Expanding the Phenotype of Intellectual Disability Caused by HIVEP2 Variants

Goel Himanshu (Orcid ID: 0000-0001-6448-6618)

1 Goldsmith et al. Expanding the Phenotype of Intellectual Disability Caused by HIVEP2 Variants

Heidi Goldsmith Hunter Genetics, Waratah, New South Wales, Australia Anna Wells University of Newcastle, Callaghan, New South Wales, Australia Maria João Nabais Sá Radboud university medical center (Radboudumc), Department of Human Genetics, Nijmegen, The Netherlands

Mark Williams Mater Research Institute, The University of Queensland, Woolloongabba, Queensland, Australia; and Genetic Pathology, Mater Pathology, South Brisbane, Queensland, Australia Helen Heussler Child Development Program, Queensland Children’s Hospital, South Brisbane, Queensland, Australia; and Child Health Research Centre, The University of Queensland, South Brisbane, Queensland, Australia Melissa Buckman Genetic Counselling Service, Tamworth, New South Wales, Australia Rolph Pfundt Radboud university medical center (Radboudumc), Department of Human Genetics, Nijmegen, The Netherlands Bert B. A. de Vries Radboud university medical center (Radboudumc), Department of Human Genetics, Nijmegen, The Netherlands Himanshu Goel Hunter Genetics, Waratah, New South Wales, Australia; and University of Newcastle, Callaghan, New South Wales, Australia

Correspondence to: Himanshu Goel, Clinical Geneticist, PO Box 84, Waratah NSW 2298, Australia Telephone: (02) 498 53100 Fax: (02) 498 53105 Email: [email protected]

Running Head: Goldsmith et al.

Running Title: HIVEP2 and New Phenotypic Presentations

This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/ajmg.a.61271

ABSTRACT

De novo pathogenic variants in the human immunodeficiency virus enhancer type I binding

protein 2 (HIVEP2) gene, a large transcription factor predominantly expressed in the brain

have previously been associated with intellectual disability and dysmorphic features in nine

patients. We describe the phenotype and genotype of two additional patients with novel de

novo pathogenic HIVEP2 variants, who have previously unreported features, including

hyperphagia and Angelman-like features. Exome sequencing was utilised in the investigation

of the patients who had previously incurred a rigorous genetic workup for their

neurodevelopmental delay, and in whom no genetic cause had been detected. Information

pertaining to phenotype and genotype for new patients was collated along with data from

previous reports, showing that the phenotypic spectrum of patients with HIVEP2 variants is broader than first noted. Additional characteristics are: an increased body mass index; and

features of Angelman-like syndromes including: intellectual disability, limited speech, post-

natal microcephaly and hypotonia. Dysmorphic features vary between patients. As yet, no

clear association between the type of gene aberration and phenotype can be concluded.

HIVEP2-related intellectual disability needs to be considered in the differential diagnosis of patients with Angelman-like phenotypes and hyperphagia, and whole exome sequencing

This article is protected by copyright. All rights reserved. 3 Goldsmith et al. should be considered in the genetic diagnostic armamentarium for patients with intellectual disability of inconclusive aetiology.

KEY WORDS

HIVEP2, intellectual disability, hyperphagia, Angelman-like syndrome, exome sequencing.

INTRODUCTION

Human immunodeficiency virus type I enhancer binding protein 2 (HIVEP2; chr6q23-q24) encodes a zinc-finger containing transcription factor protein that is expressed in the brain [Fujii et al., 2005].

HIVEP2-related intellectual disability (ID) has previously been reported in nine patients. Eight individuals had de novo loss-of-function (LoF) variants and one had a de novo missense variant [Srivastava et al., 2016; Steinfeld et al., 2016]. Clinical features in these patients were non-specific, and included moderate-to-severe ID, hypotonia, delayed speech development, microcephaly and behavioural features including hyperactivity, autism and anxiety [Srivastava et al., 2016; Steinfeld et al., 2016]. We present two patients with heterozygous de novo HIVEP2 loss-of-function variants who additionally manifested with hyperphagia and Angelman syndrome (AS)-like features. Clinical features of these two patients are described, along with current knowledge of HIVEP2 function. A summary of the

cohorts of previously described patients [Srivastava et al., 2016; Steinfeld et al., 2016] are

included in Table 1, Supplementary Table 2 and Supplementary Table 3, and complement the

additional information that has been gleaned from our cases.

