How to Cite this Article: Schönewolf-Greulich B, Ronan A, Ravn K, Baekgaard P, Lodahl M, Nielsen K, Rendtorff ND, Tranebjaerg L, Brøndum-Nielsen K, Tümer Z. 2011. Two new cases with microdeletion of 17q23.2 suggest presence of a candidate gene for sensorineural hearing loss within this region. Am J Med Genet Part A 155: 2964–2969.
Article first published online: 3 NOV 2011
Copyright © 2011 Wiley Periodicals, Inc.
American Journal of Medical Genetics Part A
Volume 155, Issue 12, pages 2964–2969, December 2011
How to Cite
Schönewolf-Greulich, B., Ronan, A., Ravn, K., Baekgaard, P., Lodahl, M., Nielsen, K., Rendtorff, N. D., Tranebjaerg, L., Brøndum-Nielsen, K. and Tümer, Z. (2011), Two new cases with microdeletion of 17q23.2 suggest presence of a candidate gene for sensorineural hearing loss within this region. Am. J. Med. Genet., 155: 2964–2969. doi: 10.1002/ajmg.a.34302
Conflict of interests: none.
Bitten Schönewolf-Greulich and Anne Ronan contributed equally to this work.
- Issue published online: 21 NOV 2011
- Article first published online: 3 NOV 2011
- Manuscript Accepted: 17 AUG 2011
- Manuscript Received: 23 FEB 2011
- sensorineural hearing loss;
- speech delay, TBX4, TBX2
Microdeletion of the 17q23.2 region has very recently been suggested as a new emerging syndrome based on the finding of 8 cases with common phenotypes including mild-to-moderate developmental delay, heart defects, microcephaly, postnatal growth retardation, and hand, foot, and limb abnormalities. In this report, we describe two new 17q23.2 deletion patients with mild intellectual disability and sensorineural hearing loss. They both had submicroscopic deletions smaller than the common deleted region for the 8 previously described 17q23.2 microdeletion cases. TBX4 was previously suggested as the responsible gene for the heart or limb defects observed in 17q23.2 deletion patients, but the present cases do not have these features despite deletion of this gene. The finding of sensorineural hearing loss in 5 of the 10 cases, including the present cases, with a microdeletion at17q23.2, strongly suggests the presence of a candidate gene for hearing loss within this region. We screened 41 patients with profound sensorineural hearing loss for mutations of TBX2 and detected no mutations. © 2011 Wiley Periodicals, Inc.
The introduction of chromosome microarray in diagnostic investigation has led to the identification of novel chromosomal imbalances in patients with intellectual disability and less specific phenotypes. The finding of unique microdeletions/duplications in patients with similar phenotypical features has enabled identification of new candidate genes for specific clinical features, as well as new microdeletion/duplication syndromes. One of the new syndromes is the 17q23.2 deletion syndrome initially described by Ballif et al.  in seven patients and later in a single case by Nimmakayalu et al. . Apart from these 8 molecularly characterized cases, there are 5 others in the literature with microscopically visible deletions covering the 17q23.2 region [Dallapiccola et al., 1993; Khalifa et al., 1993; Levin et al., 1995; Thomas et al., 1996; Marsh et al., 2000]. The common features of these cases were mild-to-moderate developmental delay (12/12), microcephaly (10/15), heart defects (10/15), limb anomalies (7/14), and hearing loss (6/12) (Table I).
Nimmakayalu et al. [2011
Ballif et al. [2010
Dallapiccola et al. [1993
Khalifa et al. [1993
Marsh et al. [2000
Levin et al. [1995
Thomas et al. [1996
|Case 1||Case 2||Case 1||7 Cases||Case 1a||Case 1||Case 1a||Case 1a||Case 1|
|Developmental delay||+||+||+||7/7||?||+||?||?||+||12/12 (100%)|
|Hearing loss||SNHL||SNHL||SNHL||2/7||?||+||?||?||−||6/12 (50%)|
|Heart defects||−||−||+||6/7||+||−||+||+||−||10/15 (67%)|
|Limb abnormalities||−||−||−||4/7||−||+||?||+||+||7/14 (50%)|
Six of the molecularly described cases had the same 2.2 Mb deletion (chromosome position: 58.0–60.2 Mb, Hg19), suggesting nonallelic homologous recombination as mediator of these rearrangements [Ballif et al., 2010]. The other deletions were 2.8 Mb (chromosome position: 57.4–60.2 Mb) and 3.75 Mb (chromosome position: 56.4–60.15 Mb) in size, respectively [Ballif et al., 2010; Nimmakayalu et al., 2011]. In this study, we present two new patients with 17q23.2 microdeletions overlapping with the 8 previously reported microdeletion cases [Ballif et al., 2010; Nimmakayalu et al., 2011] and 5 cases with microscopically visible deletions [Dallapiccola et al., 1993; Khalifa et al., 1993; Levin et al., 1995; Thomas et al., 1996; Marsh et al., 2000] (Fig. 1). The patients reported here have mild developmental delay and sensorineural hearing loss, but they do not have heart or limb defects. We also screened a population of children with hearing loss for mutations of TBX2 but found no mutations.
