anterior and/or posterior segment dysgenesis. Microphthal- create an Illumina library using standard methods. Genomic mia is a heterogeneous condition and both forms may be DNA was sheared to a size of 150 to 200 base pairs (bp) using sporadic or inherited. Microphthalmia may be isolated or a Covaris E210 system (Covaris Inc). End repair and addition occur as one of several other features in various syndromes. of an overhanging A base were performed with an NEBNext The prevalence of microphthalmia has been estimated at 0.2 DNA Laboratory prep kit (New England Biolabs). DNA frag- to 3.0 per 10 000 births.1,2 There are at least 6 X-linked loci ments were ligated to library adapters (Illumina). The in the Online Mendelian Inheritance in Man database (http: ligated fragments were then size-selected through purifica- //omim.org) that include microphthalmia in the phenotype tion using solid-phase reversible immobilization beads and description. PCR amplified to prepare the libraries. The Bioanalyzer In 1971, Goldberg and McKusick3 reported a kindred DNA1000 kit (Agilent Technologies) was used for quality from Maine with X-linked microphthalmos, microcephaly, control of the libraries to ensure adequate concentration intellectual disability, and kyphoscoliosis similar to the phe- and appropriate fragment size. Target capture was per- notype (MCOPS1 [OMIM 309800]) first described by Lenz4 formed using the SureSelect X-exome demo kit (Agilent but lacking digit or urogenital abnormalities. Forrester et al5 Technologies). Under standard procedures, biotinylated mapped the MCOPS1 locus to Xq27-28 in a different family RNA oligonucleotides were hybridized with 500 ng of the described as having Lenz microphthalmia, and Esmailpour library. Magnetic bead selection was used to capture the et al6 identified the mutation in the NAA10 gene (OMIM resulting RNA-DNA hybrids. RNA baits were removed with 300013). We were able to study the original family described ribonuclease and the remaining DNA products PCR ampli- by Goldberg and McKusick3 and identify the genetic basis as fied. Sample indexing was introduced at this step. The High an X-linked gene not previously associated with this pheno- Sensitivity DNA Analysis kit (Agilent Technologies) was type. The proband’s DNA was analyzed using 2 independent used for quality control of adequate fragment sizing and next-generation sequencing technologies, whole-genome quantity of DNA capture. sequencing and exome capture of X-linked genes followed Paired-end 100-bp reads were produced on an Illumina by next-generation sequencing. Confirmatory polymerase Genome Analyzer IIx. Raw data were processed through chain reaction (PCR) and Sanger sequencing were per- Illumina software, generating base calls and corresponding formed on the proband and his mother. In addition, PCR base-call quality scores. These data were then processed sequencing was used to evaluate the identified candidate with CIDRSeqSuite 2.0 software,7 an analysis suite that uses gene in unrelated male patients with a similar phenotype. open-source programs. Reads were aligned to hg19 using the Burrows-Wheeler Aligner (BWA), resulting in a binary alignment map (BAM) file. Postprocessing of the aligned Methods data included flagging of molecular and optical duplicates using Picard (http://sourceforge.net/projects/picard/), and Patients local realignment and base-call quality score recalibration The Johns Hopkins University School of Medicine Institu- usingGATK1.6.8 Variant calling was performed by SAMtools tional Review Board approved this study. Written informed (http://samtools.sourceforge.net) and output in VCF format consent was provided for genetic studies from the family v4.0 (http://www.1000genomes.org/node/101). Complete described by Goldberg and McKusick.3 DNA was isolated Genomics variant files were converted to VCF format using (Qiagen) from Scope mouthwash collections obtained from Complete Genomics Analysis Tools (http://cgatools.sourceforge the proband and his mother. In addition, DNA was also .net). For whole genome and exome data, we focused our obtained from 13 unrelated male patients with undiagnosed efforts on nonsynonymous coding substitutions and syndromes of microphthalmia, intellectual disability, and/or frameshift mutations on chromosome X and ranked additional malformations. Six had colobomatous microph- variants that were predicted to be damaging based on thalmia, with 4 also having intellectual disability. Two conservation,9 SIFT,10 and Polyphen211 scores. For inser- patients had bilateral microphthalmia with intellectual dis- tions and deletions (indels), all calls in exonic regions were ability. Two had microphthalmia and congenital cataracts, 1 manually reviewed in the event that the different calling of whom also had intellectual disability. One patient each methods might have placed the same indel at different had sclerocornea, bilateral anterior segment dysgenesis, or genomic positions. bilateral anophthalmia. Other malformations included con- After identification of the suspect variant in the proband, genital heart or kidney disease in 5 patients and cleft palate we used PCR and Sanger sequencing of the exon of interest to and thumb anomalies in 1 patient. confirm the mutation in him and his mother. Both PCR and Sanger sequencing were performed for each of the exons of Sequencing HMGB3 from the 13 unrelated male patients. The PCR primer Whole-genome sequencing was performed by Complete sequences for the 4 HMGB3 exons are provided in the eTable Genomics Inc using 10 μg of DNA from the proband. in the Supplement.

