CLINICAL REPORT
An Atypical 0.73 MB Microduplication of 22q11.21 and a Novel Sall4 Missense Mutation Associated With Thumb Agenesis and Radioulnar Synostosis Adam Diehl,1 Weiyi Mu,2 Denise Batista,2,3,4 and Meral Gunay-Aygun2,5* 1
School of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland
2
Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland Cytogenetics and Microarray Laboratory, Kennedy Krieger Institute, Baltimore, Maryland
3 4
Department of Pathology, Johns Hopkins University, Baltimore, Maryland
5
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland
Manuscript Received: 3 December 2014; Manuscript Accepted: 6 March 2015
We describe a 0.73 Mb duplication of chromosome 22q11.21 between LCR-B and LCR-D and a missense mutation in a conserved C2H2 zinc finger domain of SALL4 in a cognitively normal patient with multiple skeletal anomalies including radioulnar synostosis, thumb aplasia, butterfly vertebrae, rib abnormalities, and hypoplasia of the humeral and femoral epiphyses. 22q11.21 is a common site for microdeletions and their reciprocal microduplications as a result of nonallelic homologous recombination between its multiple low copy repeat regions (LCR). DiGeorge /Velocardiofacial syndrome (DG/VCFS) is classically caused by a 3 Mb deletion between LCR-A and LCR-D or a 1.5 Mb deletion between LCR-A and LCR-B. The reciprocal syndrome to DG/VCFS is the recently described 22q11.2 microduplication, which usually presents with the typical 3 Mb or 1.5 Mb duplication. Numerous atypical deletions and duplications have been reported between other LCRs. Typically, SALL4-related Duane-radial ray syndrome is caused by deletions or nonsense mutations; the only missense SALL4 mutation described prior was thought to result in gain of function and produced cranial midline defects. The skeletal anomalies presented in this report have not been previously described in association with 22q11.2 microduplication nor SALL4 mutations. Ó 2015 Wiley Periodicals, Inc.
Key words: chromosome 22q11.21 microduplication syndrome; SALL4; thumb agenesis; radioulnar synostosis
How to Cite this Article: Diehl A, Mu W, Batista D, Gunay-Aygun M. 2015. An atypical 0.73 MB microduplication of 22q11.21 and a novel SALL4 missense mutation aassociated with thumb agenesis and radioulnar synostosis. Am J Med Genet Part A 9999A:1–6.
be ascertainment bias. Many studies search for alterations at 22q11.2 in a patient cohort that exhibits DG/VCFS-like features [Ensenauer et al., 2003]. This bias led to the idea that these 22q11.2 microduplications showed partial overlap with the DG/VCFS phenotype, with features such as velopharyngeal insufficiency and cardiac malformations [Ou et al., 2008; Portnoı¨ et al., 2009]. However, the phenotype of 22q11.2 microduplications is highly variable and may be normal or near-normal [Ou et al., 2008; Pebrel-Richard et al., 2012]. Thus, many of these microduplications are likely to be missed. With the increasing use of CGH and SNP arrays in patients with developmental delay or congenital anomalies, new phenotypes for 22q11.2 microduplication are likely to be identified. Most microduplications at 22q11.2 involve the 3 Mb region from LCR-A to LCR-D, the reciprocal of the common DG/VCFS deletion, and the 1.5Mb region from LCR-A to LCR-B, the reciprocal of the common 1.5Mb nested deletion [Yobb et al., 2005; Alberti et al., 2007; Ou et al., 2008]. The major phenotypic
INTRODUCTION The chromosome 22q11.2 region is a hotspot for microdeletions and microduplications due to its eight chromosome 22 specific low copy repeat regions (LCR22A—LCR22H), which mediate nonallelic homologous recombination (NAHR) (Fig. 1). Although these deletions and duplications are reciprocal and are expected to occur in equal proportions, far fewer microduplications at 22q11.2 have been reported. One cause for this discrepancy may
Ó 2015 Wiley Periodicals, Inc.
Grant sponsor: NICHD; Grant number: 5 P30 HD024061. Correspondence to: Meral Gunay-Aygun, Johns Hopkins University School of Medicine, Department of Pediatrics, Institute of Genetic Medicine, 600 North Wolfe Street, Blalock 1008, Baltimore, MD 21287. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 00 Month 2015 DOI 10.1002/ajmg.a.37066
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AMERICAN JOURNAL OF MEDICAL GENETICS PART A
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FIG. 1. Diagram of the 22q11.2 region: Includes the position of the LCR’s and select genes, regions encompassed by the common deletions (red) and duplications (green), the region duplicated in this report, duplicated regions associated with other skeletal anomalies and reported atypical duplications between LCR-B and LCR-D. Genomic coordinates are taken from human genome build (GRCh38/hg38/December 2013). LCR ¼ low copy repeat, DECIPHER ¼ DatabasE of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources.