A female, first child to a healthy nonconsanguineous Danish couple, was born after an uncomplicated pregnancy and normal delivery (Fig. 2A). Birth weight was 3,250 g, birth length 50 cm and head circumference 34 cm. Physical examination showed asymmetry of the jaw and a slight torticollis, but no other dysmorphic features. Routine neonatal hearing screening using otoacoustic emission testing suggested mild hearing impairment (about 45 dB) and she had very narrow external ear canals. At the age of 5 months auditory brainstem response (ABR) evaluation confirmed moderate sensorineural hearing loss (50 and 60 dB hearing loss at 2,000 and 4,000 Hz, respectively). At age 11 months slight progression had occurred showing 70 dB hearing loss at the frequencies 250, 500, 1,000, and 2,000 Hz, and 85 dB hearing loss at 4,000 Hz. The audiogram was flat. Treatment with hearing aids was beneficial and her hearing has been stable since. Cranial computed tomography (CT) scan of the temporal bones in 2006 (at age 1.5 years) showed broadened semicircular canals, but was otherwise normal.
Mild developmental delay was apparent from the first year of life: she sat alone at age 9 months and walked at age 19 months. At age 3 years a Snijders-Oomen nonverbal intelligence test suggested an overall delay in cognitive functions corresponding to a level of 2½ years, especially showing speech delay and problems with social contact. Language development was assessed with the Reynell test and revealed delay in verbal comprehension. Expressive language was within the normal range, but she had verbal apraxia. Motor-perceptual development test suggested a delay of approximately 6–9 months. The brain magnetic resonance imaging and echocardiography were normal. Ophthalmological examination revealed alternating strabismus and slight hypermetropia. Clinical examination at age 5½ years showed normal extremities, with normal movements of hips and knees, and without any palpable deformities of patella. The jaw asymmetry had almost normalized without surgical intervention. All the physical measurements were within normal limits: height 118 cm, weight 20 kg, and head circumference 49 cm.
There was no family history of hearing loss, speech delay, or developmental problems.
Sequencing of the GJB2 and SLC26A4 genes did not reveal any disease related mutations. Deletion analysis of GJB6 [del(GJB-D13S1830) and del(GJB6-D13S1854)] were also normal.
This boy, the second child to a nonconsanguineous Australian couple, was born after induction at term due to prolonged labor (Fig. 2B). Birth weight was 3,050 g, length 49.5 cm, and head circumference 33 cm, all normal for gestation. Early development appeared normal; he crawled at age 7 months and walked at age 12 months. Speech delay led to assessment by Educational Support Service Profile at age 3.5 years, which showed significant delays in language, social, and fine motor skills. Gross motor skills were normal. Further assessment at age 4 years revealed bilateral SNHL of 40–50 dB which was treated with bilateral hearing aids. Despite amplification of hearing psychometric assessment at age 7 years using the Weschler nonverbal scale of ability showed that he was functioning in the mild to borderline range of intellectual disability. Currently he attends a mainstream school with considerable support, and is active and sociable with an interest in farm activities. There was no significant family history of hearing loss or intellectual difficulties.
On examination at age 7 years height and weight were above the 97th centile but head circumference was on the 25th centile with marked brachycephaly and flat occiput. He had epicanthal folds but normal interpupillary distance and was otherwise nondysmorphic, although he differed in appearance from other family members. Patellae were normal to palpation and he had full range of movement at all joints.
