Background

Copy number variations (CNV) play a significant role in the aetiology of developmental disabilities including non-syndromic intellectual impairment and/or autism [1–3], accounting for about 10–20% of non-syndromic patients [1, 4, 5]. In addition, mutations in numerous genes have been linked to the aetiology of intellectual disability (ID) [1] and autism spectrum disorders (ASD). Reduced penetrance and variable expressivity of CNVs and mutations add to the complexity of interpretation of causality. Therefore, although known to be highly heritable, the aetiology of ASD is still poorly understood with no single candidate gene accounting for a majority of ASD susceptibility [6–8]. Interpretation of the clinical significance of CNVs and evaluation of their contribution to the phenotype remains challenging, although CMAs are now widely used for diagnostic purposes.

We report on a patient with global developmental delay/intellectual disability (DD/ID) and autism for whom CMA analysis revealed the unusual finding of two microdeletions on chromosome 6q. Familial segregation analysis confirmed de novo events and cis-configuration of both deletions. Phenotype-genotype correlations of all genes within the deletions allowed to determine a likely pathogenic contribution of both deletions. Genes within both deletions are involved in ASD pathways and likely represent a contiguous gene deletion syndrome.

Case presentation

Our patient, which was last seen at 19 years of age, was the second of three children of healthy non- consanguineous Caucasian parents. The family history was unremarkable.

He was born at term after an uneventful pregnancy. Birth measurements were all above the 90th centiles with weight of 4750 g (>P 90), length of 55 cm (>P90) and occipitofrontal circumference (OFC) of 37 cm (>P 90). Neonatal and infancy periods were unremarkable. The boy said his first words at 12 months and walked unsupportedly at 15 months of age. At age two, macrocephaly (above the 97th centiles for OFC) and stagnation of expressive speech as well as stereotypic behaviour and missing eye contact were noted.

At 6 ½ years the boy was diagnosed with infantile autism and severe intellectual disability. The results of the autism diagnostic observation schedule (ADOS) were above the cut-off for autism. In the Vineland adaptive behaviour scale he showed a severe developmental delay (developmental quotient 30); his social behaviour and communication behaviour met the level of a 1 ½ year old, and everyday life skills levels were comparable with a 2 ½ year old child. The boy had a very short attention span, showed stereotypic behaviour, hardly no functional or symbolic playing, almost no eye contact and minimal adaptive social skills. Expressive and receptive language skills were limited to simple orders.

Further regular follow-up exams at different ages confirmed the clinical findings. Except for a mild hypotonia neurological exams were in normal range. The patient developed dog phobia. No aggressivity or autoaggressivity was noted. Access to specialized school education and therapies as well as a stimulating familial environment allowed a certain independence in these surroundings. The patient today is able to ride a bicycle and swim. He still needs help with body hygiene.

At the last clinical exam at age 19 dysmorphic signs were a mild dolichocephalus, a low set hairline on the neck, a broad face, hypertelorism (IPD > 97 P), medial sparse eyebrows, bilateral prominent anthelices, posteriorly rotated ears, a short wide nose, full lips, widely spaced teeth, prominent upper incisors, long hands (hand length >97 P), hyperextensible joints and a slight funnel chest. Measurements taken for OFC, weight and length were above the 97th centile. The parents and siblings physical parameters are the following: mother 167 cm, 64 kg (BMI 22.9 kg/m2), father 187 cm, 98 kg (BMI 28 kg/m2), sister 1 and 2 174 cm, 58 kg (BMI 19.2 kg/m2) and 179 cm, 65 kg (BMI 20.3 kg/m2), respectively.

Apart from frequent middle ear infections the patient was in good health. Extensive investigations in childhood which included brain magnetic resonance imaging (MRI), cranial computed tomography (CT), electroencephalogram and an abdominal ultrasound all proved unobtrusive. An X-ray of the hand performed at age 10 showed a slightly retarded bone age. Conventional chromosome analysis of lymphocytes (GTG-banding, 450 band level) did not show any numerical or structural anomalies. Specific testing for Fragile – X syndrome, Prader-Willi-, Angelman- and Beckwith-Wiedemann syndromes and for a 22q13 deletion did not show any abnormal results.