features of these patients are developmental/speech delay, behavioral problems, mild dysmorphic facial features, hearing loss, and growth delay, but may be highly variable [Portnoı¨ et al., 2009]. In addition to these more common microduplications, atypical microduplications have been identified between LCR-B and LCR-D, extending beyond LCR-D, and entirely distal to LCRD [Ensenauer et al., 2003; Fan et al., 2007; 2007; Descartes et al., 2008; Ou et al., 2008; Coppinger et al., 2009; Pebrel-Richard et al., 2012]. Sal-like protein 4 is a zinc finger transcription factor. Deletions and nonsense mutations in SALL4 result in Duane-radial ray syndrome (DRRS), also referred to as Okihiro syndrome, DR syndrome and acrorenoocular syndrome. DRRS is an autosomal dominant condition characterized by radial ray malformations associated with Duane anomaly of the eye. However, the phenotype can be quite variable and may involve GI, kidney and spinal anomalies [Kohlhase et al., 2003, 2005]. Most SALL4 mutations in DRRS are small deletions, duplications, insertions or nonsense mutations that result in haploinsufficiency [Kohlhase et al., 2005]. Only one missense mutation in SALL4 was seen in a patient with an atypical phenotype consisting of mild DRRS features associated with additional cranial midline defects [Miertus et al., 2006]. Here we describe a patient with a 0.7 Mb 22q11.2 duplication ranging from LCR-B to LCRD and a SALL4 missense mutation, who had unilateral radioulnar synostosis, thumb aplasia, butterfly vertebrae, and hypoplasia of the humeral and femoral epiphyses in association with normal cognition and a normal eye exam.
MATERIALS AND METHODS SNP Array The Human Infinium CytoSNP-850K Beadchip containing over 850,000 markers (mean spacing 3.5 kb) (Illumina, Inc. USA) was analyzed with CNV partition 2.4.4.0. Karyotype and Chromosomal Breakage Analysis Peripheral blood cells were cultured for karyotype and for breakage analysis. Cells from the patient and a control were stressed with an alkylating agent, diepoxybutane (DEB), and the number of gaps, breaks and rearrangements was determined. Similarly, cells from the patient and a control were cultured untreated with DEB and the number of rearrangements determined.
Sequencing and Deletion/Duplication Analysis Genomic DNA was PCR amplified for analysis of exons and their flanking splice sites. Bi-directional sequences were compared to the published gene sequence. Genomic DNA was examined for singleexon deletions and duplications by array based comparativegenomic hybridization (aCGH) with the ExonArrayDx array.
RESULTS Clinical Report The proband is a 3-year-old girl of Chinese descent adopted at age 2 years 3 months with no available family or birth history. She was referred to genetics for multiple congenital anomalies, including
DIEHL ET AL. left thumb agenesis, congenital scoliosis, and left in-toeing. There was no concern for developmental delay, growth retardation or behavioral abnormalities. Formal IQ studies were not performed. On physical examination, she had no craniofacial dysmorphism and eye movements were completely normal. She had agenesis of the left thumb. The right thumb was normal. However, the right thenar eminence was slightly hypoplastic. All other digits were normally formed and fully functional (Fig. 2A, B). In addition, her left palm, total hand, forearm and foot lengths were shorter than the right. A skeletal survey showed left radioulnar synostosis and left thumb aplasia with otherwise normal hands and feet (Fig. 2G, H). There were butterfly vertebrae at T5 and T7 as well as hypoplastic left pedicles at T6 and T7 (Fig. 2F). The left posterior 4th, 5th, and 6th ribs were closely opposed and fused along their lateral aspect (Fig. 2F). There were 12 ribs on the right and 13 ribs on the left (Fig. 2F). Left humeral and femoral epiphyses were smaller than the right (Fig. 2C–E). Eye examination did not show Duane anomaly. Echocardiogram, electrocardiogram and renal ultrasound were normal. The patient underwent single nucleotide polymorphism (SNP) array testing, chromosomal breakage analysis, and sequencing and deletion/duplication analyses of SALL4 and TBX5.
Genetic Testing The patient’s karyotype was 46,XX. SNP array showed a 0.73 Mb interstitial duplication on 22q11.21 at genomic location 20, 379,
3 205 to 21, 109, 441 (December 2013 GRCh38/hg38). This region maps between LCR-B and LCR-D. Chromosomal breakage analysis was normal excluding Fanconi anemia. Sequencing and deletion/ duplication testing of TBX5 were negative. No deletions or duplications were identified in SALL4. SALL4 sequencing revealed a heterozygous missense mutation c.2732G>A (p.Gly911Asp) in exon 3.
DISCUSSION Initially shown to overlap with features of DG/VCFS, the 22q11.2 microduplication syndrome phenotype has since expanded with the increasing use of SNP and CGH arrays. The phenotypes associated with 22q11.2 microduplications vary from normal to severe even within the same family, and the microduplication is often inherited from a clinically normal parent [Wentzel et al., 2008]. Because of this phenotypic variability, when a 22q11.2 microduplication is identified, it is important to search for other causative/contributing mutations in addition to fully characterizing the phenotype. We describe a patient with unilateral radioulnar synostosis, thumb aplasia, rib abnormalities, hypoplasia of the humeral and femoral epiphyses and butterfly vertebrae who had a normal eye exam and normal cognition. Other genetic disorders associated with similar features include Fanconi anemia, HoltOram syndrome and DRRS. Our patient had a normal chromo-
FIG. 2. The proband at age 30 months a: Dorsal view of the hands, showing left thumb agenesis b: Ventral view of the hands c: Radiograph of the right shoulder with normal humeral epiphysis d: Radiograph of the left shoulder with slightly hypoplastic humeral epiphysis e: Radiograph of the hips and knees showing hypoplastic left femoral epiphysis f: Chest radiograph showing scoliosis, butterfly vertebrae at T5 and T7, hypoplastic left pedicles at T6 and T7, left posterior 4th, 5th, and 6th ribs that are closely opposed and fused along their lateral aspect, and a supernumerary 13th rib on the left side g: Radiograph of the right forearm h: Radiograph of the left forearm showing radioulnar synostosis and thumb agenesis.
