RESEARCH ARTICLE

Familial Microduplication of 17q23.1–q23.2 Involving TBX4 is Associated With Congenital Clubfoot and Reduced Penetrance in Females Jess F. Peterson,1,2 Lina Ghaloul-Gonzalez,3 Suneeta Madan-Khetarpal,3 Jessica Hartman,3 Urvashi Surti,1,2,4,5 Aleksandar Rajkovic,1,2,4,5 and Svetlana A. Yatsenko1,4,5* 1

Pittsburgh Cytogenetics Laboratory, Center for Medical Genetics and Genomics, Magee-Womens Hospital of UPMC, Pittsburgh, Pennsylvania 2

Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania

3

Department of Medical Genetics, Children’s Hospital of Pittsburgh of UPMC, Pittsburgh, Pennsylvania Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

4 5

Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Manuscript Received: 10 July 2013; Manuscript Accepted: 28 August 2013

Congenital clubfoot is a heterogeneous disorder that can result in functional disability, deformity, and pain if left untreated. Although the etiology is considered multifactorial in the majority of cases, a 17q23.1–q23.2 duplication has been reported in families with congenital clubfoot characterized by variable expressivity and incomplete penetrance. The candidate gene within the duplicated region is TBX4, a T-box transcription factor required for normal hind limb development. We describe a familial 2.15 Mb duplication in the 17q23.1–q23.2 region identified in a mother, daughter, and two sons. The male proband was referred for genetic evaluation due to multiple congenital anomalies including bilateral clubfoot, dysplastic hips, multiple heart defects, microcephaly, midfacial hypoplasia, brain anomalies on MRI scan, seizure disorder, optic nerve hypoplasia, hearing loss, and bilateral vocal cord paralysis. Cytogenetic testing on family members identified the 17q23.1–q23.2 duplication in both older siblings and the mother. In this family both male siblings had clubfoot, while females were phenotypically normal. Although TBX4 remains the candidate gene for congenital clubfoot involving 17q23.1–q23.2 duplications, the explanation for variable expressivity and penetrance remains unknown. Ó 2013 Wiley Periodicals, Inc.

Key words: 17q23; congenital clubfoot; TBX4; copy-number variations (CNVs); array comparative genomic hybridization (aCGH); incomplete penetrance; sex dimorphism

INTRODUCTION Congenital unilateral or bilateral clubfoot (talipes equinovarus) is a relatively common condition with an incidence of 1–2 per 1,000 newborns that can result in long-term functional disability, deformity and pain if left untreated [Dobbs and Gurnett, 2012;

Ó 2013 Wiley Periodicals, Inc.

How to Cite this Article: Peterson JF, Ghaloul-Gonzalez L, MadanKhetarpal S, Hartman J, Surti U, Rajkovic A, Yatsenko SA. 2014. Familial microduplication of 17q23.1–q23.2 involving TBX4 is associated with congenital clubfoot and reduced penetrance in females. Am J Med Genet Part A 164A:364–369.

Gray et al., 2012]. Talipes equinovarus is characterized by a downturned foot and high medial longitudinal arch. Congenital clubfoot treatment options range from stretching and casting to surgical intervention. Although the etiology of congenital clubfoot remains largely unknown, both duplications and deletions involving the 17q23.1–q23.2 region have been associated with congenital clubfoot and other malformations [Alvarado et al., 2010; Lu et al., 2012]. The candidate gene located in 17q23.1–q23.2 includes TBX4, a T-box transcription factor required for normal early hindlimb development [Dobbs and Gurnett, 2012; King et al., 2006; Menke et al., 2008; Minguillon et al., 2009; Duboc and Logan, 2011; Naiche et al., 2011]. Conflict of interest: none.
Correspondence to: Svetlana A. Yatsenko, M.D., Pittsburgh Cytogenetics Laboratory, Center for Medical Genetics and Genomics, Magee-Womens Hospital of UPMC, 300 Halket Street, Pittsburgh, PA 15213. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 25 November 2013 DOI 10.1002/ajmg.a.36238

364

PETERSON ET AL. Four families with multiple affected individuals with isolated congenital clubfoot and 17q23.1–q23.2 duplication that includes the TBX4 gene have been described [Alvarado et al., 2010; Lu et al., 2012]. All four families demonstrate an autosomal-dominant inheritance pattern of congenital clubfoot with incomplete penetrance and variable expressivity. In addition, three of the four families demonstrate male-sex predominance, while the remaining family demonstrated equal distribution of affected males and females. We report a male proband with multiple congenital anomalies including bilateral congenital clubfoot and a 2.15 Mb duplication of the 17q23.1–q23.1 region encompassing TBX4. Interestingly, a male sibling with unilateral clubfoot, and a phenotypically normal female sibling were both confirmed carriers of the 17q23.2 duplication inherited from a phenotypically normal mother.