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Figure 1. Original Pedigree Redrawn to Indicate the Proband and His Mother

I 33 No other family members were 1 2 3-5 6-8 9 10 available at the time of this study. Squares indicate males; circles, II 3 females; filled symbols, affected 1 2 3 4 5 6 7 8 9 10 11 12-14 individuals; arrowhead, sequenced proband; diamonds, sex III undesignated; numbers within a 1234 567 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 symbol, numbers of males or females counted together; triangles, IV 22 1 2 3 4 5 6 7 8 9 10 11 12 13-14 15 16-17 miscarriages. Adapted with permission of Elsevier from Goldberg and McKusick.3

quence data obtained in our laboratory using the Agilent Results X-exome capture reagent and Illumina sequencing. The filtering cascades for whole-genome and X-exome sequences are Case History and Clinical Findings listed in Table 1. Whole-genome sequencing and X-exome cap- At the time of initial examination by Goldberg and McKusick,3 ture identified 2 possible candidates (Table 2). The first mu- the proband (IV-3) (Figure 1) was an 8-year-old boy who was tation, PORCN(OMIM 300651), is the human homologue of Dro- born at term, weighing approximately 1800 g, below the third sophila porcupine, mutations in which cause focal dermal percentile. His head circumference was 45.5 cm, which is be- hypoplasia (FDH; OMIM 305600). Focal dermal hypoplasia is low the 50th percentile for a child 3 years old. He had intellec- described as an X-linked dominant trait that affects female car- tual disability, diastema of the upper incisors, and anteverted riers. Ninety percent of patients are female, suggesting that it pinnae with minimal convolutions. He had reported congen- is generally an embryonic lethal for male fetuses. In addition, ital pes varus corrected by special shoes. 95% of cases are sporadic. Microphthalmia is reported as a fea- The proband also exhibited bilateral ptosis, pendular ture in approximately 15% of cases. nystagmus, and right esotropia. There was no light percep- The second mutation was a 2–base pair TA insertion that tion in the right eye, and best-corrected visual acuity was is predicted to cause a frameshift mutation in the fourth 12/400 OS with refraction of +1.00 + 5.00 × 065. Corneal exon of a gene (HMGB3; OMIM 300193) that codes for the diameters were 3 mm horizontally and vertically in the right evolutionarily conserved high-mobility-group protein eye and 4 mm horizontally and 6 mm vertically in the left HMGB3. The mutation had a PhastCons9 score of 1 and a eye. Both corneas were clear, and each eye had inferonasal PolyPhen211 classification of damaging. The insertion was iris coloboma. A posterior segment examination could not visualized using the Integrative Genomics Viewer12 (eFigure be performed in the right eye. However, dilated examina- 1 in the Supplement) and confirmed by Sanger sequencing tion of the left eye revealed a large chorioretinal coloboma of exon 4 (Figure 2). The variant c.477_478insTA produces that involved the optic nerve. the predicted protein change p.Lys161Ilefs*54. Sanger Three other male family members were noted to have a sequencing of the mother confirmed her carrier status similar phenotype with colobomatous microphthalmia, (eFigure 2 in the Supplement). We also carefully examined blepharoptosis, microcephaly, slow development, and short the NAA10 gene for sequence abnormalities that might have stature (III-3, III-4, and III-18) (Figure 1) but were unavail- been missed by our filtering algorithms; none were found. able for molecular analysis. The proband’s mother had left No other X-linked variants from the whole-genome or esotropia and a prominent limbal dermoid temporally in the X-exome sequencing were strong candidates in terms of left eye. potential pathogenicity.