AMERICAN JOURNAL OF MEDICAL GENETICS PART A
4 somal breakage study, and her kidney ultrasonography, eye examination, hearing test, echocardiogram, and TBX5 sequencing were normal, excluding Fanconi anemia and Holt-Oram syndrome. When attempting to rule out a mutation in SALL4, we discovered the novel missense mutation c.2732G>A that results in a glycine to aspartic acid replacement at position 911 in the most carboxy terminal C2H2-type (Kr€ uppel-like) zinc finger domain of sallike protein 4 (Fig. 3). This mutation was not observed in 6,500 individuals of European and African American ancestry from the NHLBI Exome Sequencing Project. No data are available on its frequency in the Chinese population. C2H2-type zinc fingers consist of a beta-beta-alpha motif with two cysteine and two histidine residues coordinating a zinc ion. The stability of the zinc finger structure is mainly dependent on the cysteines and histidines coordinating the zinc ion as well as the hydrophobic residues that form the hydrophobic core [Wolfe et al., 2000]. The glycine to aspartic acid change does not disrupt these conserved residues, likely leaving the zinc finger ultrastructure intact. However, this mutation lies within the alpha helix, which is inserted into the major groove of the DNA and determines the sequence specificity of zinc finger DNA binding [Wolfe et al., 2000]. Given that the first zinccoordinating histidine occupies the seventh position in the alpha helix, the mutated glycine residue then occupies position 2 within the alpha helix. In the canonical binding model of C2H2-type zinc fingers, position 2 along with residues in positions -1, 3 and 6, make the majority of base-specific contacts with the target DNA binding site[Wolfe et al., 2000]. Therefore, c.2732G>A, which replaces the small, neutral amino acid glycine with a larger, negatively charged
aspartic acid, may alter DNA binding and sequence specificity of the sal-like protein 4. However, without functional studies, whether the p.Gly911Asp mutation results in a loss or gain of function remains unknown. Haploinsufficiency of SALL4 could certainly produce some of our proband’s abnormalities, including radioulnar synostosis and thumb aplasia [Kohlhase et al., 2003]. In fact, SALL4 truncating mutations have been found in a proband with leg length discrepancy, bilateral femoral head hypoplasia, spina bifida occulta, and scoliosis as well as a proband with only 11 pairs of ribs [Kohlhase et al., 2005]. Thus, it is possible that SALL4 haploinsufficiency explains our proband’s radial ray abnormalities as well as the abnormalities of her spine, ribs and femoral epiphyses. However, butterfly vertebrae and supernumerary ribs have yet to be reported in association with SALL4 mutations. Duane-radial ray syndrome typically results from haploinsufficiency caused by protein truncating mutations in SALL4. The only previously reported missense SALL4 mutation, p.His888Arg, replaces the zinc-coordinating histidine with an arginine in the second most carboxy terminal domain (the amino terminal zinc finger of the most carboxy terminal double zinc finger domain). Modeling suggested that this mutation actually strengthened sal-like protein 4’s DNA binding affinity. The patient with p.His888Arg mutation had partial preaxial polydactyly and Duane anomaly with the additional atypical findings of cranial midline defects including single central incisor, pituitary hypoplasia, and hypotelorism [Miertus et al., 2006]. The two zinc finger motifs of the most carboxy terminal double zinc finger domain are connected by the classic TGEKP linker and likely function together to bind DNA and
FIG. 3. Diagram of sal-like protein 4: Includes the position of the c.2732G>A (p.Gly911Asp) mutation identified in this report in bold as well as the previously reported missense and nonsense mutations mentioned in the discussion. The black box within the peptide rectangle represents the glutamine rich domain thought to mediate interaction with other SALL proteins. The “I” label below the peptide rectangle marks the position of the introns in the SALL4 gene. The peptide sequences at the bottom of the figure show the consensus sequence for C2H2type zinc fingers, the wild-type SALL4 peptide sequence of the most C-terminal zinc finger domain from amino acid 898–920 (obtained from NCBI Reference Sequence: NP_065169.1), and the corresponding SALL4 sequence with the G911D mutation in bold and marked with an arrow. The c in the consensus sequence represents a hydrophobic amino acid. The asterisks mark those amino acids that coordinate with the zinc ion of the motif. The two arrows and cylinder below the peptide sequence represent the two beta sheets and single alpha helix, respectively, of the zinc finger motif. The numbers count the positions of the amino acids within the alpha helix with 1 representing the first amino acid within the alpha helix and -1 representing the first amino acid N-terminal to the alpha helix. Bolded numbers are those that are thought to mediate DNA binding in the canonical zinc finger binding model.