CLINICAL REPORT, METHODS AND RESULTS

Patient 1 is an Australian ten year old boy seen at a genetics service at the age of six for intellectual disability and microcephaly (head circumference just below 2nd percentile on

growth charts endorsed by Australasian Paediatric Endocrine Group).

He was born via a caesarean section at 35 weeks (breech presentation) following

preterm premature rupture of membranes. His growth parameters were normal at birth. Since

then, he showed deceleration of head circumference, resulting in microcephaly, and an

increase in weight relative to height. Growth parameters and dysmorphic features are detailed

in Figure 1 (left), Table 1 and Supplementary Table 3.

Patient 1 had severe intellectual delay. Expressive language skills were minimal, and

improved slowly with speech therapy. He had some three or four-word phrases, and was

often difficult to understand, even to those who knew him well. Communication was

augmented with the use of an ‘app’ on a tablet when at school. His psychological assessment

showed significant delays (below 1st percentile) across all areas. His full scale IQ was about

55. He had onset of hyperphagia at seven years of age, predominantly involving grazing

throughout the day and overfilling his mouth when eating. His caloric intake was not

formally measured but his body mass index increased significantly after age seven, from

16kg/m2 to 22.8kg/m2 (>95th percentile). AS-like features included: running with hands

raised, poor co-ordination, and inappropriate laughter. He had autistic traits: restrictive behaviours, the need for predictability and poor coping when faced with change in normal routine. He also had little to no recognition of other peoples’ personal space. On systems review, he had hypotonia, strabismus, faecal incontinence, kyphosis and required an adenoidectomy for enlarged tonsils and adenoids at the age of seven. He had received

intensive allied health input including occupational and speech therapy.

Investigations were normal and included: SNP microarray, fragile X PCR,

methylation for AS, UBE3A gene sequencing, metabolic and thyroid studies, plasma

homocysteine and lead levels, renal and liver functions, and brain MRI. A clinical diagnosis

of intellectual disability with microcephaly had been made.

Singleton whole exome sequencing (WES) was performed on Patient 1, using the

Illumina TruSeq Rapid Exome Library Preparation Kit, followed by massively-parallel

sequencing on an Illumina HiSeq 4000 with 150 bp paired-end runs. Sequencing reads were

aligned to the GRCh37/hg19 reference genome using Burrows-Wheeler Aligner (BWA-

MEM) and variants called using Genome Analysis Tool Kit (GATK). Variants were

annotated, filtered and triaged using BenchLab NGS v4.2.4 (Agilent). A novel heterozygous

de novo nonsense variant was found in the HIVEP2 gene; c.5935C>T (p.(Arg1979*)) in exon

9 (NCBI reference sequence NM_006734.3; NC_000006.11:g.143081490G>A). Parental

samples were tested using targeted PCR and Sanger sequencing.

Patient 2 is a Portuguese eight year old girl seen at a genetics service at 30 months of

age for global developmental delay, hypotonia, stereotypies (repetitive hand and mouth

movements), behavioural problems and craniofacial dysmorphic features, Figure 1 (right).

Patient 2 was delivered at 38 weeks gestation via an elective caesarean section for breech presentation. During pregnancy, fetal movements were decreased. At birth, her growth parameters were normal and no congenital abnormalities were observed. Post-natal deceleration of head circumference was noted, with a measurement at birth between the 50th

and 75th percentile and most recent recorded measurement on the 20th percentile. Patient 2

had dysmorphic features (as outlined in Table 1), and global developmental delay,

particularly in the domain of speech. She could say two syllable words but not sentences.

