Introduction

Helsmoortel–van der Aa (MIM #615873), a novel recognizable intellectual disability syndrome with autism comorbidity, is a monogenic autosomal dominant disorder caused by de novo frameshift or nonsense sequence alterations in the 3′ end of the last exon of ADNP [1]. All individuals with Helsmoortel–van der Aa syndrome had consistent clinical features, including autism spectrum disorders, mild to severe intellectual disability, and distinctive facial dysmorphism [2]. ADNP (MIM* 611386) is composed of five exons and encodes an activity-dependent neuroprotective homeobox protein. 3D genome structure analysis allows an overview of regulatory landscape and 3D genome structural units within the ADNP locus (Supplemental Figure S1). This Krüppel C2H2-type zinc-finger transcriptional factor is a component of human BAF multiprotein complexes and participates in the regulation of neuronal development through chromatin remodeling [3]. Interstitial deletions of 20q13.13 chromosome region have been rarely documented. To date, only five heterozygous interstitial deletions with different sizes ranging from 313 kb to 3.31 Mb involving (partial) ADNP gene have been recorded (Table 1 and Fig. 2a). Moreover, no cases with pure ADNP deletions have been reported so far. We report the first case exhibiting the typical features of Helsmoortel–van der Aa syndrome carrying the smallest 63 kb heterozygous interstitial deletion at 20q13.13 chromosome region. Having reviewed previously reported cases with deleterious single-point variants, the genotype–phenotype correlation of the present index-patient was established.

Table 1 Clinical features of all previously reported ADNP deleted patients
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Materials and methods

Blood lymphocytes were cultured in RPMI 1640 supplemented with PHA (Life Technologies Sas, Courtaboeuf, France) and chromosome metaphases were harvested with minor modifications according to the standard laboratory protocol as previously described. Fluorescent in situ hybridization (FISH) analysis was performed using two BAC probes: RP5-914P20 (chr19:49,495,091–49,600,424) and RP11-466J8 (chr19:2,840,928–3,027,437) according to the laboratory protocol [4]. 60K array-CGH (Agilent Technologies, Santa Clara, CA, USA) with a median probe spacing of 54 kb was carried out as previously described [5] and required at least three contiguous probes to make a call. Quantitative real-time PCR using SYBR® Green PCR Master Mix (Life Technologies, Woolston Warrington, UK) was performed and all samples were run in triplicate. The dosage of each amplicon relative to control amplicon of RPPH1 (gene ID85495) and normalized to control DNA using the 2−∆∆CT method as described [6]. Total RNA from blood sample was extracted according to Maxwell® 16 IVD instrument Nucleic Acid Extraction—Protocol Promega. One microgram of total RNA was used for reverse-transcriptase PCR (Thermo Fisher Scientific, Lithuania). Results were normalized with endogenous control gene β-2 microglobulin (B2MG) as previously described [7].

Results

Clinical phenotype

The 3 years and 10 months old boy first came to our clinic because of intellectual disability associated with pervasive developmental disorder. The pregnancy was marked with maternal hyperglycemia. He was born at 39 + 6 weeks to a healthy non-consanguineous Caucasian couple. His birth weight, length, and head circumference were, respectively, 3320 g (25th–50th centile), 47 cm (10th–25th centile), and 36 cm (>90th centile). Hypotonia was noted at birth. The family history was unremarkable, the couple already had a healthy 11-year-old child. The proband had recurrent respiratory tract infections, he suffered from congenital stridor and obstructive sleep apnea due to tonsillar hypertrophy, and underwent tonsillectomy at the age of 1 year. His developmental milestones were delayed and with kinesitherapy he could walk unsupported at 24 months. According to clinical evaluation conducted at the age of 55 months, his weight, height, and head circumference were 18 kg (75th centile), 105 cm (50th centile), and 49.8 cm (25th centile), respectively. Clinical physical evaluation showed prominent forehead with high anterior hairline, hypertelorism with inner intercanthal distance of 4 cm (97th centile), strabismus, palpebral fissure length of 30 mm, small ears with an ear length of 4 cm (<3th centile), broad nasal bridge, short nose, long/smooth philtrum, thin upper lip and protruding lower lip, hyperlaxity of the joints, and ataxic gait (Fig. 1). He had severe language delay, spoke the first word at 16 months, and combined words into short sentences at 4 years. Of note, the index-patient displayed premature primary tooth eruption (deciduous tooth eruption at the age of 2 months). He had 12 maxillary teeth, 11 mandibular teeth but lacked 1 mandibular incisor. His four 6-year molars were also prematurely present. He had difficulties in social interaction and communication, poor eye contact, and sleep disturbances. Behavioral problems and the presence of episodic mood disorders such as angry, irritable mood, and hetero-aggression were observed. His parents also noticed that he had severe chronic constipation and was treated with laxatives. Echocardiogram, renal ultrasound, chest X-ray, EEG (ElectroEncephaloGram), brain magnetic resonance imaging (MRI), ophthalmologic examination, creatine kinase (CK) dosage, and transferrin isoelectric focusing were normal.