DIEHL ET AL. determine binding site specificity [Wolfe et al., 2000]. However, our patient does not have a cranial midline defect, implying that the p.Gly911Asp mutation does not produce the same effect on sallike protein 4 function as the previously described p.His888Arg Terhal et al. [2006] reported a nonsense mutation in the 3’ end of exon 3 that truncates sal-like protein 4 seven amino acids into the most carboxy terminal zinc finger domain while likely avoiding nonsense-mediated mRNA decay. Thus, this sal-like protein 4 may be translated without a functional carboxy terminal zinc finger domain. The family members with this mutation had a wide variability of phenotypes including Duane anomaly, leg, arm, and hand length discrepancies, triphalangeal thumbs, thumb aplasia, scoliosis, hemifacial microsomia, and ear anomalies [Terhal et al., 2006]. The limb length discrepancies, thumb aplasia, and scoliosis are similar to features seen in our patient, suggesting that a similar disruption of sal-like protein 4 may occur. In summary, the c.2732G>A (p.Gly911Asp) mutation in SALL4 reported here may be contributing to our patient’s phenotype. It is not possible to definitively determine whether this missense change in SALL4 results in loss or gain of function of the transcription factor sal-like protein 4. The rarity of reported SALL4 missense mutations may stem from an inability of these mutations to cause a complete loss of function of sal-like protein 4, but rather an aberrant function and a subsequently atypical phenotype. Description of similar patients and functional studies will probably answer these questions. Thus, the contribution from the patient’s concurrent 22q11.2 microduplication must also be considered. A representation of the 22q11.2 region and selected duplications is shown in Figure 1. Other microduplications between LCR-B and LCR-D have shown a range of phenotypes including undervirilized external genitalia, mildly dysmorphic facial features, muscular hypotonia, developmental delay, oligodontia, and cleft palate [Fan et al., 2007; Fernandez et al., 2007; Ou et al., 2008; Pebrel– Richard et al., 2012]. These few reports do not include skeletal abnormalities like those reported here. A few skeletal defects have been reported in association with other 22q11.2 duplications, including proximal radioulnar synostosis and brachydactyly, [Ensenauer et al., 2003] clinodactyly and hypoplastic nails, [Yobb et al., 2005] a triphalangeal left thumb and hypoplastic toenails [Ou et al., 2008], and developmental hip dysplasia [Weisfeld-Adams et al., 2012]. Furthermore, 22q11.2 deletion syndrome can result in pre— and postaxial polydactyly of the hands, postaxial polydactyly of the feet, supernumerary ribs, butterfly vertebrae, hemivertebrae, and craniosynostosis [McDonaldMcGinn et al., 2011]. These reports, along with the findings in our proband, suggest that disruption in the dosage of genes located between LCR-B and LCR-D may impact skeletal formation. Genes involved by the 0.73 Mb microduplication in this report include: ZNF74, SCARF2, KLHL22, MED15, PI4KA, SERPIND1, SNAP29, CRKL, AIFM3, LZTR1, THAP7, P2RX6, SLC7A4,and BCRP2. Except for their hemizygous deletion in DiGeorge syndrome, most have not been associated with a disease phenotype [OMIM, 2009]. Most notably, inactivation of the SCARF2 gene leads to van den Ende–Gupta Syndrome which, in addition to blepharophimosis, abnormal facies and contractural arachnodactyly, can be characterized by radial abnormalities, radioulnar subluxation, and hypoplastic ribs [Anastasio et al., 2010]. Patients
5 with more than one genetic change are increasingly identified [Hehir-Kwa et al., 2013; Palmer et al., 2014; Agha et al., 2014]. While the interpretation of these multiple molecular genetic abnormalities is challenging, the test results allow us to better understand our patients’ features, especially those phenotypes that do not immediately fit into well-described disorders caused by a single mutation.
ACKNOWLEDGMENTS We would like to thank the family of the proband for their participation. This work was partially supported by NICHD 5 P30 HD024061. We have no competing interests.
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6 Kohlhase J, Schubert L, Liebers M, Rauch A, Becker K, Mohammed SN, Newbury-Ecob R, Reardon W. 2003. Mutations at the SALL4 locus on chromosome 20 result in a range of clinically overlapping phenotypes, including Okihiro syndrome, Holt-Oram syndrome, acro-renalocular syndrome, and patients previously reported to represent thalidomide embryopathy. J Med Genet 40:473–478. Kohlhase J, Chitayat D, Kotzot D, Ceylaner S, Froster UG, Fuchs S, Montgomery T, R€ osler B. 2005. SALL4 mutations in Okihiro syndrome (Duane-radial ray syndrome), acro-renal-ocular syndrome, and related disorders. Hum Mutat 26:176–183. McDonald-McGinn DM, Sullivan KE. 2011. Chromosome 22q11.2 deletion syndrome (DiGeorge syndrome/velocardiofacial syndrome). Medicine (Baltimore) 90:1–18. Miertus J, Borozdin W, Frecer V, Tonini G, Bertok S, Amoroso A, Miertus S, Kohlhase J. 2006. A SALL4 zinc finger missense mutation predicted to result in increased DNA binding affinity is associated with cranial midline defects and mild features of Okihiro syndrome. Hum Genet 119:154–161. 1
Online Mendelian Inheritance in Man, OMIM . McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, MD), March 15, 2014. World Wide Web URL: http://omim.org/ Ou Z, Berg JS, Yonath H, Enciso VB, Miller DT, Picker J, Lenzi T, Keegan CE, Sutton VR, Belmont J, Chinault AC, Lupski JR, Cheung SW, Roeder E, Patel A. 2008. Microduplications of 22q11.2 are frequently inherited and are associated with variable phenotypes. Genet Med 10:267–277. Palmer E, Speirs H, Taylor PJ, Mullan G, Turner G, Einfeld S, Tonge B, Mowat D. 2014. Changing interpretation of chromosomal microarray
