Recurrent Distal 7q11.23 Deletion Including HIP1 and YWHAG Identified in Patients with Intellectual Disabilities, Epilepsy, and Neurobehavioral Problems

Abstract

We report 26 individuals from ten unrelated families who exhibit variable expression and/or incomplete penetrance of epilepsy, learning difficulties, intellectual disabilities, and/or neurobehavioral abnormalities as a result of a heterozygous microdeletion distally adjacent to the Williams-Beuren syndrome region on chromosome 7q11.23. In six families with a common recurrent ∼1.2 Mb deletion that includes the Huntingtin-interacting protein 1 (HIP1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG) genes and that is flanked by large complex low-copy repeats, we identified sites for nonallelic homologous recombination in two patients. There were no cases of this ∼1.2 Mb distal 7q11.23 deletion copy number variant identified in over 20,000 control samples surveyed. Three individuals with smaller, nonrecurrent deletions (∼180–500 kb) that include HIP1 but not YWHAG suggest that deletion of HIP1 is sufficient to cause neurological disease. Mice with targeted mutation in the Hip1 gene (Hip1^−/−) develop a neurological phenotype characterized by failure to thrive, tremor, and gait ataxia. Overall, our data characterize a neurodevelopmental and epilepsy syndrome that is likely caused by recurrent and nonrecurrent deletions, including HIP1. These data do not exclude the possibility that YWHAG loss of function is also sufficient to cause neurological phenotypes. Based on the current knowledge of Hip1 protein function and its proposed role in AMPA and NMDA ionotropic glutamate receptor trafficking, we believe that HIP1 haploinsufficiency in humans will be amenable to rational drug design for improved seizure control and cognitive and behavioral function.

Main Text

The vast majority (∼95%) of patients with Williams-Beuren syndrome (WBS [MIM 194050]), a common multisystem developmental disorder characterized by supravalvular aortic stenosis, multiple peripheral pulmonary arterial stenoses, elfin face, infantile hypercalcemia, and cognitive and behavioral abnormalities, have a recurrent ∼1.6 Mb hemizygous deletion in 7q11.23.¹ In a few patients with an unusual association of WBS with infantile spasms (IS) and more severe developmental delay or intellectual disability (DD or ID), larger deletions extending distally have been reported.^2–7 Marshall et al.⁷ proposed that deletion of the membrane-associated guanylate kinase inverted-2 (MAGI2 [MIM 606382]) gene, mapping ∼3.5 Mb distal to the WBS common deletion (Figure 1), causes IS, because 15 of 16 individuals with 7q11.23-q21.1 deletions involving MAGI2 developed IS, whereas 11 of 12 subjects with 7q11.23-q21.1 deletions leaving MAGI2 intact did not. Supporting this notion, Marshall et al. found that mouse Magi2 interacts with Stargazin and that mutations in Stargazin cause epilepsy in mice.^7,8

Figure 1.

Schematic Representation of the Genomic Architecture in Distal 7q11.23

The extent of the identified deletions is shown in red, and duplications are shown in green. The NAHR sites identified in patients 6 and 8 were mapped to the directly-oriented ∼25 kb subunits (orange) with crossovers within the subunits identified in two patients (6 and 8) with recurrent deletions.

However, to date, no point mutations have been identified in MAGI2, and although no epilepsy was documented for the 12 subjects with 7q11.23-q21.1 deletions proximal to MAGI2, 11 of these subjects had severe DD or ID.^5,7,9,10 In addition, at least three patients with WBS, DD or ID, and IS with 7q11.23 deletions proximal to MAGI2 have been reported.^4,11,12 Kamoike et al.¹² suggested that in addition to deletion of MAGI2, deletion of the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, gamma (YWHAG [MIM 605356]) or Huntingtin-interacting protein 1 (HIP1 [MIM 601767]) genes, which are both normally expressed in the brain, may contribute to epilepsy and ID in these patients. To further investigate this hypothesis, they studied morpholino antisense knockdowns of ywhag and hip1 in zebrafish. hip1^−/− zebrafish had reduced or complete absence of yolk extension, narrowing along the dorsoventral axis, and severe mandibular aplasia, but there was no obvious effect on the developing brain. In ywhag^−/− zebrafish, however, increased diameter of the heart tube and reduced brain size were appreciated, leading Kamoike et al. to postulate that haploinsufficiency of YWHAG causes the IS and cardiomegaly phenotypes observed in their patients.¹²