Speech was supplemented with an invented vocabulary and 100+ signs. Her parents reported

that she had a reasonable receptive understanding of language and a good memory. Her ID

(detailed in Supplementary Table 3), was classified as severe.

Patient 2 displayed challenging behaviours; including hyperphagia, impulsivity,

aggression, hyperactivity, and anxiety. She had hand and mouth stereotypies, and walked

with a broad-based tip-toe gait. She had decreased pain sensation. On systems review, she

had excessive salivation, dysphagia and inflammation at the gastroesophageal junction. She

received regular pediatric orthopedic input for decreased hip abduction and scoliosis. She had

a myringotomy and adenoidectomy which improved her hearing significantly.

Investigations undertaken were normal, and included: high-resolution G-banded

karyotype, SNP microarray, methylation testing for Angelman/Prader-Willi syndromes,

MECP2 sequencing, metabolic profile and brain MRI. Global developmental delay with

unknown aetiology was the working diagnosis.

For Patient 2, trio WES was undertaken. Exome enrichment (Agilent SureSelectXT

Human All Exon 50Mb), sequencing (Illumina HiSeq 2000 with 150 paired-end runs), read alignment to the GRCh37/hg19 reference genome (BWA) and variant calling (GATK) was performed at BGI-Europe (Denmark). Variant annotation, selection and prioritising was performed by the Department of Human Genetics, Radboud University Medical Center

(Radboudumc, Nijmegen, Netherlands). A novel heterozygous de novo frameshift variant was found in the HIVEP2 gene; c.2956_2957del (p.(Glu986Argfs*4)) in exon 5 (NCBI reference sequence NM_006734.3; NC_000006.11:g. 143092919_ 143092920del).

Neither variant was present in the gnomAD database version 2.1 accessed on 10

March 2019 [Exome Aggregation et al., 2016]. Both variants are expected to result in a premature termination of translation. Exome sequencing for Patient 1 was undertaken as part of a study on exome sequencing (ES) research project approved by the Mater Research ethics committee. Patient 2 had a clinical sequencing. The variants have been submitted to ClinVar, with the following references: Patient 1: SUB5373516, Patient 2: SUB5430289.

DISCUSSION We contribute two patients with novel pathogenic variants (one nonsense, and one frameshift) in the HIVEP2 gene to the previous nine patients published with ID attributed to pathogenic variants in HIVEP2 [Srivastava et al., 2016; Steinfeld et al., 2016].

Features shared between Patient 1 and Patient 2 include: ID, hyperphagia (and resultant raised body mass index), AS-like features, hypotonia, hypertelorism, strabismus and hirsutism. Neither patient had a diagnosis of epilepsy. AS-like features present in both cases

This article is protected by copyright. All rights reserved. 8 Goldsmith et al. were: hypermotoric behaviours, laughing, cheerful dispositions, slow growth in head circumference (resulting in relative or absolute microcephaly), speech impairment, gastrointestinal issues and a thin upper lip. Our patients had adequate birth somatometry and no congenital abnormalities, similar to that which is observed in AS, in which newborns typically have a normal phenotype [Tan et al., 2014]. Similarities between our patients and those already in the literature [Srivastava et al., 2016; Steinfeld et al., 2016] were: ID, developmental delay (speech and gross motor), hypotonia, abnormal behaviours and gastrointestinal issues. Variable dysmorphic features were also reported in all, and are detailed in Table 1 and Supplementary Table 3. Nine patients had hypotonia. All patients had brain imaging, and varying MRI abnormalities were found in five out of eleven patients.

Hand stereotypies were found in four patients, with behavioural concerns (impulsivity and hyperactivity), and strabismus in five patients. AS was considered and methylation testing was done for one of the previously reported patients [Srivastava et al., 2016]. The gestalt evidenced in our patients (including behaviour and appearance) supports the consideration of

HIVEP2-related ID when investigating patients with AS-like features.

