Microdeletion of 6q16.1 encompassing EPHA7 in a child with mild neurological abnormalities and dysmorphic features: case report
© Traylor et al; licensee BioMed Central Ltd. 2009
Received: 5 June 2009
Accepted: 7 August 2009
Published: 7 August 2009
Of the fewer than 100 cases reported within the literature of constitutional deletions involving the long arm of chromosome 6, only five have been characterized using high-resolution microarray analysis. Reported 6q deletion patients show a high incidence of mental retardation, ear anomalies, hypotonia, and postnatal growth retardation.
We report a 16-month-old male presenting with developmental delay and dysmorphic features who was found by array-based comparative genomic hybridization (aCGH) to have a ~2.16 Mb de novo deletion within chromosome band 6q16.1 that encompasses only two genes. Expression studies of the mouse homologue of one of the genes, the ephrin receptor 7 gene (EPHA7), have shown the gene functions during murine embryogenesis to form cortical domains, determine brain size and shape, and play a role in development of the central nervous system (CNS).
Our results suggest that deletion of EPHA7 plays a role in the neurologic and dysmorphic features, including developmental delay, hypotonia, and ear malformations, observed in some 6q deletion patients.
Conventional cytogenetic analyses have identified fewer than 100 individuals with constitutional deletions within 6q. A review by Hopkin et al.  of 57 previously reported 6q deletion cases characterized cytogenetically attempted to organize phenotype/karyotype correlations into three phenotypic groups. Deletions within 6q11 to 6q16, designated Group A, showed a high incidence of hernias, upslanting palpebral fissures, and thin lips with a lower frequency of microcephaly, micrognathia, and heart malformations. Group B deletions spanned 6q15-6q25 and showed increased intrauterine growth retardation, abnormal respiration, hypertelorism, and upper limb malformations. Group C comprised deletions in 6q25 to 6qter, which presented with retinal abnormalities, cleft palate, and genital hypoplasia. Mental retardation was the only finding common among all cases of 6q deletion. The three groups also shared ear anomalies, hypotonia, and postnatal growth retardation in 90%, 82%, and 68% of cases, respectively .
Since the Hopkin et al.  review, 10 individuals with deletions encompassing 6q16.1 identified using aCGH have been reported [2–7]. Features seen among these cases are varied as the sizes of these deletions span 6–34 Mb within 6q. Here, we report an individual with a ~2.1 Mb deletion within 6q16.1 characterized by high-resolution oligonucleotide microarray analysis. Within this deletion is only one known OMIM gene, the ephrin receptor 7 gene, EPHA7. A comparison of the clinical features in the individual reported here to those of previously reported individuals suggests that deletion of EPHA7 may play a role in neurodevelopmental deficits in individuals with deletions of 6q16.1.
The proband presented at 15 months for genetic evaluation of microcephaly and developmental delay. He was born to a 21-year-old mother and 22-year-old father at 38 weeks' gestation with birth weight at the 3rd percentile and length at the 50th percentile. Choroid plexus cysts were detected prenatally by ultrasound but pregnancy and labor were uneventful.
At 6–7 months of age, the proband was not sitting and was noted to have a mild generalized hypotonia. He had received physical therapy since age 6 months and showed mild developmental delay in gross motor skills and multiple other areas. The child was physically small with microcephaly and short stature. At age 10 months, brain MRI showed questionable delayed myelination but upon a second opinion was found to be normal.
Physical examination at 12 months of age showed height at 70.4 cm (2nd percentile), weight at 7.6 kg (<3rd percentile), head circumference 43.3 cm (1st percentile).
Karyotype analysis at the 550-band level was normal. Metabolic workup, renal studies, and echocardiogram were essentially normal.
