Mutations in HIVEP2 are associated with developmental delay, intellectual disability and dysmorphic features

Abstract

HIVEP2 (human immunodeficiency virus type I enhancer binding protein 2) has been previously associated with intellectual disability and developmental delay in three patients. Here we describe six patients with developmental delay, intellectual disability and dysmorphic features with de novo likely gene damaging variants in HIVEP2 identified by whole exome sequencing (WES). HIVEP2 encodes a large transcription factor that regulates various neurodevelopmental pathways. Our findings provide further evidence that pathogenic variants in HIVEP2 lead to intellectual disabilities and developmental delay.

Introduction

HIVEP2 (human immunodeficiency virus type I enhancer binding protein 2), also referred to as MIBP1, ZAS2, ZNF40B, MBP-2, and Schnurri-2, is a gene that has been associated with intellectual disability and developmental delay [1, 2]. HIVEP2 encodes a large transcription factor that regulates various neurodevelopmental pathways. To date, three patients with loss-of-function mutations in HIVEP2 have been published, all with developmental delay, intellectual disability, hypotonia, and dysmorphic features [1]. In this paper, we present six additional patients with de novo variants in HIVEP2, identified through WES, with phenotypes similar to those previously published as well as some additional clinical features. Five of the patients have predicted loss-of-function variants while the sixth has a missense variant that is predicted to be damaging. Our cases provide further evidence that pathogenic variants in HIVEP2 cause neurodevelopmental disorders.

Methods

This study was approved by the Institutional Review Board of Columbia University. Informed consent was obtained from all individual participants included in the study. Additional informed consent was obtained from all individual participants for whom identifying information is included in this article. Genomic DNA was extracted from whole blood from the affected children and their parents. Exome sequencing for patients 1–4 was performed on exon targets isolated by capture using the Agilent Clinical Research Exome kit (Agilent Technologies, Santa Clara, CA). The sequencing methodology and variant interpretation protocol for patients 1–4 has been previously described [3]. The general assertion criteria for variant classification are publicly available on the GeneDx ClinVar submission page (http://www.ncbi.nlm.nih.gov/clinvar/submitters/26957/).

Whole exome sequencing for patient 5 was performed using the SureSelect XT Human All Exon V5 kit (Agilent) at a mean target depth of 100×. The target is defined as all coding exons of UCSC and Ensembl +/− 20bp intron flanks. Reads for patients 5 and 6 were aligned to Hg19 using BWA (BWA-MEM v0.7.5a) and variants were called using the GATK haplotype caller (v2.7-2). Detected variants were annotated, filtered and prioritized using the Bench lab NGS v.3.1.2 platform (Cartagenia, Leuven, Belgium). De novo mutation analysis, by filtering all detected variants against parental and population variants were confirmed by Sanger sequencing. For patient 6, the laboratory used the SureSelectXT Clinical Research Exome (Agilent) kit.

Results

WES was performed on 3,699 patients with neuro-developmental disorders within the study time period of patients 1–4. For those four patients, WES produced an average of ~9GB of sequence per sample. Mean coverage of captured regions was ~135X per sample, with >95% covered with at least 10x coverage, an average of >94% of base call quality of Q30 or greater, and an overall average mean quality score of >Q36. Filtering of common SNPs, manual curation, evaluation of predicted effects of rare variants and known function of the genes and associated human conditions, and examination of overlapping phenotypes of individuals with de novo variants in the same gene identified variants in HIVEP2 as likely gene damaging in four probands in the series of 3,699 patients (0.1%). Two additional patients with de novo likely gene damaging variants in HIVEP2 were identified through GeneMatcher[4].

Six unrelated patients ranging in age from 2 to 14 years of age were found to have novel, heterozygous de novo variants in HIVEP2: two nonsense, three frameshift, and one missense variant (Figure 1). Maternity and paternity were confirmed as part of quality control in the WES analysis, and alle HIVEP2 variants were confirmed by Sanger sequencing in the proband and both parents and were de novo and absent in both parents. None of the variants is present in ExAC[5] and RVIS score for this gene is −2.28 (1.36%), indicating that the gene is intolerant to variation[6]. The missense variant is a non-conservative amino acid substitution (p.Asp397Tyr) and is located at a conserved position. In silico tools SIFT, Provean, Mutation Taster, and CADD all predict it to be pathogenic[7–9]. It was classified as a likely pathogenic variant using the Richards et al. criteria[10].