Methods and results

Chromosomal microarray

Array genomic hybridization of DNA from peripheral blood lymphocytes was performed in the patient and both his parents, using the NimbleGen WG HG18 Tiling 385 K CGHv.2.0 array. The tiling array version 2.0 contains 385,000 probes with a probe spacing of 6000 bp. Labelling and hybridization of test and reference DNA was performed according to manufacturer’s protocols.

The analysis identified two deletions in 6q separated by a segment of 2 Mb. The proximal deletion (Deletion Nr.1) (Fig. 1) is an interstitial deletion localized in the chromosomal region 6q16.1q16.2, spanning ~1.4 Mb (chr6: 98,693,279-100,083,279 bp, hg19 build) and encompassing nine protein coding genes of which five are listed in the Online Mendelian Inheritance in Man (OMIM) database (Table 2).

Fig. 1
figure 1

UCSC genome browser, chromosomal region 6q16.1q16.3. The black bar marked “Patient del. Nr. 1” shows the proximal deletion of our patient, the other black bars patients with overlapping deletions as described in Table 1. RefSeq genes are depicted in blue

Full size image

The distal deletion (Deletion Nr.2), (Fig. 2) also an interstitial deletion, spans 760 kb and was assigned to the 6q16.3 chromosomal region (chr6: 102,113,307-102,873,307 bp, hg19 build). Its proximal breakpoint disrupts the GRIK2 gene in exon 3; coding exons 4–16 are therefore deleted. Results are shown in Fig. 3.

Fig. 2
figure 2

UCSC genome browser, chromosomal region 6q16.2q16.3. The black bar marked „Patient del. Nr.2“ shows the distal deletion of our patient, other black bars patients with overlapping or flanking deletions as described in Table 1. RefSeq genes are depicted in blue

Full size image
Fig. 3
figure 3

Array plot of chromosome 6q. Deletion Nr. 1 is on 6q16.1q16.2 (chr6:98693279–100083279 bp); Deletion Nr. 2 on 6q16.3 (chr6:102113307–102873307 bp). Hg version 19 (GRCh37/hg19)

Full size image

Fluorescence in situ hybridization

To further investigate the structural anomaly fluorescence-in-situ-hybridization (FISH) using the BlueGnome probes RP11-758C21 for 6q16.2 (green) and RP11-487 F5 for 6q16.3 (red) was done in the patient and his parents. The deletions were confirmed to be in cis-position in the patient. FISH analysis of the parents showed both loci to be present in correct position and orientation which proved a de novo origin of the deletions in the patient. There was no evidence for a complex structural rearrangement in the parents. FISH results are shown in Fig. 4.

Fig. 4
figure 4

Fluorescence- in-situ-hybridization (FISH) on metaphase spreads of the patient and his parents’ chromosomes 6. We used the locus-specific Bluegnome probes RP11-758C21 for 6q16.2 (green), targeting deletion Nr 1, and RP11-487 F5 for 6q16.3 (red), targeting deletion nr.2, and the Abbott centromere specific probe cep 6 (aqua) for control. a Patient, b Patient’s father, c Patient’s mother: Both loci are present in correct orientation in the parent’s FISH analysis and proved a de novo origin of the deletions in the patient. Deletions in the patient are in cis position

Full size image

Sanger sequencing of GRIK2

Because bi-allelic mutations in GRIK2 have been shown to cause autosomal recessive intellectual disability [9], we sequenced all 16 exons of the GRIK2 gene including exon-intron boundaries to confirm or rule out a compound heterozygous mutation on the second allele in GRIK2. No mutation was identified. Primer sequences can be obtained upon request.