High-Throughput Sequencing Results Targeted HMGB3 Sequencing in Male Patients Complete Genomics data were aligned to hg19 using the With Related Phenotypes Complete Genomics pipeline version 1.5. Complete Genom- Thirteen unrelated male patients diagnosed as having mi- ics called approximately 9% more bases than the X-exome crophthalmic conditions of unknown genetic basis were capture and Illumina sequencing in regions of overlap. Most screened for HMGB3 mutations by Sanger sequencing. No vari- calls were concordant. We manually inspected discordant ants were identified. positions. Sixty-three positions marked as discordant were not called in Illumina data because of insufficient depth, largely at regions that were near the ends of targeted Discussion regions. Nine positions in the Illumina data were not called because of poor mapability. We describe a mutation in a kindred with a syndrome with the The region of overlap between the whole-genome se- features of microphthalmia, microcephaly, intellectual dis- quence from Complete Genomics was compared with se- ability, and short stature. Using a combination of whole-

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genome sequencing, targeted chromosome X-exome sequenc- nucleosome to target HMGB1 to nucleosome linker DNA, a ing, and confirmatory Sanger sequencing, we found a mutation binding site for HMGB1 on chromatin.14 Morpholino knock- in the HMGB3 gene. By combining orthogonal sequencing down of the Xenopus ortholog, Xhmgb3, was reported to methods, our ability to identify pathogenic variants was sig- have a significant role in retinal progenitor proliferation nificantly improved over the use of a single technique. As noted during eye development and to produce reduced eye and above, no other variants with predicted damaging effects were brain sizes in developing embryos.15 As can be seen in found in the whole-genome or targeted exon sequencing data Figure 3, the carboxyl terminus of the HMGB3 protein is from the proband. remarkably conserved in vertebrates. The predicted substi- We suggest that the mutation arose in or was passed from tution of the acidic polyglutamic tail with a basic polyargi- the great grandmother (I-1), who had 8 daughters and 1 son. nine domain is expected to have a significant effect on The skewed female to male ratio of offspring may have re- HMGB3 function. Although we cannot entirely rule out that sulted from the loss of affected males during pregnancy. We the variant seen in PORCN may be causative and segregate have been unable to identify HMGB3 mutations in other pa- as an X-linked recessive, we believe that the milder tients with microphthalmia phenotypes, and thus the condi- phenotype seen in our patient, the neutral Condel16 score, tion reported here is rare. Consequently, we propose that this and the mode of inheritance make it significantly particular disorder be named Maine microphthalmia (re- less likely than the clearly deleterious frameshift in cently designated OMIM 300915) to distinguish it from re- HMGB3. lated phenotypes and as originally suggested by Goldberg and There are 110 clinical synopses in the OMIM database that McKusick.3 include the matching term microphthalmia, of which at least 46 HMGB3 is a member of a subfamily of genes thought to have known mutations that cause more than 50 clinical enti- play a key role in DNA replication, nucleosome assembly, ties with microphthalmia. Among them, several phenotypes in- and transcription.13 For the related protein HMGB1, the clude clinical features seen in Maine microphthalmia. MCOPS2 acidic tail was found to interact with histone H3 in the (OMIM 300166) is characterized with microphthalmia, intellectual disability, external ear malformations, scoliosis, and geni- Table 1. Filtering Results From Whole-Genome and X-Exome Sequencing tourinary malformations caused by abnormalities in the BCOR 17 of the Proband gene (OMIM 300485) at chromosome Xp11.4. MCOPS3 (OMIM 206900) is characterized by microphthalmia, external ear mal- Complete Genomics Agilent/Illumina Identified Variants Whole Genome X-Exome formations, genitourinary malformations, and tracheo- Total variants 726 238 858 esophageal malformations caused by mutations in SOX2 (OMIM Chromosome X only 17 684 858 184429) at 3q26.2.18 MCOPS9 (OMIM 601186) is characterized by Not in dbSNP 138 10 032 70 microphthalmia, intellectual disability, and genitourinary, car- Frameshift or missense 20 7 diac, and pulmonary malformations caused by mutations in 19 Highly conserved 6 5 STRA6 (OMIM 610745) at 15q24. BCOR is a repressor of BCL6 Final manual review 2 2 (OMIM 109565), a transcription repressor required for germi- nal center formation.20 SOX2 is a member of the SRY-related Abbreviation: dbSNP 138, Single Nucleotide Polymorphism Database build 138. HMG-box family of transcription factors required for gene regu-

