Background

Chromosome microarray analysis of subjects with NDD has uncovered a large number of rare copy number variations (CNVs); nevertheless, some pathogenic and putatively pathogenic CNVs detected in patients cannot completely explain complex patient phenotypes, particularly when an unaffected parent carries the same submicroscopic imbalance. One example of a susceptibility locus for NDD is the 16p11.2 region with ~600 kb deletions and duplications observed in ~1 % of autism and 1.5 % of children diagnosed with significant developmental or language delays compared to 0.04–0.07 % amongst control populations [1, 2]. Carriers of 16p11.2 CNV manifest a broad spectrum of neurocognitive phenotypes, ranging from ID [1, 3, 4], autism spectrum disorder (ASD) [5, 6], schizophrenia [7], congenital anomalies [4, 8] to individuals without a specific phenotype [3, 4, 8]. There is familial coincidence of both phenotypically affected and unaffected carriers in some families [1, 7, 8]. The estimated penetrance of 16p11.2 deletion and duplication are 46.8 % and 27.2 %, respectively [2]. Multiple studies demonstrated the power of WES to find the genetic etiology of clinical variability among such patients. WES helped to discover that the presence of variants on the non-CNV containing homolog chromosome may unmask biallelic mutations in an autosomal recessive condition [9, 10], or that damaging variants in other parts of the genome may contribute to such variable expressivity [11]. The results of these studies suggest that inconsistent phenotypes in patients with known pathogenic CNVs or with CNVs inherited from an unaffected parent may indicate the co-occurrence of secondary genomic events elsewhere in the genome.

In this study, we report that pathogenic variants of VPS13B located at chromosome 8 in a boy with NDD carrying a familial dup16p11.2 contribute to the clinical variability in this family.

Case presentation

The proband is an 11 year old boy introduced to our clinic with global developmental delay and verbal apraxia at the age of four. He is the third of four-children of non-consanguineous parents of Chinese descent. His mother and his paternal grand-mother have a history of recurrent spontaneous pregnancy losses with unknown cause. His parents and three siblings are apparently healthy (Fig. 1a). The proband was born after 39 weeks of uneventful pregnancy via caesarean section for fetal distress with Apgar scores of 8 and 9 at one and five minutes after birth, respectively. His birth weight was 2175 gram (<3rd percentile (%ile)), length was 47 cm (10th %ile) and occipito-frontal circumference (OFC) was 34 cm (25th %ile). The patient exhibited feeding difficulty, low muscle tone, bilateral ptosis, club foot, bilateral undescended testes, and flexion contracture of hand and wrist. The proband’s laboratory diagnostic workup was normal and included routine karyotype, subtelomeric FISH, fragile X, biochemical assessment, cranial MRI and CT scan. Affymetrix Genome-Wide Human SNP Array 6.0 revealed a 709.2 kb duplication of 16p11.2 (29,425,199–30,134,432) in the proband, confirmed by FISH and parental studies indicating maternal inheritance. The proband’s siblings were not tested for dup16p11.2 per the family’s request.

Fig. 1
figure 1

a Family pedigree. b Sanger sequencing analysis of VPS13B variants. I) Proband and his mother are carriers of splicing mutation of c.1426-1G > A. II) Proband and his father are carriers of splicing mutation of c.4157 + 1G > T (sequences of reverse strands are shown)

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We examined the mother who is a carrier of dup16p11.2 for the possibility that apparently healthy carrier parents might have some unnoticed clinical features, and for the presence of phenotypic commonality with his child. She showed no sign of ID, ASD, psychiatric disorder (anxiety, depression, obsessive-compulsive disorders (OCD)), underweight or microcephaly. She was also negative for history of other dup16p11.2 features including epilepsy, speech and motor delay, and congenital anomalies.

Genetic testing

DNA samples of family trios were sent to PerkinElmer Company for exome enrichment using the TruSeq Exome Enrichment Kit (Agilent v5 + UTR), followed by paired-end sequencing (Illumina HiSeq 2000, read length of 100 bp). Using Golden Helix (GH) software (SNP & Variation Suite 7.7.8), the WES data from a single VCF file for sequenced family members was analyzed (Additional file 1: Figure S1). Two novel splicing mutations of VPS13B (8q22.2) with compound heterozygous inheritance were identified in the proband and subsequently confirmed by Sanger sequencing (Fig. 1b). A sequence variant of c.1426-1G > A located in the acceptor splice site of intron 10 was identified in Proband A and his mother. The second variant, a nucleotide change of G > T at c.4157 + 1 situated in the donor site of intron 27, was inherited from his father. Mutations and/or CNVs in the VPS13B gene lead to a rare autosomal recessive condition called Cohen syndrome (CS) [12].

Functional prediction tools used for WES data analysis anticipate the effect of non-synonymous variants (coding region). However, both variants of VPS13B are located at canonical splice sites. ALAMUT software predicted that two intronic variants of VPS13B would result in skipping of the exon 11 and 27. To confirm this prediction, we performed PCR on cDNA samples of proband and a control using two separate sets of primers covering exons 9–12 and 26–29 of VPS13B, followed by Sanger sequencing of the PCR products. This confirmed that both variants abolish the canonical splice sites and create aberrant RNA sequences (Fig. 2). Real-time quantitative PCR (qPCR) for VPS13B demonstrated reduced expression in the proband compared to two controls (Additional file 1: Material and methods). The mother also showed reduced RNA expression compared to one control (Fig. 3). Other family members were not available for VPS13B or dup16p11.2 testing.