CLINICAL REPORT The propositus was the third child of nonconsanguineous Caucasian parents. Prenatal ultrasound identified bilateral clubfoot. He was born to a 26-year-old, G3P2A0 mother and a 28-year-old father. Labor was induced due to intrauterine growth retardation and poor biophysical profile at 37 weeks gestation, and he was delivered vaginally. The patient was noted to be in respiratory distress. Apgar scores were 4 and 6 at 1 and 5 min, respectively, and he was placed on mechanical ventilation.

365 His birth weight was 1.8 kg (3.74 SD), length 42.5 cm (3.99 SD), and OFC was 28.5 cm (4.8 SD). Dysmorphic facial features included dolichocephaly, bitemporal narrowing with overriding sutures and small anterior fontanel, small palpebral fissures, posteriorly angulated ears, broad nasal bridge, skin tag on the neck, and microretrognathia (Fig. 1). He had a short sternum and shield chest appearance and fisted hands with clenched fingers bilaterally. He had camptodactyly of both thumbs and left index finger, joint contractures of fingers, absence of the distal crease on the index and left 5th fingers, single transverse creases bilaterally, left ulnar deviation, bilateral clubfoot, limited extension of the right knee and multiple joint contractures. He had vocal cord paralysis, underwent tracheostomy and was ventilator and gastrostomy tube dependent. He also underwent Nissen fundoplication due to gastroesophageal reflux and aspiration. Brain MRI showed findings consistent with microcephaly, generalized volume loss, and simplified gyral pattern, which was likely secondary due to arrested brain development. Echocardiography revealed an atrial septal defect (ASD) and patent ductus arteriosus (PDA), both resolved spontaneously. Orthopedic evaluation showed dysplastic hips with dislocation on the left, bilateral short hindfeet, overriding fingers, and severe bilateral clubfoot, corrected by serial Ponseti castings. At age 2 months the patient developed a seizure disorder later diagnosed as complex partial epilepsy, managed medically with

FIG. 1. Proband at birth. A: Frontal view. Note the posteriorly angulated ears, microretrognathia, and clubfoot. B: Fist deformities included long thumbs located underneath clenched fingers. C: Appearance of clubfoot. D: Proband at age 5 months. [Color figure can be seen in the online version of this article, available at http://wileyonlinelibrary.com/journal/ajmga]

366

AMERICAN JOURNAL OF MEDICAL GENETICS PART A

phenobarbital, levetiracetam, and oxcarbazepine. Renal ultrasonography at age 3 months demonstrated mild bilateral renal pelviectasis, small bladder, Hutch diverticulum, and congenital phimosis. Audiologic assessment at age 3.5 months showed bilateral moderate to severe hearing loss, and an ophthalmologic exam revealed bilateral optic nerve hypoplasia, hyperopia and exotropia. Due to an undescended atrophic left testicle, a simple left orchiectomy was performed at age 7 months. Family history was significant for a brother with unilateral clubfoot (no additional phenotypic abnormalities), phenotypically normal sisters and parents, all with normal intelligence (Fig. 2). Extended family history is only remarkable for a maternal aunt with clenched feet and hands.

METHODS Array comparative genomic hybridization (aCGH) analysis was performed on purified genomic DNA from the proband using a 135K oligonucleotide array (Roche NimbleGen, Madison, WI), and scanned with a DNA Microarray Scanner (Agilent Technologies, Santa Clara, CA). Results were displayed by Genoglyphix v3.0 software (Signature Genomic Laboratories, Spokane, WA). Fluorescence in situ hybridization (FISH) was performed on cultured peripheral blood interphase cells from the proband, two siblings, parents and maternal grandmother using the BAC probe (Invitrogen, Carlsbad, CA) RP11-905P16 (chr17: 56,823,202– 57,029,161; hg18), located in the duplicated region of 17q23.1– q23.2. FISH analysis and image capture was performed using Isis FISH Imaging System v5.3 software (MetaSystems, North Royalton, OH). FISH and aCGH procedures were performed according to manufacturer protocols.