Table 2. The 2 Genes Identified by Each Filtering Cascade on Chromosome X

Position Ref Alter Type Gene Change Condel Scorea Comments 48 370 741 C A SNV PORCN Missense Ala>Glu 0.36-0.46 Condel neutral 150 156 265 NA TA Indel HMGB3 Frameshift NA See text

Abbreviations: Alter, alternative nucleoside; Indel, insertion/deletion; NA, not applicable; Ref, human reference sequence; SNV, single-nucleotide variant. a Condel16 scores were calculated using the online web tool at http://bg.upf.edu/condel/analysis.

Figure 2. Alignment of Sanger Sequence From the Proband Against the Human HMGB3 Reference cDNA NM_005342.2

C T G A C T A T A T A A G T C G A A A G

Exon 4 was amplified by polymerase chain reaction and sequenced. The Proband AGGATGTTGCTGACTATATAAGTCGAAAGGAAAGTTTGATGGTGCAAAGGGTCCTGCTAA resulting sequence was in agreement with the insertion/deletion identified NM_005342.2 556 AGGATGTTGCTGAC - - TATAAGTCGAAAGGAAAGTTTGATGGTGCAAAGGGTCCTGCTAA 613 from the Illumina X-exome and Ly sAs pVa l A l aAs p Ty r Ly s Se r Ly sG l yLy s PheAs pG l yA l aLy sG l yP r oA l aLy s Complete Genomics whole-genome sequences.

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Figure 3. Conservation of HMGB3 Protein Sequences

HUMAN ------MAKGDPKKP P GKMSAYA F FVQ TCREEHKKKNPEVPVNFAEFSKKCSERWKTM S GKEK S KFD E MAK PROBAND ------MAKGDPKKPKGKMSAYA F FVQ TCREEHKKKNPEVPVNFAEFSKKCSERWKTM S GKEK S KFD E MAK ORANGUTAN ------MAKGDPKKPKGKMSAYA F FVQ TCREEHKKKNPEVPVNFAEFSKKCSERWKTM S GKEK S KFD E MAK MACAQUE ------MAKGDPKKPKGKMSAYA F FVQ TCREEHKKKNPEVPVNFAEFSKKCSERWKTM S GKEK S KFD E MAK MARMOSET ------MAKGDPKKPKGKMSAYA F FVQ TCREEHKKKNPEVPVNFAEFSKKCSERWKTM S GKEK S KFD E MAK MOUSE ------MAKGDPKKPKGKMSAYA F FVQ TCREEHKKKNPEVPVNFAEFSKKCSERWKTM S S KEK S KFD E MAK PIG ------MAKGDPKKPKGKMSAYA F FVQ TCREEHKKKNPEVPVNFAEFSKKCSERWKTM S GKEK S KFD E MAK OPOSSUM ------GDPKKPKGKMSAYA F FVQ TCREEHKKKNPEVPVNFAEFSKKCSERWKTM S GKEK S KFD E MAK PLATYPUS ------MAKGDPKKPKGKMSAYA F FVQ TCREEHKKKNPEVPVNFAEFSKKCSERWKTM S GKEK S KFD E MAK CHICKEN MAQLLPHCRQQY RV RMAKGDPKKPKGKMSAYA F FVQ TCREEHKKKNPEVPVNFAEFSKKCSERWKTMA S KEKAKFD E MAK XENOPUS ------MAK R DPKKPKGKMSAYAYFVQ TCREEHKKKNPE I PVNFAEFSKKCSERWRSM S GKEK S KF E D L AK