We queried our chromosome microarray database for individuals with distal 7q11.23 deletions or duplications that include the HIP1 and/or YWHAG genes. Three small nonrecurrent distal 7q11.23 deletions, six common recurrent distal 7q11.23 deletions, and one larger-sized deletion harboring HIP1 were found in ∼29,510 individuals referred for chromosomal microarray analysis (CMA) at the Baylor College of Medicine Medical Genetics Laboratories (BCM MGL), yielding a frequency of 1 in 3279, comparable with the 1 in 2960 frequency of the 680 kb CHRNA7 (MIM 118511) deletion.¹³ Our collection of subjects referred for CMA includes mostly children with various combinations of DD, ID, autism, and birth defects; a small percentage has epilepsy, but rarely as an isolated finding.

Initial array CGH analyses were performed with microarrays designed by BCM MGL and manufactured by Agilent Technology as previously described.^14,15 We received DNA samples from all probands and their family members after obtaining informed consent via protocols approved by the Institutional Review Board for Human Subject Research at Baylor College of Medicine (except patient 5, who is deceased, and patient 6, for whom no forwarding contact information was available). Patients' genomic DNA was extracted from peripheral blood via the Puregene DNA isolation kit (Gentra System). To fine map the size and the extent of the genomic deletions and duplications, we performed oligonucleotide microarray CGH analyses with custom-designed 7q11.23 region-specific high-resolution oligonucleotide microarrays: 135K (NimbleGen Systems) according to the manufacturers' instructions. Microarrays were scanned on the NimbleGen MS 200 scanner. Scans were processed with NimbleScan version 2.5 and analyzed with SignalMap version 1.9. Confirmatory and parental fluorescence in situ hybridization analyses in patients and their parents were performed with chromosome 7q11.23-specific bacterial artificial chromosome clones CTB-139P11, RP11-229D13, RP11-622P13, and RP11-845K6 via standard procedures. Clinical histories were obtained from parents and from physicians. For the patients with epilepsy, medical records including EEG reports were reviewed, and physicians and families were interviewed about clinical seizure semiology, thereby allowing us to classify the epilepsy in most cases as generalized, localization related, or mixed.

Overall, we identified 26 individuals from ten unrelated families who exhibit variable expression and/or incomplete penetrance of epilepsy, learning difficulties, intellectual disabilities, and/or neurobehavioral abnormalities as a result of hemizygous genomic deletions, including HIP1. The deletions mapped between the WBS region and MAGI2 ranged in size from ∼4 Mb to ∼180 kb and included apparent recurrent, as well as nonrecurrent, rearrangements (Figure 2). Our subjects presented with a range of neurological and neurodevelopmental phenotypes (Table 1). Epilepsy was common among probands with distal 7q11.23 deletion with 8 of 10 (80%) affected; however, among all family members with the deletion, epilepsy was present in 13 of 26 (50%). Generalized epilepsy was most common, occurring in 6 of 13 (46%); localization-related epilepsy occurred in 4 of 13 (31%), mixed epilepsy in 1 of 13 (8%), epilepsy not otherwise specified in 1 of 13 (8%), and neonatal epilepsy in 1 of 13 (8%). Routine clinical brain MRI exams were typically interpreted as normal, but one subject (patient 4) had a dysembryoplastic neuroectodermal tumor and a temporal lobe cortical dysplasia that were identified on pathological examination status post epilepsy surgery (temporal lobectomy) for medically refractory symptomatic localization-related epilepsy, and a second subject (patient 3) had a mild Chiari 1 malformation.

Figure 2.

Results of Array CGH Analysis with a 7q11.23 Region-Specific High-Resolution Oligonucleotide Microarray, Including the WBS Common Deletion Region

The extents of the identified deletions (red) and duplications (green) (135K, NimbleGen). Patients 1, 3, 6–8, and 10 had a common ∼1.2 Mb deletion.