over time in a community cohort with intellectual disability. Am J Med Genet Part A 164A:377–385. Pebrel-Richard C, Kemeny S, Gouas L, Eymard-Pierre E, Blanc N, Francannet C, Tchirkov A, Goumy C, Vago P. 2012. An atypical 0.8 Mb inherited duplication of 22q11.2 associated with psychomotor impairment. Eur J Med Genet 55:650–655. Portnoı¨ MF. 2009. Microduplication 22q11.2: A new chromosomal syndrome. Eur J Med Genet 52:88–93. Terhal P, R€ osler B, Kohlhase J. 2006. A family with features overlapping Okihiro syndrome, hemifacial microsomia and isolated Duane anomaly caused by a novel SALL4 mutation. Am J Med Genet A 140:222–226. Weisfeld-Adams JD, Edelmann L, Gadi IK, Mehta L. 2012. Phenotypic heterogeneity in a family with a small atypical microduplication of chromosome 22q11.2 involving T BX1. Eur J Med Genet 55:732–736. Wentzel C, Fernstr€ om M, Ohrner Y, Anneren G, Thuresson AC. 2008. Clinical variability of the 22q11.2 duplication syndrome. Eur J Med Genet 51:501–510. Wolfe SA, Nekludova L, Pabo CO. 2000. DNA recognition by Cys2His2 zinc finger proteins. Annu Rev Biophys Biomol Struct 29:183–212. Yobb TM, Somerville MJ, Willatt L, Firth HV, Harrison K, MacKenzie J, Gallo N, Morrow BE, Shaffer LG, Babcock M, Chernos J, Bernier F, Sprysak K, Christiansen J, Haase S, Elyas B, Lilley M, Bamforth S, McDermid HE. 2005. Microduplication and triplication of 22q11.2: A highly variable syndrome. Am J Hum Genet 76:865–876.
An Atypical 0.73 MB Microduplication of 22q11.21 and a Novel Sall4 Missense Mutation Associated With Thumb Agenesis and Radioulnar Synostosis Adam Diehl,1 Weiyi Mu,2 Denise Batista,2,3,4 and Meral Gunay-Aygun2,5* 1
School of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland
2
Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland Cytogenetics and Microarray Laboratory, Kennedy Krieger Institute, Baltimore, Maryland
3 4
Department of Pathology, Johns Hopkins University, Baltimore, Maryland
5
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland
Manuscript Received: 3 December 2014; Manuscript Accepted: 6 March 2015
We describe a 0.73 Mb duplication of chromosome 22q11.21 between LCR-B and LCR-D and a missense mutation in a conserved C2H2 zinc finger domain of SALL4 in a cognitively normal patient with multiple skeletal anomalies including radioulnar synostosis, thumb aplasia, butterfly vertebrae, rib abnormalities, and hypoplasia of the humeral and femoral epiphyses. 22q11.21 is a common site for microdeletions and their reciprocal microduplications as a result of nonallelic homologous recombination between its multiple low copy repeat regions (LCR). DiGeorge /Velocardiofacial syndrome (DG/VCFS) is classically caused by a 3 Mb deletion between LCR-A and LCR-D or a 1.5 Mb deletion between LCR-A and LCR-B. The reciprocal syndrome to DG/VCFS is the recently described 22q11.2 microduplication, which usually presents with the typical 3 Mb or 1.5 Mb duplication. Numerous atypical deletions and duplications have been reported between other LCRs. Typically, SALL4-related Duane-radial ray syndrome is caused by deletions or nonsense mutations; the only missense SALL4 mutation described prior was thought to result in gain of function and produced cranial midline defects. The skeletal anomalies presented in this report have not been previously described in association with 22q11.2 microduplication nor SALL4 mutations. Ó 2015 Wiley Periodicals, Inc.
Key words: chromosome 22q11.21 microduplication syndrome; SALL4; thumb agenesis; radioulnar synostosis
How to Cite this Article: Diehl A, Mu W, Batista D, Gunay-Aygun M. 2015. An atypical 0.73 MB microduplication of 22q11.21 and a novel SALL4 missense mutation aassociated with thumb agenesis and radioulnar synostosis. Am J Med Genet Part A 9999A:1–6.
be ascertainment bias. Many studies search for alterations at 22q11.2 in a patient cohort that exhibits DG/VCFS-like features [Ensenauer et al., 2003]. This bias led to the idea that these 22q11.2 microduplications showed partial overlap with the DG/VCFS phenotype, with features such as velopharyngeal insufficiency and cardiac malformations [Ou et al., 2008; Portnoı¨ et al., 2009]. However, the phenotype of 22q11.2 microduplications is highly variable and may be normal or near-normal [Ou et al., 2008; Pebrel-Richard et al., 2012]. Thus, many of these microduplications are likely to be missed. With the increasing use of CGH and SNP arrays in patients with developmental delay or congenital anomalies, new phenotypes for 22q11.2 microduplication are likely to be identified. Most microduplications at 22q11.2 involve the 3 Mb region from LCR-A to LCR-D, the reciprocal of the common DG/VCFS deletion, and the 1.5Mb region from LCR-A to LCR-B, the reciprocal of the common 1.5Mb nested deletion [Yobb et al., 2005; Alberti et al., 2007; Ou et al., 2008]. The major phenotypic
INTRODUCTION The chromosome 22q11.2 region is a hotspot for microdeletions and microduplications due to its eight chromosome 22 specific low copy repeat regions (LCR22A—LCR22H), which mediate nonallelic homologous recombination (NAHR) (Fig. 1). Although these deletions and duplications are reciprocal and are expected to occur in equal proportions, far fewer microduplications at 22q11.2 have been reported. One cause for this discrepancy may
Ó 2015 Wiley Periodicals, Inc.