Fig. 1.

Predicted pathogenic variants in HIVEP2. Variants in the solid symbols are reported in this manuscript. Previously published cases are included in checkered symbols.

All six patients have developmental delay, intellectual disabilities, and mild dysmorphic features (Figure 2). Four of the children have hypotonia, and one has hypertonia. Gross motor milestones were delayed. Two of the children are nonverbal while another has only three words. One child can speak in full sentences but also imitates and is difficult to understand, and the six year old child speaks in only 3–4 word sentences. IQ was not consistently available but ranged from 50–75. The clinical course was largely one of severe delay in development but some gains in developmental milestones. Two of the children have seizures. Behavioral issues are common and include autism, hyperactivity, anxiety, and oppositional behaviors. MRI of the brain did not generally demonstrate structural anomalies although there was mild volume loss in patient and another patient had incomplete myelination at 4 years old. Notably, three of the children are microcephalic and another child has a head circumference < 10%. Medical issues are not common, and gastrointestinal issues of constipation and gastroesophageal reflux disease are reported. Ophthalmologic issues were reported in three patients and included strabismus and amblyopia.

Fig. 2.

Facial features of Patient 4 which include mild dolichocephaly with a tall forehead, mild hypertelorism, and a glabellar nevus flammeus.

Five individuals with chromosomal deletions of 6q24.2 ranging in size from 6 to 20 Mb and including HIVEP2 have been described [11]. All five individuals have neurodevelopmental delay, and some have additional features including short stature and/or craniosynostosis, some features of which may be due to deletion of neighboring genes.

Discussion

We describe six patients with common clinical features of neurodevelopmental disorders including developmental delay/intellectual disabilities and dysmorphic features with heterozygous de novo likely gene damaging novel variants in HIVEP2, identified through WES. Our series include one de novo missense variant, p.D397Y, which is predicted to be damaging but has not been functionally assessed. The phenotype of patient 4 who carries the p.D397Y variant overlaps with the other patients in our series but is nonspecific. Most of the children are nonverbal or minimally verbal. Additional less consistent features in our series include microcephaly without associated brain malformations, seizures and a range of behavioral problems commonly observed in individuals with intellectual disabilities including autism, attention deficit, oppositional defiance, and anxiety. Our series is consistent with and expands the phenotype previously reported by Srivastava et al describing three patients with loss of function de novo variants in HIVEP2[1]. Common features across all nine patients include hypotonia, developmental delay, intellectual disability, and dysmorphic features (Table 1) [1] [2].

Table 1.

Clinical features of individuals with likely gene damaging variants in HIVEP2, including previously published cases.