Table 1.

Phenotypic Features of 26 Individuals from Ten Unrelated Families with an HIP1 Deletion and Four Individuals from Two Unrelated Families with an HIP1 Duplication

	Age at Diagnosis	Sex	Ancestrya	CNV	Cognitive Function, Other Problems	Epilepsy	Inheritance	Method	Other CMA Abnormalities V8
Pt 1	1 mo.	Female	E	Com del	34 wga, cystic encephalomalacia, death in neonatal period	Neonatal seizures	Pat	V5.0, V8.0	None
Pt 1's father	34 yr.	Male	E	Com del	Normal	Generalized epilepsy	Unknown	FISH	Unknown
Pt 2	4 yr.	Male	E/H	Small del	Global DD, microcephaly, PDA	None	Mat	V6.1, V8.0	Xp22.31dupb
Pt 2's mother	30 yr.	Female	H	Small del	Normal	None	Unknown	FISH	Unknown
Pt 3	4 yr.	Male	E	Com del	Global DD, ID, ASD, Chiari 1	Febrile seizures, generalized epilepsy	Mat	V6.1, V8.0	None
Pt 3's mother	45 yr.	Female	E	Com del	Struggled in school, miscarriages (singleton and twin)	None	Unknown	FISH	Unknown
Pt 3's sister	7 yr.	Female	E	Com del	ASD	None	Mat	V5.0	None
Pt 4	13 yr.	Female	E	Small del	Severe ID	Medically refractory generalized epilepsy s/p VNS placement	Mat	V6.2, V8.0	5q31.2delc
Pt 4's mother	38 yr.	Female	E	Small del	Difficulty with reading	None	Unknown	FISH	Unknown
Pt 4's brother	14 yr.	Male	E	Small del	Severe ID, aggressive, hyperactive	Generalized epilepsy	Mat	V6.2	None
Pt 5	3 mo.	Female	H	Large del	WBS, congenital heart disease	None	De novo	V6.2, V8.0	None
Pt 6	4 yr.	Female	E	Com del	Moderate ID, hyperactivity	Mixed epilepsy	Pat	V6.3, V8.0	None
Pt 6's father	27 yr.	Male	E	Com del	Learning disabilities, special education	Epilepsy	Unknown	FISH	Unknown
Pt 6's brother	1.5 yr.	Male	E	Com del	Mild ID, hyperactivity	None	Pat	V6.4	None
Pt 7	9 yr.	Female	AA	Com del	Learning disabilities	Medically intractable localization-related epilepsy	Pat	V8.0	Xp11.22dupd
Pt 7's sister	11 yr.	Female	AA	Com del	Learning disabilities	Localization-related epilepsy	Pat	V8.1	Xp11.22dupd
Pt 7's father	38 yr.	Male	AA	Com del	Struggled in math	Localization-related epilepsy	Unknown	FISH	Unknown
Pt 8	13 yr.	Female	E	Com del	Mild ID, ADHD, depression, mood disorder, aggressive	Generalized epilepsy	Unknown/adopted	V8.0	None
Pt 9	3 yr.	Male	AA	Small del	Normal	Localization-related epilepsy	Mat	V8.1	None
Pt 9's mother	18 yr.	Female	AA	Small del	Normal	None	Pat	FISH	Unknown
Pt 9's MGF	38 yr.	Male	AA	Small del	Normal	Single seizure event as adult	Unknown	FISH	Unknown
Pt 10	19 yr.	Male	E	Com del	Severe/moderate ID	Generalized epilepsy	Mat	V8.0	None
Pt 10's mother	40 yr.	Female	E	Com del	Mild ID, special education	None	Mat	FISH	Unknown
Pt 10's MGM	70 yr.	Female	E	Com del	Normal	None	Unknown	FISH	Unknown
Pt 10's uncle	30 yr.	Male	E	Com del	Mild ID, special education	None	Mat	FISH	Unknown
Pt 10's aunt	41 yr.	Female	E	Com del	Mild ID	None	Mat	FISH	Unknown
Pt 11	3 mo.	Male	E	Small dup	Large posterior encephalocele/Chiari III malformation, hydrocephalus VPS	None	Pat	V8.0	None
Pt 11's father	34 yr.	Male	E	Small dup	Spinal cord schwannoma	None	Unknown	FISH	Unknown
Pt 12	16 yr.	Male	E	Com dup	Behavior/mood disorder (diagnosed with bipolar disorder), ADHD, aggressive	None	Not mate	V8.0	None
Pt 12's sister	5 yr.	Female	E	Com dup	Speech delay, ADHD, aggressive	None	Not mate	FISH, V8.1	11p14.3p14.2delf