The HIVEP2 gene encodes a transcription factor that is a member of the ZAS protein family, which is involved in growth and development [Wu L. C., 2002]. Somatostatin receptor type 2 (SSTR2), a G-protein-couple receptor, may participate in neuron development and maturation during brain development by stimulating neuronal migration and axon outgrowth [Le Verche et al., 2009]. The expression of SSTR2 is activated by HIVEP2 transcription factor and its binding partner Transcription Factor 4 (TCF4) [Rauch et al.,

2012]. SSTR2 is expressed in the cortex during nervous system development, as are HIVEP2

and TCF4 which are co-expressed in the frontal cortex and hippocampus during development

[Le Verche et al., 2009; Dörflinger et al., 1999]. Pathogenic variants in the TCF4 gene are

described in Pitt-Hopkins syndrome (PHS). PHS presents with features including global

developmental delay, severe speech delay, autism, seizures, sleep disturbances, stereotypic

hand movements and myopia. These features have overlap with AS; hence PHS is often on

the differential for AS. Hyperventilation is typically present in PHS, and has not been seen in

patients with HIVEP2 variants.

Takao et al. [2013] studied HIVEP2-knockout mice, who were reported to

demonstrate evidence of central nervous system inflammation; a decreased number of

hippocampal dentate gyrus neurons with more immature neurons compared to mature

neurons; thinner cortices (including prelimbic and primary visual cortices); and a decreased level of dopamine D1 receptors in the dentate nucleus. Biochemically, Takao et al. [2013] found that gene expression of the HIVEP2 mutated mice resembled that within the brain of patients with schizophrenia, but also shared similarities with patients with neurodegenerative disorders (such as Alzheimer’s or Parkinson’s Disease), despite an absence of apoptosis or neurodegeneration in the mice with the HIVEP2 mutation. Phenotypically, HIVEP2-knockout mice demonstrated anxiety, hyperactivity, memory deficits, impaired sociability and decreased pre-pulse inhibition leading to decreased startle reflex [Takao et al., 2013]. Some of these characteristics mirror the behaviours that are reported in Patient 1 and Patient 2, with their AS-like phenotype, hyperactivity, and challenging social behaviours.

The molecular mechanism of how HIVEP2 LoF manifests as hyperphagia has not

been ascertained using cellular or animal models. Neither Patient 1 nor Patient 2 showed

pituitary axis disturbances. Their MRI brain did not reveal hypothalamic or pituitary

pathology. AS patients commonly display hyperphagic behaviour [Mertz et al., 2014]. We

used gene-gene interaction database GeneMANIA [Warde-Farley et al., 2010] to investigate

the interaction between HIVEP2 and UBE3A, and this did not show any strong interactions.

This neurodevelopmental disorder is likely the result of haploinsufficiency of the

HIVEP2 gene. The Residual Variation Intolerance Score is -2.28 (1.37%) and the probability of the gene being LoF intolerant (pLI) score is 1.0 [Petrovski et al., 2013; Exome

Aggregation et al., 2016]. These computational predictions indicate that HIVEP2 is intolerant to genetic variation. Moreover, while not functionally assessed, one mechanism of disease could be a dominant-negative effect, suggesting that rare de novo missense variants in the

HIVEP2 gene may play a role in this syndrome. Further evaluation using cellular or animal models, is required to help further elucidate their role.

HIVEP2 gene variants have been shown to result in a phenotype which displays neurodevelopmental changes and broad dysmorphic features. It is evident that LoF variants cause HIVEP2 related ID, but there is no clear genotype-phenotype trend regarding the severity of presentation, with all of the variants in this report likely to be deleterious. Further patients with HIVEP2 variants would need to be examined and functional studies undertaken to determine a true genotypic-phenotypic correlation.

The addition of these two patients to the already documented cohort of individuals with likely pathogenic HIVEP2 variants indicates that the phenotypic spectrum may be wider than first described. Furthermore, the use of WES earlier in the genetic diagnostic journey for patients with clinical presentation of ID and AS-like features could allow for expedited

diagnosis. This is particularly pertinent as most clinical AS gene panels do not include the

HIVEP2 gene, and therefore utilising WES for these presentations may be warranted. This

would ultimately cost less both financially and emotionally for the patients and their families

who embark upon a lengthy process whilst seeking a diagnosis for ID.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the patients’ families for generously giving permission to write about their loved ones.