Investigations including renal and liver function, thyroid function, urine metabolic screen, intrauterine infection screen, electrocardiography, karyotype, and Fragile X analysis were all normal. Skull X-ray at age 6 years suggested early fusion of coronal sutures, but this was not identified by CT scan and neurosurgical opinion was that no treatment was needed. Screening of exon 7 of FGFR3 for the p.Pro250Arg mutation associated with Muenke syndrome of deafness and craniosynostosis was normal. CT scan of the temporal bones did not show inner ear abnormality. Analyses of GJB2 and the common GJB6 deletions for genetic causes of SNHL were normal. X-rays of the pelvis and femoral bone, and CT scan of spine, chest and abdomen after a motor vehicle accident at age 9 years showed no anomalies. Echocardiogram at age 10 years was normal.
Array CGH, qPCR, and FISH Studies
Array CGH was carried out using CytoChip BAC array (BlueGnome Ltd., Cambridge, UK) v2.0.1 (Patient 1; Fig. 3A) or v3.01 (Patient 2). To verify the deletion of Patient 1 and to investigate the parents we performed quantitative PCR (qPCR) using SYBR Green I detection chemistry and the 7500 Fast Real-Time PCR System according to manufacturer's instructions (Applied Biosystems, Foster City, CA). qPCR was also used for further mapping of the deletion breakpoints of this patient. In Patient 2, fluorescence in situ hybridization (FISH) was performed to verify the deletion and to investigate the parents. Fine mapping of the deletion breakpoints were carried out using 60k Oligo array in this patient (BlueGnome Ltd., Cambridge, UK; Fig. 3B). The chromosome positions are given according to the UCSC Human Genome Browser Feb 2009 release (NCBI37/Hg19 assembly; http://genome.ucsc.edu).
Mutation Screening of the TBX2 Gene in 41 Unrelated Patients With Hearing Loss
Primers were designed to PCR amplify exons 2–7 and the flanking intronic regions of TBX2 (RefSeq NM_005994.3). Exon 1 which includes the 5′ untranslated sequences, the ATG start 395 bp coding sequence could not be PCR amplified due to high GC content. Primer sequences and PCR conditions are available upon request. PCR products were sequenced using BigDye Terminator chemistry (Applied Biosystems) and separated on an ABI 3130XL genetic analyzer (Applied Biosystems) according to the manufacturer's instructions. Identified base pair changes were checked against the dbSNP Short Genetic Variations Database (www.ncbi.nlm.nih.gov/snp).
Mutation screening was carried out in 41 unrelated Danish children with congenital profound hearing loss. Most of the patients belonged to nuclear families with one affected child and unrelated parents with normal hearing capacity. Two of the families had multiple affected sibs, and none of the families were suitable for preselecting based on SNP or linkage analysis hinting to 17q. All the patients have previously been screened for mutations of the GJB2 and SLC26A4 genes and deletions of the GJB6 gene, without finding any disease-related variations.
In Silico Screening of Genes Expressed in Human Inner Ear
The genes mapping to the shortest region of deletion overlap were compared against a set of ear gene expression database obtained from fetal cochlear cDNA library (Morton inner ear cDNA library) and EST database (updated as of 2002) of the Morton Hearing Research Group (http://www.ncbi.nlm.nih.gov/UniGene/library.cgi?ORG=Hs&LID=371).
Analysis of Two Patients
In Patient 1, array CGH revealed a 2–3 Mb deletion at 17q23.1-q23.2 and the deletion was further mapped with qPCR to chromosome position: 58,121,080–60,118,579, which includes 14 RefSeq genes. In Patient 2 array CGH revealed an app. 2 Mb deletion at 17q23.2 and the deletion was further mapped with 60k Oligo array to chromosome position: 58,172,730–60,315,273. The deleted region included 13 RefSeq genes. In both cases the parental studies were normal and the deletions were evaluated to be de novo of origin. No other de novo CNVs were detected in the patients.
Sequencing of the TBX2 gene in 41 unrelated Danish children with congenital profound hearing loss did not reveal any disease-causing mutations, but two previously described SNPs (rs1057987 and rs61751978). However, we cannot exclude disease-related mutations in exon 1, which could not be amplified due to high GC content.