The following abbreviations are used: ADHD, attention deficit hyperactivity disorder; ASD, autism spectrum disorder; com, common; del, deletion; dup, duplication; FISH, fluorescence in situ hybridization; mat, maternal; MGF, maternal grandfather; MGM, maternal grandmother; mo., months old; pat, paternal; PDA, patent ductus arteriosus; pt, patient; s/p, status post; V, version; VNS, vagus nerve stimulator; yr., years old; VPS, ventriculoperitoneal shunt; wga, weeks of gestational age.

^aAA, African American; E, European; H, Hispanic.

^barrXp22.31(6876449-8075153)x2.

^carr5q31.2(138439046- 138591992)x1.

^darrXp11.22(53081560-53332267)x3.

^eFather in prison.

^farr11p14.3p14.2(25518340-26613145)x1. (Coordinates according to NCBI build 36, March 2006.)

The intellectual spectrum associated with distal 7q11.23 deletion is broad. Of the 12 surviving children, (eight surviving probands, including patient 10, and their four siblings; patients 1 and 5 are deceased), 7 of 12 (58%) had moderate to severe global DD or ID and/or were on the autistic spectrum, 2 of 12 (17%) had mild ID, 2 of 12 (17%) were diagnosed with learning disabilities only, and 1 of 12 (8%) was developmentally on target. Of the 12 adults, 3 of 12 (25%) had mild ID and had required special education, 3 of 12 (25%) expressed concern that they had really struggled in school or disproportionately with certain subjects, and 6 of 12 (50%) reported no cognitive difficulties, no special needs in the educational system, and no history of neuropsychiatric disease. Behavioral problems were common among children with distal 7q11.23 deletion and include inattention, hyperactivity, impulsivity, and aggression. Depression and self-abusive behaviors were reported in one patient. Autism was diagnosed in 2 of 12 children (siblings, 17%).

We have also identified two reciprocal microduplications inclusive of HIP1 in three children from two families (Figure 2; Table 1). Although none of these children have developed epilepsy, 1 of 3 (33%) has an expressive language disorder, 2 of 3 (67%) have attention deficit hyperactivity disorder and problems with aggression, 1 of 3 (33%) was formally diagnosed with bipolar disorder, and 1 of 3 (33%) had a large posterior encephalocele (Chiari III malformation).

In contrast to other disease-associated recurrent deletion and duplication CNVs that typically arise de novo (e.g., 16p11.2 and 1q21.1),^16–19 we found that our patients inherited the common or small distal 7q11.23 deletion or duplication in all cases for which parental origin could be determined. Deletions of distal 7q11.23 are therefore reminiscent of the typically inherited 15q13.3 microdeletions that cause epilepsy and other neurodevelopmental phenotypes but exhibit incomplete penetrance and variable expressivity.^20–24 The incomplete penetrance of epilepsy and ID in apparently clinically unaffected transmitting parents can potentially be explained by a two-hit model,^25,26 single nucleotide variants of the second allele,²⁷ or a combination thereof. The observed increase in severity of phenotypes among probands is likely explained by ascertainment bias.