Summary of clinical features of individuals with 6q16.1 deletions encompassing EPHA7 characterized by aCGH
Klein et al Patient 3
Le Caignec et al
Zherebstov et al
Current Growth Parameters
Head MRI results
CT: subdural hemorrhage due to significant brain atrophy
Questionable brain atrophy/Questionable hemorrhage
Mild on left
Poor visual motor development
Present (-3 SD)
Present (-2 SD)
Flat occiput/heart-shaped face
Downslanting palpebral fissures
Low-set/posteriorly rotated with hypoplastic helices
Cupped ears, periauricular tags
Bilateral echogenic kidneys/pelvicaliectasis of the right kidney
Tetrology of Fallot
The deletion identified in our case encompasses two genes. One gene, TSG1, a predicted tumor suppressor gene , is not a likely candidate for the clinical features in our patient. The remaining gene, ephrin receptor 7 (EPHA7) (chr6:94,006,458–94,186,021, UCSC build March 2006 – hg18) is part of the Eph/ephrin family of receptor tyrosine kinases (RTK), cell-surface-bound proteins whose signaling mediates cell-to-cell communication during development and directs the migration and positioning of cells within many tissue types . The complexity of Eph/ephrin signaling is demonstrated by the proteins' involvement in bidirectional signaling, participation in a broad spectrum of developmental processes, and as a player in other communicative pathways . Murine studies have shown EphA7 functions during embryogenesis to form cortical domains, determine brain size and shape, and play a role in development of the CNS [8–10].
This current case is the smallest deletion encompassing 6q16 reported, to the best of our knowledge, within the literature to date. Phenotypic overlap between previously reported 6q16 deletion cases that have been characterized at the molecular level by array CGH with this case indicates that in humans EPHA7 plays a role in neurological and dysmorphic features such as developmental delay, hypotonia, and ear malformations. The characterization of additional cases at the molecular level will assist with accurate genotype/phenotype correlations and allow for the identification of additional features associated with 6q16 deletions.
Materials and methods
Oligonucleotide-based microarray analysis was performed using a 105K-feature whole-genome microarray (SignatureChip Oligo Solution®, made for Signature Genomic Laboratories by Agilent Technologies) with one probe every 10 kb in targeted regions – microdeletion/microduplication syndromes, the pericentromeric regions, subtelomeres and genes in important developmental pathways – and an average probe spacing of one probe every 35 kb throughout the rest of the genome. Two oligonucleotide probes cover the EPHA7 gene which spans ~178 kb. Genomic DNA was labeled with Alexa Fluor dyes 555 or 647 using a BioPrime Total DNA labeling kit (Invitrogen Corp, Carlsbad, CA). Array hybridization and washing were performed as specified by the manufacturer (Agilent Technologies). Arrays were scanned using an Axon 4000B scanner (Molecular Devices, Sunnyvale, CA) and analyzed using Agilent Feature Extraction software v9.5.1 and Agilent CGH Analytics software v3.5.14. Results were then displayed using custom oligonucleotide aCGH analysis software (Oligoglyphix™; Signature Genomic Laboratories).
The deletion was confirmed and visualized by metaphase fluorescence in situ hybridization (FISH) using a bacterial artificial chromosome (BAC), clone RP11-270O11, which encompasses the EPHA7 locus. Metaphase chromosomes were obtained from the proband's sample by using standard culturing procedures, and fixed cell suspensions were dropped onto clean microscope slides. After a 10 min 2× SSC soak the slides were dehydrated in a series of ethanol washes (70%, 90%, and 95%) for 2 min each wash. The slides were then air-dried. The chromosomes were denatured in a 70% formamide, 2 × SSC solution at 70°C for 2 min, immediately placed in 70% ethanol at -20°C for 2 min, then transferred to 80%, 90%, and 100% ethanol at -20°C for 2 min each, and then air-dried. DNA was labeled by nick translation (Abbott Molecular Inc., Des Plaines, IL, USA) with Enzo Red-dUTP or Enzo Green-dUTP (Abbott). Each was diluted in a 50% formamide hybridization solution. The double stranded DNA probes were denatured at 70°C for 10 min and were applied to the prepared slides. Slides were placed in a moist chamber and incubated overnight at 37°C. The slides were washed at 43°C in 0.4× SSC/0.3% Tween solution for 2 min, placed in 2× SSC/0.1% Tween for 1 min. The slides were counterstained with DAPI. Cells were examined with a Zeiss Axioplan II, Imager.M1, or Imager.Z1 fluorescence microscope equipped with a triple-bandpass filter that allows multiple colors to be visualized simultaneously. Digital images were captured and stored with Isis software V 3.4.0 (Metasystems, Altlussheim, Germany).
This case report is presented with the consent of the patient's family.
We thank Tricia Matz, Scott Sulpizio and Nick Neill (Signature Genomic Laboratories) for their help with Figures 3 and 4. We also thank the clinical FISH group at Signature Genomic Laboratories for their assistance with the FISH methodology. We thank Pierre Vanderhaeghen (University of Brussels, Brussels, Belgium) for helpful discussions.
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