FIG. 2. Pedigree. The proband has multiple congenital anomalies (MCA) including bilateral clubfoot. The mother and her three children all carry the 17q23.1–q23.2 duplication. Arrow indicates the proband. Black squares indicate males with clubfoot.

RESULTS Microarray analysis revealed a gain in DNA copy number involving a 2.15 Mb segment (chr17: 55,471,610–57,621,696; hg18) in the 17q23.1–q23.2 region that contained 11 OMIM genes (CA4, USP32, APPBP2, PPM1D, BCAS3, TBX2, TBX4, NACA2, BRIP1, INTS2, MED13) and 7 RefSeq genes (MIR4737, HEATR6, LOC645638, LOC653653, SCARNA20, C17orf64, C17orf82; Fig. 3) in the proband. FISH analysis using a RP11-905P16 probe (17q23.2) was performed on the proband to confirm aCGH findings and to determine the physical location of the duplicated segment. In addition, FISH analysis was performed on two older siblings, parents, and the maternal grandmother to determine carrier status. The 17q23.1–q23.2 duplication was confirmed by FISH analysis in the proband, both siblings and the mother (Fig. S1 in Supporting Information Online). FISH analyses of the father and maternal grandmother were negative for the 17q23 duplication. Therefore, all three children inherited the 17q23 duplication from their phenotypically normal mother.

DISCUSSION Immediately flanked by highly homologous segmental duplications, 17q23 was identified as a chromosome region prone to unequal crossing-over between paralogous segments that may result in clinically relevant copy-number variations (CNVs) [Rudd et al., 2009]. Indeed, 17q23 deletions and duplications have been repeatedly associated with human congenital malformation syndromes involving craniofacial, cardiovascular, and skeletal structures [King et al., 2006; Alvarado et al., 2010; Ballif et al., 2010; Nimmakayalu et al., 2010; Radio et al., 2010; Lu et al., 2012; Scho¨newolf-Greulich et al., 2011]. More specifically, duplication

FIG. 3. Array CGH profile showing duplication of 17q23.1–q23.2 in the proband (red rectangle). Below, a magnified view of the duplicated region (pink shaded area) that includes TBX4. Genes are displayed as blue rectangles with an arrow indicating a direction of transcription. At the bottom, duplication boundaries (black filled bars) reported by Alvarado et al. [2010] and Lu et al. [2012] are illustrated for comparison. [Color figure can be seen in the online version of this article, available at http:// wileyonlinelibrary.com/journal/ajmga]

PETERSON ET AL.

367

of the 17q23.1–q23.2 region has been reported to result in congenital clubfoot with variable penetrance and expressivity [Alvarado et al., 2010; Lu et al., 2012]. Of the genes located within the duplication, TBX4 has been identified as the candidate gene for congenital clubfoot as it plays a critical role in hindlimb initiation and development [Dobbs and Gurnett, 2012; King et al., 2006; Menke et al., 2008; Minguillon et al., 2009; Duboc and Logan, 2011; Naiche et al., 2011]. To date, individuals from five families, including our family, underwent genetic testing for 17q23.1–q23.2 duplication carrier determination (Table I). The 17q23 duplication across four of the five families with congenital clubfoot encompasses approximately the same 2.2 Mb segment and includes 18 genes. One family, reported by Lu et al. [2012], carried a 350 kb duplication that included three genes: TBX4, NACA2 and the 30 portion of BRIP1 (Fig. 3). Common to all five families is the duplicated 17q23.2 region including TBX4. A total of 32 patients (20 males, 12 females) from five families were confirmed carriers of the 17q23.1–23.2 duplication. However, only 20 patients had congenital clubfoot, 15/20 (75%) males and 5/12 (42%) females (Table I). Moreover, all five affected females were members of the same family with the 350 kb duplication of 17q23 [Lu et al., 2012]. Of the 12 non-clubfoot individuals carrying the 17q23.1–q23.2 duplication, seven patients (four males, three females) had unusually short wide feet with toe anomalies, 1 female had developmental dysplasia of the hip, and four individuals (one male, three females) were phenotypically normal. These findings demonstrate incomplete penetrance and variable expressivity in patients with TBX4 duplication. Furthermore, the penetrance and expressivity of clubfoot in offspring was not predicated on the penetrance or expressivity of the transmitting parent. Based on analysis of five families, there appears to be a male preponderance for the clubfoot phenotype among duplication carriers, including our family with two affected males and two unaffected females (Table I). Similarly, in family 5377 [Alvarado et al., 2010] females with 17q23 duplication had a normal phenotype, while males were affected. Kruse et al. [2008] hypothesized that the male predominance of idiopathic clubfoot could be explained by the Carter effect, a multifactorial threshold model with sex dimorphism for liability. This model proposes that females