HUMAN A D K V R Y D R E M K D Y G P A K G G K K K K D P N A P K R P P S G F F L F C S E F R P K I K S T N P G I S I G D V A K K L G E M N N N L N D S E K Q P Y I T K PROBAND A D K V R Y D R E M K D Y G P A K G G K K K K D P N A P K R P P S G F F L F C S E F R P K I K S T N P G I S I G D V A K K L G E M N N N L N D S E K Q P Y I T K ORANGUTAN A D K V R Y D R E M K D Y G P A K G G K K K K D P N A P K R P P S G F F L F C S E F R P K I K S T N P G I S I G D V A K K L G E M N N N L N D S E K Q P Y I T K MACAQUE A D K V R Y D R E M K D Y G P A K G G K K K K D P N A P K R P P S G F F L F C S E F R P K I K S T N P G I S I G D V A K K L G E M N N N L N D S E K Q P Y I T K MARMOSET A D K V R Y D R E M K D Y G P A K G G K K K K D P N A P K R P P S G F F L F C S E F R P K I K S T N P G I S I G D V A K K L G E M N N N L N D S E K Q P Y I T K MOUSE A D K V R Y D R E M K D Y G P A K G G K K K K D P N A P K R P P S G F F L F C S E F R P K I K S T N P G I S I G D V A K K L G E M N N N L S D N E K Q P Y V T K PIG A D K V R Y D R E M K D Y G P A K G G K K K K D P N A P K R P P S G F F L F C S E F R P K I K S T N P G I S I G D V A K K L G E M N N N L S D S E K Q P Y I N K OPOSSUM A D K V R Y D R E M K D Y G P A K G G K K K K D P N A P K R P P S G F F L F C S E F R P K I K S T N P G I S I G D V A K K L G E M N N N L S D N E K Q P Y N N K PLATYPUS A D K V R Y D R E M K D Y G P A K G G K K K K D P N A P K R P P S G F F L F C S E F R P K I K S T N P G I S I G D V A K K L G E M N N N L S D S E K Q P Y N N K CHICKEN A D K V R Y D R E M K D Y G P A K G G K K K K D P N A P K R P P S A F F L F C S E F R P K I K S T N P G I S I G D V A K K L G E M N N N L S D G E K Q P Y N N K XENOPUS A D K V R Y D R E M K D F G P V K K G K R N K D P N A P K R P P S G F F L F C S E F R P K I K S T N P G I S I G D I A K K L G E M N N N L S D G E K Q P Y N N K

HUMAN AAKLKEKYEKDVADYKSKGKFD G A K GP A - KVARK K V E E E E E E E E E E E E E E E E E E D E - - - 200 PROBAND AAKLKEKYEKDVADY I SRK ES L MV QRV L L K L L GKRW K R KMK K R R R K K R R R R R R RMNK E TVYL S PCEYLES* ORANGUTAN AAKLKEKYEKDVADYKSKGKFD G A K GP A - KVARK K V E E E D E E E E E E E E E E E E E G G - - - - 199 MACAQUE AAKLKEKYEKDVADYKSKGKFD G A K GP A - KVARK K V E E E D E E E E E E E E E E E E E E E D E - - 201 MARMOSET AAKLKEKYEKDVADYKSKGKFD G A K GP A - KVARK K V E E E D E DD E E E E E E E E E E E E E E DE 203 MOUSE AAKLKEKYEKDVADYKSKGKFD G A K GP A - KVARK K V E E E E E E E E E E E E E E E E E E D E - - - 200 PIG AAKLKEKYEKDVADYKSKGKFD G A K GP A - KVARK K V E E D D E D E DDDDDD E E E E E E E D E - 202 OPOSSUM AAKLKEKYEKDVADYKSKGKFD G V K GT A - KA S RK K V E E E E E E E E E E E E E E E E ------193 PLATYPUS AAKLKEKYEKDVADYKSKGKFD G A K GA A - KAARK K V E E E D E E D E E E D E E D E D E D D D E - - 201 CHICKEN AAKLKEKYEKDVADYKSKGKFD G A K GA ATKAARK K V E E E D E E E E E D E E E E D E DD D D E - - 217 XENOPUS AAKLKEKYEKDVADYKSKGKFD G A K GA P - K L ARK K E E D Y D DD E E E E E D E E D E E E D D E - - 201