Grant sponsor: NICHD; Grant number: 5 P30 HD024061. Correspondence to: Meral Gunay-Aygun, Johns Hopkins University School of Medicine, Department of Pediatrics, Institute of Genetic Medicine, 600 North Wolfe Street, Blalock 1008, Baltimore, MD 21287. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 00 Month 2015 DOI 10.1002/ajmg.a.37066
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AMERICAN JOURNAL OF MEDICAL GENETICS PART A
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FIG. 1. Diagram of the 22q11.2 region: Includes the position of the LCR’s and select genes, regions encompassed by the common deletions (red) and duplications (green), the region duplicated in this report, duplicated regions associated with other skeletal anomalies and reported atypical duplications between LCR-B and LCR-D. Genomic coordinates are taken from human genome build (GRCh38/hg38/December 2013). LCR ¼ low copy repeat, DECIPHER ¼ DatabasE of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources.
features of these patients are developmental/speech delay, behavioral problems, mild dysmorphic facial features, hearing loss, and growth delay, but may be highly variable [Portnoı¨ et al., 2009]. In addition to these more common microduplications, atypical microduplications have been identified between LCR-B and LCR-D, extending beyond LCR-D, and entirely distal to LCRD [Ensenauer et al., 2003; Fan et al., 2007; 2007; Descartes et al., 2008; Ou et al., 2008; Coppinger et al., 2009; Pebrel-Richard et al., 2012]. Sal-like protein 4 is a zinc finger transcription factor. Deletions and nonsense mutations in SALL4 result in Duane-radial ray syndrome (DRRS), also referred to as Okihiro syndrome, DR syndrome and acrorenoocular syndrome. DRRS is an autosomal dominant condition characterized by radial ray malformations associated with Duane anomaly of the eye. However, the phenotype can be quite variable and may involve GI, kidney and spinal anomalies [Kohlhase et al., 2003, 2005]. Most SALL4 mutations in DRRS are small deletions, duplications, insertions or nonsense mutations that result in haploinsufficiency [Kohlhase et al., 2005]. Only one missense mutation in SALL4 was seen in a patient with an atypical phenotype consisting of mild DRRS features associated with additional cranial midline defects [Miertus et al., 2006]. Here we describe a patient with a 0.7 Mb 22q11.2 duplication ranging from LCR-B to LCRD and a SALL4 missense mutation, who had unilateral radioulnar synostosis, thumb aplasia, butterfly vertebrae, and hypoplasia of the humeral and femoral epiphyses in association with normal cognition and a normal eye exam.
MATERIALS AND METHODS SNP Array The Human Infinium CytoSNP-850K Beadchip containing over 850,000 markers (mean spacing 3.5 kb) (Illumina, Inc. USA) was analyzed with CNV partition 2.4.4.0. Karyotype and Chromosomal Breakage Analysis Peripheral blood cells were cultured for karyotype and for breakage analysis. Cells from the patient and a control were stressed with an alkylating agent, diepoxybutane (DEB), and the number of gaps, breaks and rearrangements was determined. Similarly, cells from the patient and a control were cultured untreated with DEB and the number of rearrangements determined.
Sequencing and Deletion/Duplication Analysis Genomic DNA was PCR amplified for analysis of exons and their flanking splice sites. Bi-directional sequences were compared to the published gene sequence. Genomic DNA was examined for singleexon deletions and duplications by array based comparativegenomic hybridization (aCGH) with the ExonArrayDx array.
RESULTS Clinical Report The proband is a 3-year-old girl of Chinese descent adopted at age 2 years 3 months with no available family or birth history. She was referred to genetics for multiple congenital anomalies, including
DIEHL ET AL. left thumb agenesis, congenital scoliosis, and left in-toeing. There was no concern for developmental delay, growth retardation or behavioral abnormalities. Formal IQ studies were not performed. On physical examination, she had no craniofacial dysmorphism and eye movements were completely normal. She had agenesis of the left thumb. The right thumb was normal. However, the right thenar eminence was slightly hypoplastic. All other digits were normally formed and fully functional (Fig. 2A, B). In addition, her left palm, total hand, forearm and foot lengths were shorter than the right. A skeletal survey showed left radioulnar synostosis and left thumb aplasia with otherwise normal hands and feet (Fig. 2G, H). There were butterfly vertebrae at T5 and T7 as well as hypoplastic left pedicles at T6 and T7 (Fig. 2F). The left posterior 4th, 5th, and 6th ribs were closely opposed and fused along their lateral aspect (Fig. 2F). There were 12 ribs on the right and 13 ribs on the left (Fig. 2F). Left humeral and femoral epiphyses were smaller than the right (Fig. 2C–E). Eye examination did not show Duane anomaly. Echocardiogram, electrocardiogram and renal ultrasound were normal. The patient underwent single nucleotide polymorphism (SNP) array testing, chromosomal breakage analysis, and sequencing and deletion/duplication analyses of SALL4 and TBX5.