require a greater load of susceptibility genes, in addition to other environmental and/or hormonal factors, and are thus less likely to develop clubfoot. Lu et al. [2012] reported the only family with clubfoot present in equal sex ratios (M: 60% [6/10], F: 62.5% [5/8]), and when compared to Alvarado et al. [2010] and our family, has a substantially smaller duplication (350 kb versus 2.2 Mb). Among 14 individuals (ten males, four females) with a 2.2 Mb duplication, nine males (90%) were affected, and none of the females had clubfoot. This suggests that in addition to TBX4, other clubfoot susceptibility genes could be located within the 2.2 Mb duplication segment (excluding the 350 kb duplicated region; Fig. 3), which increase predisposition to clubfoot in males. Variation in penetrance can be caused by gene(s) modifier(s) with sex-biased expression, position effect or imbalance in dosage of the neighboring genes due to 17q23 duplication, resulting in different expression patterns in males versus females. Within the 2.2 Mb duplication one such gene, USP32, has been found to be expressed in all tissues except ovary, with the highest expression in testes [Paulding et al., 2003]. However, little is known about this gene and further investigation would be required. In addition, TBX2 is another potential clubfoot susceptibility gene. The duplication of TBX2 has been reported in a patient with complex heart defects and skeletal malformations [Radio et al., 2010]. Our family with 17q23.1–q23.2 duplication demonstrates variable expressivity and penetrance of congenital clubfoot. The transmitting mother is phenotypically normal and all three offspring (two males, one female) inherited the duplication. The daughter is phenotypically normal while both sons were born with congenital clubfoot. Although the elder male sibling was born with unilateral clubfoot, the younger proband was born with bilateral clubfoot and additional congenital anomalies. Four families with the 17q23 duplication reported previously were ascertained from screening a cohort of patients with familial isolated clubfoot. However, detailed clinical evaluation of individuals with 17q23 duplication revealed additional abnormities in some, such as hip dysplasia and short, wide feet [Alvarado et al., 2010; Lu et al., 2012]. In our family all three children carry the same 17q23 duplication, but present with different phenotypes. The female is unaffected, the male sibling has isolated clubfoot, while the proband has bilateral clubfoot, hip

TABLE I. Summary of Clinical Features in Families With 17q23.1–q23.2 Duplication Features Size Location (hg18)

TBX4 involvement Penetrance Additional findings in non-clubfoot duplication carriers a

Family 5377 [Alvarado et al., 2010] 2.2 Mb chr17: 55,457, 520–57,693,617 Yes Males: 100% (2/2) Females: 0% (0/2) Sister: bilateral hip dysplasia Mother: none

Family 5103 [Alvarado et al., 2010] 2.2 Mb chr17: 55,457, 520–57,693,617 Yes Males: 100% (2/2) Females: none —

Family 5035 [Alvarado et al., 2010] Not reporteda 17q23.1–q23.2a Yes Males: 75% (3/4) Females: none Father: none

17q23.1q23.2 duplications identified with TaqMan copy-number assays (Hs01196629_cn and Hs02252963_cn). Converted from assembly hg19.

b

Lu et al. [2012] 350 kb chr17: 56,842, 225–57,190,554b Yes Males: 60% (6/10) Females: 62.5% (5/8) 6 family members (4 males, 2 females) w/short, wide feet

Family reported here 2.15 Mb chr17: 55,471, 610–57,621,696 Yes Males: 100% (2/2) Females: 0% (0/2) Sister and mother: none