The predicted frameshifted protein is highlighted in red. Species differences from human are italicized.

lation in the central nervous system and in many other tissues networks involved in microphthalmia. On the basis of this throughout development.21 STRA6 encodes a membrane pro- study, we believe that HMGB3 should be added to the list tein involved in the metabolism of retinol.22 Further func- of candidate genes to be investigated in future studies of tional and animal studies of these and other genes will be nec- this and other inherited ocular disorders, especially in male essary to fully understand the developmental pathways and patients.

ARTICLE INFORMATION Critical revision of the manuscript for important Additional Contributions: Marla O'Neill, MD, MPH, Submitted for Publication: November 26, 2013; intellectual content: Mohr, Kasch, Barton, Pittiglio, and Joanna Amberger, BA, the Johns Hopkins final revision received March 20, 2014; accepted Ingersoll, Craig, Marosy, Doheny, Bromley, University School of Medicine, discussed the March 21, 2014. Chassaing, Calvas, Prabhu, Jabs. phenotype and reviewed the manuscript. They Statistical analysis: Mohr. were not compensated for their work. Published Online: July 3, 2014. Administrative, technical, or material support: Scott, doi:10.1001/jamaophthalmol.2014.1731. Mohr, Kasch, Barton, Pittiglio, Ingersoll, Craig, REFERENCES Author Contributions: Dr Scott had full access to Marosy, Bromley, Chassaing, Calvas, Prabhu, Jabs. 1. Shaw GM, Carmichael SL, Yang W, Harris JA, all the data in this study and takes responsibility for Study supervision: Scott, Doheny, Jabs. Finnell RH, Lammer EJ. Epidemiologic characteris- the integrity of the data and the accuracy of the Conflict of Interest Disclosures: None reported. tics of anophthalmia and bilateral microphthalmia data analysis. Additional Information: This work is dedicated to among 2.5 million births in California, 1989-1997. Study concept and design: Scott, Mohr, Barton, Am J Med Genet A. 2005;137(1):36-40. Pittiglio, Roderick, Chassaing, Calvas, Jabs. the memory of Victor A. McKusick, MD, the Johns Acquisition, analysis, or interpretation of data: Scott, Hopkins University School of Medicine, and Thomas 2. Shah SP, Taylor AE, Sowden JC, et al; Mohr, Kasch, Barton, Pittiglio, Ingersoll, Craig, Marosy, H. Roderick, PhD, The Jackson Laboratory, Center Surveillance of Eye Anomalies Special Interest Doheny, Bromley, Chassaing, Calvas, Prabhu, Jabs. for Human Genetics. We thank the family who Group. Anophthalmos, microphthalmos, and Drafting of the manuscript: Scott, Mohr, Barton, provided samples and have waited so many years coloboma in the United Kingdom: clinical features, Pittiglio, Roderick, Chassaing, Calvas, Prabhu, Jabs. to learn the cause of their disorder. results of investigations, and early management. Ophthalmology. 2012;119(2):362-368.