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ACCOMPANYING IMAGES

Table 1: Summary of features in patients with HIVEP2 variants.

Figure 1: Patient photographs. Patient 1 (left): at 6 years old, had a broad forehead, straight eyebrows, mild hypertelorism, bulbous nasal tip, widened mouth with thin upper lip.

Patient 2 (right): at 5 years old, had an elongated face, arched eyebrows, wide-set eyes, slightly downslanted palpebral fissures, high nasal bridge, low set columnella and wide mouth with thin upper lip.

Table 1: Summary of features in patients with HIVEP2 variants.

Patient 1 Patient 2 Steinfeld et al combined: 6 Srivastava et al combined: 3 patients patients Variant † c.[5935C>T];[=] c.[2956_2957del];[=] 2 nonsense 2 nonsense p.[(Arg1979*)];[(=)] p.[(Glu986Argfs*4)];[(=)] 3 frameshift 1 frameshift 1 missense Inheritance de novo de novo All de novo All de novo Age at time of 10 8 5, 9, 10, 13, 14 and 17 6, 7 and 24 publishing (years) Gender Male Female 3 females and 3 males 2 females and 1 male Birth Weight and length: 25th Weight: 25-50th All within normal range OFC: 1 patient <3rd anthropometry OFC: 50th Height: 10-25th (percentile) OFC: 50-75th Most recent 10 years, 2 months: 7 years 1 month: Weight: 1 patient: <5th OFC: 1 patient <3rd anthropometry Weight: 85th Weight: 90th Height: 2 patients: <5th (percentile) Height: 5th Height: 75th OFC: 3 patients < 3rd BMI: >95th BMI: >90th OFC: 3rd OFC: 20th Dysmorphic Microcephaly Low anterior hairline Various. Various. facial features Brachycephalic Elongated, narrow face Each patient had different Each patient had different Broad forehead Micrognathia features; however, the features, however, the Mild hypertelorism Widely set eyes following features appeared following features appeared Widened mouth, thin upper Slightly down slanted palpebral more than once: more than once: lip fissures Retrognathia (2 patients), Widely set eyes (2 patients), Frontal upsweep Arched eyebrows Dental abnormalities (2 Broad nasal root (2 patients) Mild synophrys patients), Broad nasal root and high nasal Helical changes (2 patients), bridge High nasal bridge (2 patients) Low set columella Small, widely spaced teeth Thin upper lip Squared superior portion of helix Other Hypertrichosis Short neck ? Tapering fingers (2 patients) dysmorphic Clinodactyly of fifth finger features Pes planus Age at walking 24 months 20 months Range: 24 months – 5years Range: 24 months – 7 years Age at talking 24 months 18 months Range: 10 – 24 months; Range: 24 months – 7 years 2 patients non-verbal Current speech Can talk in three to four Says two syllable words but does Range: from no speech to full Range: all patients have speech abilities word phrases with variable not talk in sentences. Uses more sentences (but difficult to impairment of varying degrees clarity than 100 gestures. Has her own understand) vocabulary Hypotonia + + + (4/6) + (3/3) Autism - - + (1/6) ? Intellectual + + + (5/6) + (3/3) disability Abnormal - - + (2/6) + (3/3) brain MRI / CT results Abnormal + + + + behaviors Gastro- + + + + intestinal / growth issues Strabismus + + + (3/6) + (1/3)

Epilepsy - - + (3/6) - Table adapted from Steinfeld et al. [2016] Key: - absent, no, or normal + present, yes, or abnormal ? unknown Footnote: further explanation of features introduced in Table 1 can be found in Table 3, Supplementary Material. † Variants are described as per HGVS nomenclature version 15.11 (varnomen.hgvs.org), using the reference sequence: RefSeq (NM_006734.3, NP_006725.3) and Ensembl (ENST00000367603.7).