	Patient 1	Patient 2	Patient 3	Patient 4	Patient 5	Patient 6	Srivastava et al. #1	Srivastava et al. #2	Srivastava et al. #3
Mutation	c.G6475T p.G2159X	c.G2857T p.E953X	c.5614dupG p.E1872Gfs*16	c.G1189T p.D397Y	c.6625dup p.Y2209fs	c.3434delC p.P1145fs	c.C2827T p.R943X	c.C3556T p.Q1186X	c.5737delGp.D1 913Mfs*15
Current Age (years)	7	14	10	2	11	6	4	3	21
Gender	Female	Female	Male	Male	Female	Male	Female	Male	Female
Prenatal issues	Premature contractions, decreased fetal movement	None	First trimester vaginal bleeding, decreased fetal movement	Decreased amniotic fluid	None	None	First-trimester vaginal bleeding	None	None
Congenital anomalies	N	N	N	N	N	N	N	N	Umbilical hernia, mild pulmonary stenosis
Birth Weight, Height, Head circumference (OFC) centiles	WT= 3997 g (90%) HT = 53.34 cm (90%), OFC UNK	WT= 3062 g (25%) HT=48.26 cm (25–50%), OFC = 32.4 cm (Z=−1.8)	WT = 3856 g (~75%), HT = 53.3 cm (~90%), OFC UNK	WT= 3770 g (50 – 75%), HT & OFC UNK	WT = 3880 g (75–90%), HT & OFC UNK	WT= 4100 g (90%)HT & OFC UNK	WT= 3150 g (25–50 %)	HT= 53cm (75th–90th centile), WT= 3330g (25th–50th centile), OFC= 35cm (Z=−0.4)	HT= 50cm (25th centile), WT= 2750g (10th centile), OFC= 32cm (Z=−2)
Current Weight, Height, Head circumference (OFC) centiles	WT= 22.8 kg (50 %) HT= 125 cm (75 %), OFC= 48.5 cm (Z=−2.4)	WT= 47.5 kg (25–50%), HT= 149.5 cm (5 %) OFC= 51 cm (Z=−2.6)	WT= 31.4 kg (25–50%), HT= 140.9 cm (50–75%) OFC= 48.7 cm (Z=−3.2)	WT= 10.6 kg (<5 %), HT= 80.8 cm (<3%), OFC= 46.7 cm (Z=−1.7	WT = 44 kg (50–75 %), HT = 152 cm (50–75%). OFC at 3yo10mos = 50.8 (Z=0.7)	WT= 24 kg (50–75 %), HT= 120 cm (50 %), OFC= 52cm (Z=0.3)	WT= 17.1 kg (85%), HT= 100 cm (70%), OFC= 48 cm (Z=−1.5)	WT= 11 kg (25–50 %), HT= 80 cm (25 %), OFC= 48 cm (Z=−1.4)	WT= 59kg (25–50%), HT= 164 cm (25 –50 %), OFC= 52.2 cm (Z=−2)
Dysmorphic features	Mild retrognathia	Elongated and narrow face	Low anterior hairline, hirsute, prominent eyebrows, synophrys, epicanthal folds, mildly thickened helices & simple antihelices in ears, mild dental crowding of lower jaw	Small hands and feet	Somewhat high nasal bridge, broad mouth, rather flat philtrum, mild bifrontal narrowing, mildly broad halluces, sacral dimple, broad thorax, mild finger webbing	Square face, high/broad forehead, unilateral strabismus, high nasal bridge, columella under alae nasi, small square ears with transverse crease, small square teeth, microretrognat hia	Widely set eyes, broad nasal root, slightly upturned nose, high-arched palate	High forehead, medial eyebrow flare, widely set eyes, broad nasal root, small mouth, slightly tapering fingers, flat feet	Upslanting palpebral fissures, mild synophrys, small ears with attached earlobes, prominent nose with high nasal bridge and columella extending below the alae nasi, short philtrum, thin upper lip, overbite, hypertrophic gingiva, tapering fingers, radial deviation of 4th fingers, wide feet with short toes, hirsutism
Developmental delay	Y	Y	Y	Y	Y	Y	Y	Y	Y
Age at sitting (mos)	6	12	28	7	14	9	UNK	13	9
Age at walking (mos)	24	36	With walker, 30–36 months; without walker 4 to 5 years	Not yet	30	22	36	30	36
Age at talking	18 mos	N/A	N/A	24 mos	10 mos	18 mos	24 mos	24 mos	7 yo
Current speech abilities	Speaks in short sentences; had 100 words by 3 yo	No verbal speech	No verbal speech	3 words	Full sentences and imitation; difficult to understand	Sentences with 3–4 words	Language age equivalent of 15mos		Partially slurred articulation
Hypo/hyperton ia	Hypotonia	Hypotonia	Hypertonia	Hypotonia	Hypotonia	N	Hypotonia	Hypotonia	Hypotonia
Autism (Y/N)	N	Y	N	N	N	N	Unavailable	Unavailable	Unavailable
Intellectual disabilities (Y/N)	Borderline	Y	Y	N/A	Y	Y	Y	Y	Y
Full scale IQ	75	UNK	UNK	N/A	50	50–70	Unavailable	Unavailable	Unavailable
Other DSM diagnoses	Sensory processing disorder	N	Sensory integration disorder	N/A		N	Unavailable	Unavailable	Unavailable
Brain MRI/CT results	Normal	Mild volume loss	Normal structure MRI at age 6 yo; abnormal MR spectroscopy. Normal structure MRI at age 9 yo	Normal	Incomplete myelination at age 4 yo	Normal	Slightly thin corpus callosum	Hypoplasia of the corpus callosum	Mild frontal atrophy
Abnormal behaviors	Hand-wringing, oppositional/de fiant behaviors	Hands in hair, hands up like puppet, wringing of hands	Tic like head jerking at one point. Impulsive, distractible	N	Sensitive to stimuli, requires structure	Hyperactivity, concentration problems, anxiety	Hyperactivity, impulsivity, distractibility	None	Aggression, impulsivity, self-stimulation, hyperactivity
Gastrointestinal/Growth issues	Abdominal pain, GERD	Constipation, GERD	GERD, milk protein intolerance as infant	Short stature	N	N	Gastroparesis and projectile vomiting	None	Constipation
Ophthalmologic issues	N	Strabismus	Strabismus (esotropia) s/p eye muscle surgery at age 6 yo	N	N	Amblyopia and strabismus, high hypermetropia (+5/+6 diopters)	N	Hypermetropia	N
Seizures (Y/N)	Possible	Y	Y	N	N	N	N	N	N
Seizure type/EEG results	Normal EEG	Mostly tonic seizures, history of myoclonic and complex partial seizures EEG: multiple tonic seizures, focal seizures, spike and slow wave- bifrontal	Febrile (one) and afebrile (one)	EEG abnormality: left occipital sharp wave and spike/wave discharges that increase as he progresses into sleep.	N/A	N/A	Normal EEG	Normal EEG	Normal EEG
Seizure frequency	Occasional	Daily, Lennox Gastaut Syndrome	Rare (2 in 10 years)	N/A	N/A	N/A	N/A	N/A	N/A
Other neurological disorder(s)	Static encephalopathy, dystonia, exercise intolerance/easily fatigued	Ataxia, spasticity, cerebral palsy, quadriplegia, muscle weakness, dystonia	Spasticity, muscle weakness, tremors, progressive Parkinsonism, right hemiparesis, tongue fasciculations	N	Dyspraxia	N	Frequent head tilt and leftward eye movements with preserved ability to fix/follow objects, subtle left facial weakness, wide based gait	Clumsy gait	Ataxia, dystonia
Other significant medical problems	Temperature instability	Iron deficiency anemia, nephrocalcinosis on renal US, hip dysplasia, irregular menstrual periods, syncope, dyspnea	Frequent tonsillitis, improved after T & A at age 9	Reactive airway disease; AFOs primarily for ankles rolling due to hypotonia	Hypermobility of fingers, increased inversion of feet and decreased eversion	Bronchial hyperreactivity, breath holding spells	Asymmetric cry, unilateral hip dysplasia, dysphagia	Significant salivation	Hypertrophic gingiva with frequent bleeding, high TSH levels