Fig. 2
figure 2

Sanger sequencing of RT-PCR products of proband and control, using primers covering exons 9–12 and 26–29 of VPS13B. a The variant of c.1426-1G > A disrupted the following sequences and caused frameshift in the proband. The orange arrow shows the first bp of exon 11 in the normal control. b The variant of 4157 + 1G > T disrupted following sequences, and caused frameshift in the proband. The orange arrow shows the first bp of exon 27 in the normal control

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Fig. 3
figure 3

Expression study of VPS13B gene. The mean RNA expression of VPS13B calculated from three different time-series of RNA extraction in the proband, his mother and two normal controls. The relative expression of VPS13B is <0.5 fold in the proband and >0.6 fold in his mother. Error bars indicate standard errors from three replicates

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The VPS13B gene, also known as COH1 (OMIM: 607817), is approximately 864 kb in length and located on chromosome 8q22.2. It consists of 62 exons encoding a transmembrane protein of 4022 amino acids [12]. VPS13B is a peripheral membrane protein that is required for function, orientation and structural integrity of the Golgi apparatus and thus plays a role in vesicle-mediated sorting and intracellular protein transport [13, 14]. Homozygous or compound heterozygous mutations/CNVs of VPS13B cause CS [12]. 

Intronic point mutations within donor and acceptor sites at mRNA splice junctions typically cause mRNA mis-splicing, leading to subsequent nonsense-mediated mRNA decay (NMD), and altered protein with effect on the clinical phenotype [15]. Indeed, Sanger sequencing of RT-PCR product corresponding to each specific VPS13B variant demonstrated that both variants create aberrant RNA sequences and frameshift and thus probably lead to NMD. Moreover, the RNA expression level of VPS13B in the proband was significantly reduced compared to two controls. VPS13B expression in his mother was intermediate between the proband and one control, suggesting that partial loss-of-function in carriers of autosomal recessive disorders is not sufficient to produce a complete disease phenotype.

Absence of dup16p11.2 -related phenotype in the mother, presence of some CS features in the proband, and the discovery of pathogenic VPS13B mutations warranted re-evaluation of our patient at 10 years of age. CS has a broad clinical phenotype spectrum including ID, microcephaly, hypotonia, dysmorphic facial features, truncal obesity, slender extremities, joint hypermobility, myopia, retinal dystrophy, intermittent isolated neutropenia, and happy personality. Neutropenia is characterized as a neutrophil count of <1.5 × 109/L in children and <1.8 × 109/L in adults [16]. The facial gestalt includes down-slanting palpebral fissures, wave-shaped eyelids, thick eyebrows and eyelashes, low hairline, prominent and beak-shaped nose, malar hypoplasia, short philtrum, high-arched palate, maxillary prognathia and prominent central incisors [17–19]. Patients with CS grimace when they are asked to smile [12, 20]. Other signs and symptoms include short stature and scoliosis [12, 20]. In addition, individuals with CS have high rates of ASD or autistic features [21]. The estimated prevalence of CS is 1:105,000 [22], however, its frequency may be considerably higher due to the fact that patients are often not diagnosed until they reach their teenage or adult years. The early diagnosis of CS is challenging because facial features are less noticeable in pre-school age, truncal obesity may evolve in late-childhood, neutropenia is rarely identified due to its intermittent pattern and absence of clinical consequences, and diagnosis of retinal dystrophy usually occurs in later childhood [16, 17, 20].

Reverse phenotyping of our patient at 10 years of age unequivocally confirmed a pattern of features consistent with CS (Table 1). Table 1 shows the presence or absence of clinical features observed in our proband relative to patients with CS [17, 18, 20–24], or dup16p11.2 [4, 8, 25–27].

Table 1 Clinical phenotypes of proband, and their presence/absence among reported cases of CS and dup16p11.2
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Being underweight is a known feature of dup 16p11.2. Although the proband was underweight at birth, his weight changed with age to the 5–10th %ile at the age of 10. He also developed truncal obesity with slender extremities, mild scoliosis, and evolving facial gestalt consistent with CS. Similar to the report by El Chehadeh-Djebbar et al. [17], our study suggests that some CS features are age-dependent and evolve later in childhood (Table 2).

Table 2 Evolving clinical features of proband
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Conclusion

Inherited dup16p11.2 by itself cannot explain the variable expressivity among NDD patients when their carrier parents are unaffected. We utilized WES in a family with a child presenting with NDD carrying dup16p11.2 inherited from his unaffected mother, and searched for sequence changes that could explain this clinical variability. We discovered that compound heterozygous variants of VPS13B contribute to the proband’s phenotypic features. The new CS diagnosis helps in screening and earlier management of scoliosis, periodontal disease and tooth loss, early cataract, vision loss, and premature aging [24] in the proband; and provides more informed genetic counselling for the family.

Our study suggests that NDD patients with dup16p11.2 may show additional pathogenic SNVs in their genome, which significantly influence phenotype heterogeneity and the genetic counselling of families with putatively pathogenic CNVs showing variable expressivity and incomplete penetrance. Genomic microarray is a valuable first-tier test for the postnatal evaluation of individuals with NDD including ID, ASD, and/or multiple congenital anomalies. However, coupling of microarray with WES or whole genome data analyses will facilitate a more comprehensive and accurate analysis of genetic causes of NDD, heighten understanding of the etiology of variable expressivity among NDD patients, and optimize clinically-informed and effective genetic counselling and personalized management options.