Fig. 1
figure 1

Photographs of the face (a, c) and right profile (b) of the proband showed typical facial dysmorphic features, including: frontal bossing, high anterior hairline, broad nasal bridge, hypertelorism, wide palpebral fissures, short nose, smooth/long philtrum, small and widely spaced teeth, thin upper lip, and protruding lower lip

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Conventional karyotype and molecular characterization of an interstitial 20q13.13 microdeletion

Conventional cytogenetics showed a normal male karyotype 46,XY. Agilent 60K array-CGH analysis showed a heterozygous interstitial deletion at 20q13.13 chromosome with three probes (A_16_P03530666, A_14_P134776, and A_14_P104282), log2ratio = −0.85, encompassing two genes: ADNP and DPM1, chr20:g.(49,457,856_49,508,676)_(49,571,808_49,620,163)del (hg19, NC_000020.10) (NG_034200.1) (Fig. 2b). Quantitative real-time PCR with primers in exons 1 and 5 of ADNP gene did not detect the deletion in the parental DNA (Fig. 2c). FISH analysis showed that the deletion occurred de novo in the proband and excluded parental balanced insertional translocation (Supplemental Figure S2). Quantitative reverse-transcriptase PCR showed that ADNP expression was decreased approximately 40% in the proband in comparison to his mother and two normal controls (Supplemental Figure S3).

Fig. 2
figure 2

a Overview of 1 Mb at 20q13.13 chromosome region with five reported overlapping deletions in medical literature: Decipher309, Decipher265392, Decipher257542, Decipher258702, Patient01975-5515, and index-patient. Gray shadow highlights the smallest region of overlap (SRO); the proband carried the smallest 63 kb deletion at 20q13.13 chromosome region harboring two genes ADNP and DPM1. The ADNP gene in Helsmoortel–van der Aa syndrome is highlighted in yellow. b 60K array-CGH showed a 63 kb interstitial deletion at chromosome 20q13.13 containing ADNP and DPM1. c Quantitative real-time PCR showed that the deletion occurred de novo in the proband with primers in exons 1 and 5 of ADNP gene (index-patient: purple color; mother: pink color; father: blue color; pool of male control: light blue color; pool of female control: yellow color)

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Discussion

Hitherto, 18 patients with Helsmoortel–van der Aa syndrome have been reported [8]. Novel additional clinical features associated with ADNP syndrome, including blepharophimosis, craniosynostosis, primary hypothyroidism, hypertension, lack of autistic behaviors, cleft palate, and early deciduous tooth eruption were also recorded [9,10,11]. All frameshift and nonsense ADNP sequence variations were gain-of-function variants except for a stop-codon variant in the fourth exon with predicted loss of ADNP function. The index-patient carried a de novo 63 kb heterozygous interstitial deletion at 20q13.13 chromosome region containing two genes: ADNP and DPM1. The deletion has never been reported in the databases of genomic variants (DGV at http://dgv.tcag.ca/dgv) (Supplemental Figure S4). DPM1 (MIM* 603503) encodes a key enzyme dolichol phosphate mannose (DPM) synthase which plays an important function in protein glycosylation. Disease-associated sequence variants of DPM1 cause congenital disorder of glycosylation type Ie (MIM #608799). To date, nine reported patients had typical features, including: seizure (8/9), postnatal microcephaly (8/9), abnormal MRI (8/9), and increased serum CK levels (7/9) [12]. Based on the detailed analysis of clinical findings, it is worth noting that the clinical features as well as paraclinical examinations of the reported proband are not consistent with the diagnosis of congenital disorder of glycosylation type Ie. Of interest, the proband displayed typical findings of Helsmoortel–van der Aa syndrome. Additionally, he had severe chronic constipation. Only 4 of 18 reported patients had constipation, one carrying a de novo c.2491_2494delTTAA p.Lys831Ilefs*81 variant had intermittent constipation, while other three patients had the same sequence alteration c.2157C>G p.Tyr719* in ADNP gene. Up to date, all reported disease-associated variants apparently escaped from nonsense-mediated mRNA decay which showed an increased aberrant ADNP mRNA. Unlike dominant-negative mutational mechanism, ADNP loss-of-function variants or deletion would produce an amorphic allele leading to gene dosage imbalance hypothesis. Normal BAF complex function requires the physical interaction of distinct subunits which ideally are mutually balanced. The presence of multiple heterozygous variants in genes encoding different components of BAF complex would lead to synergistic effects that could drive the multimer concentration and/or activity below a critical thresold required for a normal phenotype or otherwise, ADNP haploinsufficiency associated with hypomorphic alleles of other protein partners in BAF complex could cause a reduced complexes activities below a critical thresold resulting in an abnormal phenotype [13]. Moreover, regarding the probability of loss-of-function intolerance (pLI) score, ADNP has a high pLI score of 1.0 which is particularly intolerant to loss-of-function variants, dosage sensitive and predicted to have a high probability of being relevant to haploinsufficiency (pLI ≥ 0.9), while low DPM1 pLI score of 0 is loss-of-function tolerant. The ADNP gene region on 8 × 60K array-CGH platform is inadequately covered and contains only two probes: A_16_P03530666 and A_14_P134776 which could not detect small intragenic ADNP rearrangements during routine array-CGH testing. High-resolution techniques should be preferentially performed in patients with unexplained intellectual disability in order to tackle these issues [14]. In summary, we report the first case of ADNP haploinsufficiency associated with Helsmoortel–van der Aa syndrome and highlight the importance of high-resolution techniques such as high-density oligonucleotide array-CGH and whole-genome sequencing in clinical diagnostic workup when all available conventional and specific gene tests did not reveal causative abnormalities.