Genetic Testing The patient’s karyotype was 46,XX. SNP array showed a 0.73 Mb interstitial duplication on 22q11.21 at genomic location 20, 379,
3 205 to 21, 109, 441 (December 2013 GRCh38/hg38). This region maps between LCR-B and LCR-D. Chromosomal breakage analysis was normal excluding Fanconi anemia. Sequencing and deletion/ duplication testing of TBX5 were negative. No deletions or duplications were identified in SALL4. SALL4 sequencing revealed a heterozygous missense mutation c.2732G>A (p.Gly911Asp) in exon 3.
DISCUSSION Initially shown to overlap with features of DG/VCFS, the 22q11.2 microduplication syndrome phenotype has since expanded with the increasing use of SNP and CGH arrays. The phenotypes associated with 22q11.2 microduplications vary from normal to severe even within the same family, and the microduplication is often inherited from a clinically normal parent [Wentzel et al., 2008]. Because of this phenotypic variability, when a 22q11.2 microduplication is identified, it is important to search for other causative/contributing mutations in addition to fully characterizing the phenotype. We describe a patient with unilateral radioulnar synostosis, thumb aplasia, rib abnormalities, hypoplasia of the humeral and femoral epiphyses and butterfly vertebrae who had a normal eye exam and normal cognition. Other genetic disorders associated with similar features include Fanconi anemia, HoltOram syndrome and DRRS. Our patient had a normal chromo-
FIG. 2. The proband at age 30 months a: Dorsal view of the hands, showing left thumb agenesis b: Ventral view of the hands c: Radiograph of the right shoulder with normal humeral epiphysis d: Radiograph of the left shoulder with slightly hypoplastic humeral epiphysis e: Radiograph of the hips and knees showing hypoplastic left femoral epiphysis f: Chest radiograph showing scoliosis, butterfly vertebrae at T5 and T7, hypoplastic left pedicles at T6 and T7, left posterior 4th, 5th, and 6th ribs that are closely opposed and fused along their lateral aspect, and a supernumerary 13th rib on the left side g: Radiograph of the right forearm h: Radiograph of the left forearm showing radioulnar synostosis and thumb agenesis.
AMERICAN JOURNAL OF MEDICAL GENETICS PART A
4 somal breakage study, and her kidney ultrasonography, eye examination, hearing test, echocardiogram, and TBX5 sequencing were normal, excluding Fanconi anemia and Holt-Oram syndrome. When attempting to rule out a mutation in SALL4, we discovered the novel missense mutation c.2732G>A that results in a glycine to aspartic acid replacement at position 911 in the most carboxy terminal C2H2-type (Kr€ uppel-like) zinc finger domain of sallike protein 4 (Fig. 3). This mutation was not observed in 6,500 individuals of European and African American ancestry from the NHLBI Exome Sequencing Project. No data are available on its frequency in the Chinese population. C2H2-type zinc fingers consist of a beta-beta-alpha motif with two cysteine and two histidine residues coordinating a zinc ion. The stability of the zinc finger structure is mainly dependent on the cysteines and histidines coordinating the zinc ion as well as the hydrophobic residues that form the hydrophobic core [Wolfe et al., 2000]. The glycine to aspartic acid change does not disrupt these conserved residues, likely leaving the zinc finger ultrastructure intact. However, this mutation lies within the alpha helix, which is inserted into the major groove of the DNA and determines the sequence specificity of zinc finger DNA binding [Wolfe et al., 2000]. Given that the first zinccoordinating histidine occupies the seventh position in the alpha helix, the mutated glycine residue then occupies position 2 within the alpha helix. In the canonical binding model of C2H2-type zinc fingers, position 2 along with residues in positions -1, 3 and 6, make the majority of base-specific contacts with the target DNA binding site[Wolfe et al., 2000]. Therefore, c.2732G>A, which replaces the small, neutral amino acid glycine with a larger, negatively charged
aspartic acid, may alter DNA binding and sequence specificity of the sal-like protein 4. However, without functional studies, whether the p.Gly911Asp mutation results in a loss or gain of function remains unknown. Haploinsufficiency of SALL4 could certainly produce some of our proband’s abnormalities, including radioulnar synostosis and thumb aplasia [Kohlhase et al., 2003]. In fact, SALL4 truncating mutations have been found in a proband with leg length discrepancy, bilateral femoral head hypoplasia, spina bifida occulta, and scoliosis as well as a proband with only 11 pairs of ribs [Kohlhase et al., 2005]. Thus, it is possible that SALL4 haploinsufficiency explains our proband’s radial ray abnormalities as well as the abnormalities of her spine, ribs and femoral epiphyses. However, butterfly vertebrae and supernumerary ribs have yet to be reported in association with SALL4 mutations. Duane-radial ray syndrome typically results from haploinsufficiency caused by protein truncating mutations in SALL4. The only previously reported missense SALL4 mutation, p.His888Arg, replaces the zinc-coordinating histidine with an arginine in the second most carboxy terminal domain (the amino terminal zinc finger of the most carboxy terminal double zinc finger domain). Modeling suggested that this mutation actually strengthened sal-like protein 4’s DNA binding affinity. The patient with p.His888Arg mutation had partial preaxial polydactyly and Duane anomaly with the additional atypical findings of cranial midline defects including single central incisor, pituitary hypoplasia, and hypotelorism [Miertus et al., 2006]. The two zinc finger motifs of the most carboxy terminal double zinc finger domain are connected by the classic TGEKP linker and likely function together to bind DNA and
FIG. 3. Diagram of sal-like protein 4: Includes the position of the c.2732G>A (p.Gly911Asp) mutation identified in this report in bold as well as the previously reported missense and nonsense mutations mentioned in the discussion. The black box within the peptide rectangle represents the glutamine rich domain thought to mediate interaction with other SALL proteins. The “I” label below the peptide rectangle marks the position of the introns in the SALL4 gene. The peptide sequences at the bottom of the figure show the consensus sequence for C2H2type zinc fingers, the wild-type SALL4 peptide sequence of the most C-terminal zinc finger domain from amino acid 898–920 (obtained from NCBI Reference Sequence: NP_065169.1), and the corresponding SALL4 sequence with the G911D mutation in bold and marked with an arrow. The c in the consensus sequence represents a hydrophobic amino acid. The asterisks mark those amino acids that coordinate with the zinc ion of the motif. The two arrows and cylinder below the peptide sequence represent the two beta sheets and single alpha helix, respectively, of the zinc finger motif. The numbers count the positions of the amino acids within the alpha helix with 1 representing the first amino acid within the alpha helix and -1 representing the first amino acid N-terminal to the alpha helix. Bolded numbers are those that are thought to mediate DNA binding in the canonical zinc finger binding model.