Genotype-phenotype correlations

We searched current databases, specifically Decipher [10], ClinGen [11] and DGV [12] for overlapping copy number variations in patients or healthy individuals. Results of the overlapping pathogenic deletions are shown in Table 1. We performed an extended literature and database search on all genes included in the deleted regions, their functions and related phenotypes using PubMed [13] and OMIM [14]. Results are summarized in Table 2.

Table 1 Overlapping deletions
Full size table
Table 2 Involved genes
Full size table

Discussion

Chromosomal microarray is now widely used as a diagnostic tool to elucidate the aetiology of developmental delay, intellectual disability and autism spectrum disorders. The genetic heterogeneity, reduced penetrance and variable expressivity as well as suggested polygenic versus monogenic inheritance often renders the interpretation of the causal contribution of single rare CNVs to these phenotypes difficult. Here, we report on a now adult patient with global developmental delay/intellectual disability and autism for which CMA analysis has revealed the unusual finding of two interstitial de novo microdeletions in cis-position on chromosome 6q. Both deletions, spanning ~1.39 Mb and ~0.7 Mb respectively, separated by a genomic segment of ~2 Mb, have not been described before, but phenotype-genotype correlations with overlapping deletions mentioned in the medical literature and databases as well as haploinsufficiency of genes part of neuronal networks encompassed in both deletions, likely suggest their pathogenic contribution. Notably, Kasher et al. reported on several small heterozygous deletions in 6q16.1 presenting with variable developmental delay, intellectual disability and susceptibility to obesity. In one of their patients the deletion of 1.04 Mb includes the same nine genes as does the deletion Nr. 1 in the patient we describe [15]. The authors suggest a common critical region in their patients encompassing POU3F2.

POU3F2/Brn2 belongs to a family of transcription factors that share a highly homologous POU domain and is highly expressed in the central nervous system [16], especially the human hypothalamus and hippocampus [15]. POU3F2 upregulates proneuronal genes [16], promotes neurogenesis in the neocortex and is required for cortical neural migration [17]. Kasher et al. showed that in zebrafish, POU3F2 functions as a downstream target of SIM1 in the leptin – melanocortin – SIM1 pathway and plays a role in the regulation of oxytocin expression in the hypothalamus. They suggest POU3F2 being the likely causal mechanism affecting hypothalamic functions such as the control of food intake and social behaviour or learning, compatible with the human phenotypes in 6q16.1 deletions [15].

Previously, Bonaglia et al. [18], Griswold et al. [5], El Khattabi el al. [19] and Le Caignec et al. [20] reported on patients with mostly larger heterozygous deletions in 6q16 (in a range between 1.7 and 14 Mb) which do not allow specific phenotype-genotype correlations. However, their patients, the patients summarized in Table 1 and our patient share phenotypic findings such as hypotonia, intellectual disability, developmental delay as well as autistic features or behavioural anomalies reminiscent of ASD. Macrocephaly and tall stature and/or obesity are shared physical findings in some patients compatible with a contribution of the genomic region to growth regulation. El Khattabi et al. suggested an involvement of the MCHR2 and SIM1 genes in behavioural disorders and SIM1 in obesity [19]. Both genes are not located within the deleted region of our patient. The normal BMI and feeding habits in our patient are in keeping with the finding of an incomplete obesity phenotype in the cohort of El Khattabi et al.

Of further interest, each of the two deletions in our patient span genes which have been described to play a significant role in the pathogenesis of autism [5, 6, 18, 21] and intellectual disability [22] due to their common roles in ASD associated pathways (Table 2). Particularly, the genes GRIK2, disrupted and partially deleted by deletion 2 in our patient, and PRDM13, encompassed by deletion 1, are functionally linked.