In this study, we describe two children with sensorineural hearing loss, speech delay, and mild intellectual disability, and a microdeletion at 17q23.1-q23.2. One of the present patients (Patient 2) has the smallest deletion of the region, which enables us to delineate the shortest region of overlap for the 17q23.2 deletion syndrome to 1.95 Mb (chromosome position: 58,172,730–60,118,579; Fig. 1). This region includes 13 RefSeq genes and 11 of these genes (CA4, USP32, APPBP2, PPM1D, BCAS3, TBX2, TBX4, NACA2, BRIP1, INTS2, MED13) are annotated in the OMIM Morbid Map (http://www.ncbi.nlm.nih.gov/Omim/getmorbid.cgi). Four of the OMIM genes (CA4, PPM1D, TBX4, BRIP1) are disease-associated: Heterozygous point mutations in the CA4 (carbonic anhydrase IV) gene have been described in few families with autosomal dominant retinitis pigmentosa (RP), but the present cases do not have any signs of RP. The PPM1D gene (protein phosphatase, magnesium-dependent, 1D, alias WIP1) may contribute to growth inhibitory pathways activated in response to DNA damage [Fiscella et al., 1997] and both PPM1D and BCAS3 (Breast carcinoma amplified sequence 3), have been implicated in breast cancer development [Barlund et al., 2002; Li et al., 2002]. The BRIP1 gene (BRCA1-interacting protein 1) is one of the genes in the Fanconi anemia pathway [Levitus et al., 2005] and it is also a binding partner of the breast cancer tumor suppressor BRCA1 [Levran et al., 2005]. These genes are unlikely to contribute to the problems observed in the present cases.
Loss of function mutations in the TBX4 gene (T-box 4) are associated with the autosomal dominant small patella syndrome (SPS, OMIM 147891), characterized by small or absent patellae, abnormal ossification of the ischio-pubic junction, and abnormalities of the feet [Bongers et al., 2004]. TBX4 duplications are also described as causative for familial isolated clubfoot [Alvarado et al., 2010]. Five of the 7 patients described by Ballif et al.  were thought to have musculoskeletal abnormalities related to SPS, but only 3 had patella or acetabulum abnormalities, more specific signs of SPS. Neither the case described by Nimmakayalu et al.  nor the present cases have any clinical signs of SPS or foot abnormalities.
Together with the two present cases, hearing loss has been observed in 6 out of the 12 individuals with a 17q23.2 deletion, suggesting this region as a candidate locus for hearing impairment. To identify potential candidate genes for hearing impairment, we searched the “Morton” inner ear cDNA library for the presence of the 11 RefSeq genes residing within the common deleted region. cDNA of the three genes, USP32 (ubiquitin specific peptidase 32), APPBP2 (amyloid beta precursor protein (cytoplasmic tail) binding protein 2), and MED13 (protein phosphatase 1D magnesium-dependent, delta isoform) were present in this library. These three genes together with BCAS3, PPM1D, TBX2, TBX4, and BRIP1, are also expressed in mouse inner ear (http://www.ncbi.nlm.nih.gov/unigene; http://www.informatics.jax.org/) and may be potential candidate genes for hearing loss. One of these genes, TBX2, which is expressed in the brain and in embryonic tissue (www.ncbi.nlm.nih.gov/unigene), has previously been related to retinoic acid (RA). In a study investigating the effect of excess RA on the gene expression profile of neural crest-derived tissue, TBX2 was among the genes, which were induced by RA at all times [Williams et al., 2004]. RA is a derivative of vitamin A and deviations in optimal retinoid levels, either excess or deficiency, result in inner ear dysmorphogenesis [Romand et al., 2006]. This is a recognized teratogenic effect of vitamin A deficiency during pregnancy [Romand et al., 2006] as well as intake or even topical exposure to tretinoin (all-trans RA) during first trimester [Selcen et al., 2000]. These studies suggested that TBX2 could be a potential candidate gene for hearing loss. Even though an inner ear phenotype has not been reported for the TBX2 mouse model (http://www.informatics.jax.org/) [Harrelson et al., 2004], we investigated this gene further for mutations, as a number of genes known to cause hearing loss in human, lack a reported hearing phenotype in mice. However, sequencing of exons 2–7 in 41 children with congenital profound hearing impairment did not reveal any disease mutations. Even though we cannot exclude mutations in exon 1, the present results do not support the hypothesis that TBX2 is a candidate gene for hearing loss.
So far, 46 genes have been shown to be involved in nonsyndromic hearing loss [Hilgert et al., 2009]. There are 59 further loci where the causative genes are not identified, and 17q23 is not reported among these (www.hereditaryhearingloss.org). In conclusion the finding that 50% of patients with a deletion of 17q23.1-q23.2 have SNHL suggests that this region may be a candidate locus for hearing loss.
None of the genes within the minimum region of deletion overlap are known to be associated with intellectual disability. The cognitive problems observed in these patients might be the result of the chromosomal imbalance per se rather than being associated with a specific gene, or may be related to one or more of the deleted genes with yet unknown functions and disease associations.
We thank the families for participating and giving consent to publishing this article.
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