DIEHL ET AL. determine binding site specificity [Wolfe et al., 2000]. However, our patient does not have a cranial midline defect, implying that the p.Gly911Asp mutation does not produce the same effect on sallike protein 4 function as the previously described p.His888Arg Terhal et al. [2006] reported a nonsense mutation in the 3’ end of exon 3 that truncates sal-like protein 4 seven amino acids into the most carboxy terminal zinc finger domain while likely avoiding nonsense-mediated mRNA decay. Thus, this sal-like protein 4 may be translated without a functional carboxy terminal zinc finger domain. The family members with this mutation had a wide variability of phenotypes including Duane anomaly, leg, arm, and hand length discrepancies, triphalangeal thumbs, thumb aplasia, scoliosis, hemifacial microsomia, and ear anomalies [Terhal et al., 2006]. The limb length discrepancies, thumb aplasia, and scoliosis are similar to features seen in our patient, suggesting that a similar disruption of sal-like protein 4 may occur. In summary, the c.2732G>A (p.Gly911Asp) mutation in SALL4 reported here may be contributing to our patient’s phenotype. It is not possible to definitively determine whether this missense change in SALL4 results in loss or gain of function of the transcription factor sal-like protein 4. The rarity of reported SALL4 missense mutations may stem from an inability of these mutations to cause a complete loss of function of sal-like protein 4, but rather an aberrant function and a subsequently atypical phenotype. Description of similar patients and functional studies will probably answer these questions. Thus, the contribution from the patient’s concurrent 22q11.2 microduplication must also be considered. A representation of the 22q11.2 region and selected duplications is shown in Figure 1. Other microduplications between LCR-B and LCR-D have shown a range of phenotypes including undervirilized external genitalia, mildly dysmorphic facial features, muscular hypotonia, developmental delay, oligodontia, and cleft palate [Fan et al., 2007; Fernandez et al., 2007; Ou et al., 2008; Pebrel– Richard et al., 2012]. These few reports do not include skeletal abnormalities like those reported here. A few skeletal defects have been reported in association with other 22q11.2 duplications, including proximal radioulnar synostosis and brachydactyly, [Ensenauer et al., 2003] clinodactyly and hypoplastic nails, [Yobb et al., 2005] a triphalangeal left thumb and hypoplastic toenails [Ou et al., 2008], and developmental hip dysplasia [Weisfeld-Adams et al., 2012]. Furthermore, 22q11.2 deletion syndrome can result in pre— and postaxial polydactyly of the hands, postaxial polydactyly of the feet, supernumerary ribs, butterfly vertebrae, hemivertebrae, and craniosynostosis [McDonaldMcGinn et al., 2011]. These reports, along with the findings in our proband, suggest that disruption in the dosage of genes located between LCR-B and LCR-D may impact skeletal formation. Genes involved by the 0.73 Mb microduplication in this report include: ZNF74, SCARF2, KLHL22, MED15, PI4KA, SERPIND1, SNAP29, CRKL, AIFM3, LZTR1, THAP7, P2RX6, SLC7A4,and BCRP2. Except for their hemizygous deletion in DiGeorge syndrome, most have not been associated with a disease phenotype [OMIM, 2009]. Most notably, inactivation of the SCARF2 gene leads to van den Ende–Gupta Syndrome which, in addition to blepharophimosis, abnormal facies and contractural arachnodactyly, can be characterized by radial abnormalities, radioulnar subluxation, and hypoplastic ribs [Anastasio et al., 2010]. Patients
5 with more than one genetic change are increasingly identified [Hehir-Kwa et al., 2013; Palmer et al., 2014; Agha et al., 2014]. While the interpretation of these multiple molecular genetic abnormalities is challenging, the test results allow us to better understand our patients’ features, especially those phenotypes that do not immediately fit into well-described disorders caused by a single mutation.
ACKNOWLEDGMENTS We would like to thank the family of the proband for their participation. This work was partially supported by NICHD 5 P30 HD024061. We have no competing interests.
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