Barbon and co-workers showed experimental evidence that several different splicing variants exist, and splicing mechanisms lead to truncated subunit isoforms suggesting a complex GluR system [23]. Zhawar et al. found GluR6 receptor subunits to be differentially expressed with expression of the GluR6A variant specifically in brain [24]. Studies on mice lacking the GluK2 subunit of kainate receptors, for which GRIK2 encodes, observed reduced social interaction and altered executive functions. The homozygous knockout of the GRIK2 gene produces a lower capacity for pattern separation and an increased propensity for pattern completion [8]. Patients with homozygous mutations in GRIK2 present with autosomal recessive intellectual disability [22]. In addition, studies on CNVs in ASD phenotypes suggest a contributory role of GRIK2 haploinsufficiency in ASD [5, 18, 20, 21]. The small size of the deletion reported by Griswold et al. supports the hypothesis of GRIK2 being a candidate gene for ASD.

GRIK2 is linked to the expression of PRDM13, a gene involved in neuronal development. PRDM13, a transcription factor, is a critical component in neuronal diversity and balance of inhibitory and exhibitory neurons in the dorsal spinal cord [25]. Most interestingly, cells of the inhibitory neuronal lineage in the dorsal spinal cord express excitatory receptor genes/subunits, among them GRIK2, indicating an interaction between excitatory and inhibitory neurons [26] (Fig. 5).

Fig. 5
figure 5

In the dorsal spinal cord PRDM13 is required to produce the correct number of pax2+/GABAergic neurons [25]. Excitatory and inhibitory neurons of the dorsal spinal cord emerge from progenitor populations within the dorsal neural tube. Depending on the transcription factors that are expressed progenitors can become neurons in either class. To redirect cell fate such a transcription factor activates lineage specific gene expression of one lineage and represses the expression of genes in the alternate lineage. The inhibitory (GABAergic) lineage of neurons for example is specified by the homeodomain (HD) transcription factor Pax2. Pax2 and PRDM13 are required for the specification of inhibitory cell fate in the dorsal spinal cord and are direct downstream targets of Ptf1a. The excitatory (glutamatergic) lineage is specified by the HD factors Tlx1/3. PRDM13 actively represses excitatory cell fate by binding to regulatory sequences near Tlx1/Tlx3 genes, suppressing their expression. Ptf1a, a transcription activator induces factor Pax2 while suppressing the expression of Tlx1/3 which leads – through PRDM13- to an increase of inhibitory neurons at the expense of excitatory neurons. Adapted from Guo et al. [26] and Chang et al. [25]

Full size image

Based on these findings haploinsufficiency of all three genes POU3F2, GRIK2 and PRDM13 indicate a contribution of both deletions to our patient’s phenotype and suggest their functional interaction as part of a contiguous gene deletion syndrome. However, the specific contribution of PRDM13 to human disease phenotypes remains to be further elucidated.

Griswold et al. identified several novel candidate genes in molecular pathways or genetic networks implicated in autism aetiology in a large genome-wide SNP array in a European ancestry case–control data set [5]. ASD candidate genes and their functional interactions are tightly linked with general dysregulation of gene expression, disturbances of neuronal maturation and pattering and may be key in conferring susceptibility to autism spectrum conditions [27]. Multi-dimensional co-expression analysis of ASD candidate genes in the normal developing human brain suggests the heterogeneous set of ASD candidates to share transcriptional networks related to synapse formation and elimination, protein turnover, and mitochondrial function [28]. The majority of genes encompassed in deletion Nr.1 (Table 2), in particular FBXL4, CCNC, POU3F2, PRDM13 and COQ3, can be considered part of these networks [16, 17, 25, 26, 29–31].

Conclusions

Recent research into gene interaction and networks in autism spectrum disorders and intellectual disability suggest that phenotype-related gene products are integral to neuronal development. It often remains difficult to draw firm conclusions as to the pathogenic contribution of each single CNV or mutation of these genes. However, we suggest that the two neighbouring deletions on 6q in our patient are likely to cause his ID/ASD phenotype since they harbour several genes implicated in different linked pathways of neuronal development and function.