368 dysplasia, additional anomalies (craniofacial, chest, fist, fingers), as well as brain defects. The family presented herein demonstrates high phenotypic variability and incomplete penetrance. Interestingly, 17q23.1–q23.2 deletion phenotypes are highly variable and include microcephaly, hearing loss, pulmonary hypertension, developmental delay, postnatal onset growth delay, heart defects, and limb abnormalities [Ballif et al., 2010; Nimmakayalu et al., 2010; Scho¨newolf-Greulich et al., 2011]. Remarkably, Alvarado et al. [2010] described a familial case of clubfoot in siblings with a 17q23.1–q23.2 deletion. Of the clinical features seen in deletion phenotypes, our proband has microcephaly, hearing loss, postnatal onset growth delay, heart defects, and clubfoot. Although multiple congenital anomalies were observed in only one patient with the 17q23 duplication, we speculate that these abnormalities could represent a spectrum of the 17q23 duplication phenotype. The TBX4 dosage appears to play a critical role in clubfoot, however, other genes such as TBX2 and USP32 within the duplicated 17q23 region or elsewhere may have a modifying effect. In contrast, the complex phenotype observed in the proband could be due to a combined effect of other genetic alterations in addition to the 17q23 duplication. Mechanisms explaining CNVs as they relate to human disease remain elusive. This is clearly demonstrated by the phenotypic variability and sex dimorphism associated with copy number alterations of the 17q23 region. In general, duplications of DNA segment can lead to dosage effects due to increased protein production, yet they can also affect expression of genes located at distances of up to several hundred kilobases due to the disruption of highly conserved regulatory landscapes [Montavon et al., 2012]. However, these mechanisms cannot explain the inter- and intrafamilial phenotypic variability of congenital clubfoot considering that the reported 17q23.1–q23.2 duplications are approximately of the same size (with the exception of one family with a 350 kb duplication). Multiple, large (>500 kb) CNVs (including those of unknown clinical significance) in individuals have also been reported to compound, resulting in variable phenotypic outcomes. Girirajan et al. [2012] hypothesized that a primary mutation may sensitize a person to disease from secondary mutational events, which compound at the molecular level to modify the outcome and severity. Furthermore, Girirajan et al. [2012] suggested that the CNV size and gene content, in addition to the mode of inheritance of the primary and secondary mutations, alters phenotypic variation. Based on these conclusions, the complex phenotype in our proband may result from a 17q23.1–q23.2 duplication (primary mutation) and modifier gene(s) outside of the 17q23 region, giving rise to a complex trait. Additional CNVs or mutations in other genes in association with the 17q23.1–q23.2 duplication were not reported, therefore such families may benefit from whole genome sequencing studies to decipher other genetic and epigenetic factors contributing to congenital clubfoot. In conclusion, we describe a fifth family with a 2.15 Mb duplication in the 17q23.1–q23.2 region associated with congenital clubfoot and multiple congenital anomalies. A phenotypically normal transmitting mother and affected offspring demonstrate highly variable expressivity and penetrance of the duplication. Although TBX4 is the critical gene for clubfoot, the clinical significance of other genes in the 17q23 region and mechanisms explaining the clinical heterogeneity involving this CNV remain to be elucidated.