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3. Goldberg MF, McKusick VA. X-linked insect, worm, and yeast genomes. Genome Res. 16. González-Pérez A, López-Bigas N. Improving colobomatous microphthalmos and other 2005;15(8):1034-1050. the assessment of the outcome of nonsynonymous congenital anomalies: a disorder resembling Lenz’s 10. Ng PC, Henikoff S. SIFT: Predicting amino acid SNVs with a consensus deleteriousness score, dysmorphogenetic syndrome. Am J Ophthalmol. changes that affect protein function. Nucleic Acids Condel. Am J Hum Genet. 2011;88(4):440-449. 1971;71(5):1128-1133. Res. 2003;31(13):3812-3814. 17. Ng D, Thakker N, Corcoran CM, et al. 4. Lenz W. Recessive, sex-limited microphthalmia 11. Adzhubei IA, Schmidt S, Peshkin L, et al. Oculofaciocardiodental and Lenz microphthalmia with multiple abnormalities [in German]. A method and server for predicting damaging syndromes result from distinct classes of mutations Z Kinderheilkd. 1955;77(4):384-390. missense mutations. Nat Methods. 2010;7(4): in BCOR. Nat Genet. 2004;36(4):411-416. 5. Forrester S, Kovach MJ, Reynolds NM, Urban R, 248-249. 18. Fantes J, Ragge NK, Lynch SA, et al. Mutations Kimonis V. Manifestations in four males with and an 12. Thorvaldsdóttir H, Robinson JT, Mesirov JP. in SOX2 cause anophthalmia. Nat Genet. 2003;33 obligate carrier of the Lenz microphthalmia Integrative Genomics Viewer (IGV): (4):461-463. syndrome. Am J Med Genet. 2001;98(1):92-100. high-performance genomics data visualization and 19. Pasutto F, Sticht H, Hammersen G, et al. 6. Esmailpour T, Riazifar H, Liu L, et al. A splice exploration. Brief Bioinform. 2013;14(2):178-192. Mutations in STRA6 cause a broad spectrum of donor mutation in NAA10 results in the 13. Wilke K, Wiemann S, Gaul R, Gong W, Poustka A. malformations including anophthalmia, congenital dysregulation of the retinoic acid signalling Isolation of human and mouse HMG2a cDNAs: heart defects, diaphragmatic hernia, alveolar pathway and causes Lenz microphthalmia evidence for an HMG2a-specific 3′ untranslated capillary dysplasia, lung hypoplasia, and mental syndrome. J Med Genet. 2014;51(3):185-196. region. Gene. 1997;198(1-2):269-274. retardation. Am J Hum Genet. 2007;80(3):550-560. 7. Barnhart MG, Hetrick K, Goldstein J, et al. 14. Ueda T, Chou H, Kawase T, Shirakawa H, 20. Huynh KD, Fischle W, Verdin E, Bardwell VJ. CIDRSeqSuite 2.0: an automated analysis pipeline Yoshida M. Acidic C-tail of HMGB1 is required for its BCoR, a novel corepressor involved in BCL-6 for next-generation sequencing [abstract 805]. In: target binding to nucleosome linker DNA and repression. Genes Dev. 2000;14(14):1810-1823. Proceedings and abstracts of the 61st Annual Meeting transcription stimulation. Biochemistry. 2004;43 21. Sarkar A, Hochedlinger K. The Sox family of tran- of the American Society for Human Genetics; October (30):9901-9908. scription factors: versatile regulators of stem and 12, 2011; Montreal, Quebec, Canada. 15. Terada K, Kitayama A, Kanamoto T, Ueno N, progenitor cell fate. Cell Stem Cell. 2013;12(1):15-30. 8. McKenna A, Hanna M, Banks E, et al. The Furukawa T. Nucleosome regulator Xhmgb3 is 22. Kawaguchi R, Yu J, Honda J, et al. A membrane Genome Analysis Toolkit: a MapReduce framework required for cell proliferation of the eye and brain as receptor for retinol binding protein mediates for analyzing next-generation DNA sequencing a downstream target of Xenopus rax/Rx1. Dev Biol. cellular uptake of vitamin A. Science. 2007;315 data. Genome Res. 2010;20(9):1297-1303. 2006;291(2):398-412. (5813):820-825. 9. Siepel A, Bejerano G, Pedersen JS, et al. Evolutionarily conserved elements in vertebrate,

OPHTHALMIC IMAGES

Multiple Serous Retinal Detachments Seen on Wide-Field Imaging in a Patient With Sympathetic Ophthalmia

Bryn M. Burkholder, MD; James P. Dunn, MD

A B

A 20-year-old healthy patient with a history of corneal perforation from chronic herpetic disease in the left eye presented with decreased vision in the right eye. Wide-field fluorescein angiography showed punctate areas of leakage and multiple lobular serous retinal detachments in the posterior pole of the right eye (A), consistent with a diagnosis of sympathetic ophthalmia; the subretinal fluid had resolved 2 months later, after treatment with oral prednisone and initiation of mycophenolate mofetil (B).

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