WT=weight, HT=heights, OFC=occipital frontal circumference, UNK=unknown, GERD=gastroesophageal reflux disease, s/p = status post, T & A = tonsillectomy & adenoidectomy, mos = months old, yo = years old

HIVEP2 is hypothesized to cause disease through its involvement in the somatostatin receptor pathway, which has been previously associated with intellectual disability[2]. Our six additional patients provide further evidence that loss-of-function mutations in HIVEP2 cause neurodevelopmental disease.

HIVEP2 is located on 6q23–q24[12]. It is expressed in the brain and encodes a 2446 amino acid protein that is a member of the ZAS protein family, along with HIVEP1 and HIVEP3. ZAS proteins all contain two zinc finger pairs, a serine/threonine-rich sequence and an acidic region, and encode large transcription factors involved in growth and development [13]. With specificity provided by the zinc fingers, HIVEP2 binds to the DNA sequence 5′-GGGACTTTCC-3′ in enhancer elements of viral and gene promoters.

The predominance of neurodevelopmental features observed in our six patients, along with the three previously published patients, may be explained by HIVEP2’s role in brain development. HIVEP2 has been shown to enhance expression of somatostatin receptor type II (SSRT-2) in the brain. HIVEP2 binds with the initiator-binding protein TCF4 to a TC-rich site in the SSRT-2 promoter and activates transcription. SSRT-2 and HIVEP2 are co-expressed in the frontal cortex and hippocampus [14]. HIVEP2 is also known to repress transcription of key developmental genes, including c-myc, a transcription factor that regulates cell growth, differentiation and apoptosis [15]as well as genes in the NF-κB pathway, which play a role in neuronal development, function, and survival [16, 17].

Furthermore, mouse models demonstrate the gene’s role in neurologic function, primarily in the hippocampus. Hivep2-knockout mice show decreased maturation of neurons and expression of dopamine receptors in the dentate gyrus region of the hippocampus. Phenotypically, Hivep2-knockout mice demonstrate behavioral abnormalities including anxiety, hyperactivity, working memory deficits, reduced pre-pulse inhibition, and impaired sociability, features consistent with those observed in our patients. [1, 18]

The six patients in our series support the hypothesis that loss of function variants in HIVEP2 are a rare cause of neurodevelopmental abnormalities.