We did not study a matched control set of healthy children, but we performed two analyses to consider the frequency of the common recurrent ∼1.2 Mb distal 7q11.23 deletion in normal controls from the studies that surveyed CNV incidence. We did not find this deletion in approximately 20,000 individuals from different world populations^28–34 (dbGaP; M. Hurles, personal communication). To further refine our analysis, we calculated a p value for the HIP1 deletion event among patients with an indication of epilepsy on their submitting diagnosis. Of the ten distinct families with the HIP1 deletion, eight had epilepsy as an indication and two did not. In our CMA database as a whole, ∼5,000 patients have epilepsy as an indication, whereas ∼24,500 do not. Together, these values suggest that the incidence of epilepsy as an indication is significantly higher in the HIP1 deletion subjects, with a Fisher's exact p value of 2.224 × 10⁻⁵ and a 95% confidence interval of between 3.91 and 189.12.

Three smaller-sized overlapping deletions (∼180–500 kb) in patients 2, 4, and 9 (Figure 2) enabled us to narrow the critical region to a single gene, HIP1 encoding Huntingtin-interacting protein 1,³⁵ that has a role in clathrin-mediated endocytosis.^36,37 HIP1 has been implicated in the pathogenesis of Huntington disease (MIM 143100)³⁸ and prostate and colon cancers.³⁹ A balanced reciprocal translocation t(5;7)(q33;q11.2) resulting in a HIP1 and platelet-derived growth factor β receptor (PDGFβR [MIM 173410]) fusion protein was identified in a patient with chronic myelomonocytic leukemia.⁴⁰

Hip1^−/− mice developed premature testicular degeneration, severe spinal deformities, abnormal hematopoiesis, and cataracts.^41,42 Reportedly, these animals had no gross structural abnormalities of the central nervous system; however, both Hip1^−/− and Hip1^+/− mice develop epilepsy (T. Ross, personal communication). In a second loss-of-function model, Metzler et al.⁴³ showed that by 3 months of age, Hip1^−/− mice develop neurological problems characterized by failure to thrive, tremor, and gait ataxia (likely secondary to a rigid thoracolumbar kyphosis). Hip1^−/− mice have defects in presynaptic function, as demonstrated by a reduction in paired-pulse facilitation in hippocampal brain slices. In addition, Hip1^−/− mice demonstrate delayed recovery from chemically induced long-term depression, as well as altered α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor function.^43,44

Hip1 colocalizes in hippocampal and cortical neurons with NMDA ionotropic glutamate receptors (iGluRs), and Hip1^−/− mice have a profound decrease in NMDA-induced AMPA-type iGluR clathrin-mediated internalization.^38,43 Endocytic trafficking and recycling maintains a pool of mobile surface AMPA receptors required for synaptic potentiation.⁴⁵ Interestingly, the increased internalization of ionotropic receptors for glutamate has been demonstrated to be pathogenic in mouse and Drosophila models of Fragile X syndrome (MIM 300624).⁴⁶ Mutations in the ionotropic glutamate receptor 3 gene (GRIA3 [MIM 305915]) have been found in patients with X-linked mental retardation, seizures, and autistic behavior (MRX94 [MIM 300699]).⁴⁷ Moreover, AMPA receptors have also been implicated in patients with clinical diagnoses of schizophrenia (MIM 181500), Alzheimer disease (MIM 104300), amyotrophic lateral sclerosis (MIM 105400), stroke, Parkinson disease (MIM 168600), depression, epilepsy, and Rasmussen encephalitis.⁴⁸