AMERICAN JOURNAL OF MEDICAL GENETICS PART A

REFERENCES Alvarado DM, Aferol H, McCall K, Huang JB, Techy M, Buchan J, Cady J, Gonzales PR, Dobbs MB, Gurnett CA. 2010. Familial isolated clubfoot is associated with recurrent chromosome 17q23.1q23.2 microduplications containing TBX4. Am J Hum Genet 87:154–160. Ballif BC, Theisen A, Rosenfeld JA, Traylor RN, Gastier-Foster J, Thrush DL, Astbury C, Bartholomew D, McBride KL, Pyatt RE, Shane K, Smith WE, Banks V, Gallentine WB, Brock P, Rudd MK, Adam MP, Keene JA, Phillips JA III, Pfotenhauer JP, Gowans GC, Stankiewicz P, Bejjani BA, Shaffer LG. 2010. Identification of a recurrent microdeletion at 17q23.1q23.2 flanked by segmental duplications associated with heart defects and limb abnormalities. Am J Hum Genet 86:454–461. Dobbs MB, Gurnett CA. 2012. Genetics of clubfoot. J Pediatr Orthop B 21:7–9. Duboc V, Logan MP. 2011. Pitx1 is necessary for normal initiation of hindlimb outgrowth through regulation of Tbx4 expression and shapes hindlimb morphologies via targeted growth control. Development 138:5301–5309. Girirajan S, Rosenfeld JA, Coe BP, Parikh S, Friedman N, Goldstein A, Filipink RA, McConnell JS, Angle B, Meschino WS, Nezarati MM, Asamoah A, Jackson KE, Gowans GC, Martin JA, Carmany EP, Stockton DW, Schnur RE, Penney LS, Martin DM, Raskin S, Leppig K, Thiese H, Smith R, Aberg E, Niyazov DM, Escobar LF, El-Khechen D, Johnson KD, Lebel RR, Siefkas K, Ball S, Shur N, McGuire M, Brasington CK, Spence JE, Martin LS, Clericuzio C, Ballif BC, Shaffer LG, Eichler EE. 2012. Phenotypic heterogeneity of genomic disorders and rare copy-number variants. N Engl J Med 367:1321–1331. Gray K, Pacey V, Gibbons P, Little D, Frost C, Burns J. 2012. Interventions for congenital talipes equinovarus (clubfoot). Cochrane Database of Systematic Reviews 2012. King M, Arnold JS, Shanske A, Morrow BE. 2006. T-genes and limb bud development. Am J Med Genet Part A 140A:1407–1413. Kruse LM, Dobbs MB, Gurnett CA. 2008. Polygenic threshold model with sex dimorphism in clubfoot inheritance: The Carter effect. J Bone Joint Surg Am 90:2688–2694. Lu W, Bacino CA, Richards BS, Alvarez C, VanderMeer JE, Vella M, Ahituv N, Sikka N, Dietz FR, Blanton SH, Hecht JT. 2012. Studies of TBX4 and chromosome 17q23.1q23.2: An uncommon cause of nonsyndromic clubfoot. Am J Med Genet Part A 158A:1620–1627. Menke DB, Guenther C, Kingsley DM. 2008. Dual hindlimb control elements in the Tbx4 gene and region-specific control of bone size in vertebrate limbs. Development 135:2543–2553. Minguillon C, Gibson-Brown JJ, Logan MP. 2009. Tbx4/5 gene duplication and the origin of vertebrate paired appendages. Proc Natl Acad Sci USA 106:21726–21730. Montavon T, Thevenet L, Duboule D. 2012. Impact of copy number variations (CNVs) on long-range gene regulation at the HoxD locus. Proc Natl Acad Sci 109:20204–20211. Naiche LA, Arora R, Kania A, Lewandoski M, Papaioannou VE. 2011. Identity and fate of Tbx4-expressing cells reveal developmental cell fate decisions in the allantois, limb, and external genitalia. Dev Dyn 240:2290–2300. Nimmakayalu M, Major H, Sheffield V, Solomon DH, Smith RJ, Patil SR, Shchelochkov OA. 2010. Microdeletion of 17q22q23.2 encompassing TBX2 and TBX4 in a patient with congenital microcephaly, thyroid duct cyst, sensorineural hearing loss, and pulmonary hypertension. Am J Med Genet Part A 155A:418–423. Paulding CA, Ruvolo M, Haber DA. 2003. The Tre2 (USP6) oncogene is a hominoid-specific gene. Proc Natl Acad Sci 100:2507–2511. Radio FC, Bernardini L, Loddo S, Bottillo I, Novelli A, Mingarelli R, Dallapiccola B. 2010. TBX2 gene duplication associated with complex

PETERSON ET AL. heart defect and skeletal malformations. Am J Med Genet Part A 152A:2061–2066. Rudd MK, Keene J, Bunke B, Kaminsky EB, Adam MP, Mulle JG, Ledbetter DH, Martin CL. 2009. Segmental duplications mediate novel, clinically relevant chromosome rearrangements. Hum Mol Genet 18:2957–2962. Scho¨newolf-Greulich B, Ronan A, Ravn K, Baekgaard P, Lodahl M, Nielsen K, Rendtorff ND, Tranebjaerg L, Brøndum-Nielsen K, Tu¨mer Z. 2011. Two new cases with microdeletion of 17q23.2 suggest presence of a

369 candidate gene for sensorineural hearing loss within this region. Am J Med Genet Part A 155A:2964–2969.

SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher’s web-site.