Given that in six families the common recurrent ∼1.2 Mb deletions are flanked by large complex low-copy repeats (HIP1-LCRs), we hypothesized that they have likely arisen via nonallelic homologous recombination (NAHR). To address this hypothesis, we performed computational analyses of the proximal and distal HIP1-LCR copies. We found that they are organized as clusters of subunits, two of which are in direct orientation (Figure 1). Long-range PCR primers were designed to be specific to regions directly flanking each of these two paralogous subunit pairs in both HIP1-LCR copies and to each subunit (when possible) in order to amplify the predicted junction between the proximal and distal subunits (orange) of HIP1-LCRs. Amplification of the 4–17 kb fragments was performed with Takara LA Taq polymerase (TaKaRa Bio USA) following the manufacturer's protocol. Amplification of the GC-rich region in HIP1-LCRs was done in the presence of 4% dimethyl sulfoxide, with the following conditions: 94°C for 1 min 30 s, 61°C for 30 s, 72°C for 6 min. PCR products were treated with ExoSAP-IT (USB) to remove unconsumed dNTPs and primers and were directly sequenced by Sanger method with the initial primers and primers specific for both paralogous subunits in the proximal and distal copies of the HIP1-LCRs (Lone Star Labs). The genomic sequences based on the oligonucleotide coordinates from the array CGH experiments were downloaded from the UCSC Genome Browser (NCBI build 37, February 2009) and assembled with Sequencher version 4.2 software (Gene Codes). Interspersed repeat sequences were identified with RepeatMasker). DNA GC content was analyzed with CPGPlot. Using long-range PCR primers F: 5′-CTCTATGGAGCACAGATTCCATGCTAGACC, specific to the region centromeric and adjacent to the ∼25 kb subunit in the proximal HIP1-LCR copy, and R: 5′-AGATCAGAGATCCTCTATTTTCCCCCTTAATC, specific to both subunits, we identified NAHR sites in patients 6 and 8 between the cis-morphic nucleotides or paralogous sequence variants in a 368 bp GC-rich interval (hg19; chr. 7:75,071,539-75,071,906; chr. 7:76,256,141-76,256,508 in the proximal and distal HIP1-LCRs, respectively; Figure 3).⁴⁹

Figure 3.

Genomic Structure of the ∼25 kb Subunits of 99.2% DNA Sequence Identity from the Proximal and Distal HIP1-LCRs, in which the NAHR Sites Were Mapped in Patients 6 and 8

(A) The NAHR site regions were narrowed in patients 6 and 8 to a 0.4 kb interval by sequencing the long-range PCR products obtained with primer F, specific to the region adjacent to the proximal LCR, and the reverse primer R, specific for both proximal and distal LCRs (arrows depicting primers are not shown to scale).

(B) Schematic representation of GC content of the HIP1-LCRs. The unique feature of the NAHR sites reveals remarkably high GC content (black boxes in A mark regions with GC content of at least 70% within a 100 bp window). Polypurine and pyrimidine sequences with GC content of at least 50% have been associated with human recombination hot spots.⁴⁹

These collective data suggest that haploinsufficiency of HIP1 is sufficient to alter neuronal homeostasis in a manner that predisposes the human brain to both focal and generalized epilepsies, as well as to a broad range of cognitive dysfunction and neurobehavioral abnormalities, including mild to severe intellectual disabilities and inattention, hyperactivity, impulsivity, and aggression through a mechanism involving abnormal AMPA and NMDA iGluR trafficking. However, these data do not exclude haploinsufficiency of YWHAG as a contributing factor to the neurological phenotypes in patients with deletions of both HIP1 and YWHAG, nor do they exclude YWHAG as a candidate gene that, when altered, is also sufficient to cause neurological dysfunction.

Future studies should include sequence analysis of both the HIP1 and YWHAG genes in patients with unexplained epilepsy and neurodevelopmental or neurobehavioral syndromes, natural history protocols to document the full spectrum of human phenotypes associated with HIP1 and/or YWHAG loss or gain of function, complete neurological and neurobehavioral characterization of Hip1 and Ywhag function in mice, and preclinical drug studies in these mouse models.

Web Resources

The URLs for data presented herein are as follows:

• Baylor Medical Genetics Laboratories, http://www.bcm.edu/geneticlabs/?pmid=16207

• CPGPlot, http://www.ebi.ac.uk/Tools/emboss/cpgplot

• Database of Genotypes and Phenotypes (dbGaP), http://www.ncbi.nlm.nih.gov/gap

• Online Mendelian Inheritance in Man, http://www.ncbi.nlm.nih.gov/Omim/

• RepeatMasker, http://www.repeatmasker.org

• UCSC Genome Browser, http://genome.ucsc.edu/

Accession Numbers

Array data have been deposited in Gene Expression Omnibus database under accession number GSE23834. The NAHR site sequences have been deposited in the GenBank database with the accession number HQ148673.