Case report | Open | Open Peer Review | Published:
Exome sequencing identifies pathogenic variants of VPS13B in a patient with familial 16p11.2 duplication
BMC Medical Geneticsvolume 17, Article number: 78 (2016)
The recurrent microduplication of 16p11.2 (dup16p11.2) is associated with a broad spectrum of neurodevelopmental disorders (NDD) confounded by incomplete penetrance and variable expressivity. This inter- and intra-familial clinical variability highlights the importance of personalized genetic counselling in individuals at-risk.
In this study, we performed whole exome sequencing (WES) to look for other genomic alterations that could explain the clinical variability in a family with a boy presenting with NDD who inherited the dup16p11.2 from his apparently healthy mother. We identified novel splicing variants of VPS13B (8q22.2) in the proband with compound heterozygous inheritance. Two VPS13B mutations abolished the canonical splice sites resulting in low RNA expression in transformed lymphoblasts of the proband. VPS13B mutation causes Cohen syndrome (CS) consistent with the proband’s phenotype (intellectual disability (ID), microcephaly, facial gestalt, retinal dystrophy, joint hypermobility and neutropenia).
The new diagnosis of CS has important health implication for the proband, provides the opportunity for more meaningful and accurate genetic counselling for the family; and underscores the importance of longitudinally following patients for evolving phenotypic features.
This is the first report of a co-occurrence of pathogenic variants with familial dup16p11.2. Our finding suggests that the variable expressivity among carriers of rare putatively pathogenic CNVs such as dup16p11.2 warrants further study by WES and individualized genetic counselling of families with such CNVs.
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 , 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 . 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 . 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.
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.
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.
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) .
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.
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 . 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 .
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 . 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 . 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 . The estimated prevalence of CS is 1:105,000 , 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].
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. , our study suggests that some CS features are age-dependent and evolve later in childhood (Table 2).
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  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.
Autism spectrum disorder
Copy number variation
Nonsense-mediated mRNA decay
Real-time quantitative PCR
Whole exome sequencing
Weiss LA, Shen Y, Korn JM, et al. Association between microdeletion and microduplication at 16p11.2 and autism. N Engl J Med. 2008;358(7):667–75.
Rosenfeld JA, Coe BP, Eichler EE, et al. Estimates of penetrance for recurrent pathogenic copy-number variations. Genet Med. 2013;15(6):478–81.
Bijlsma EK, Gijsbers AC, Schuurs-Hoeijmakers JH, et al. Extending the phenotype of recurrent rearrangements of 16p11.2: deletions in mentally retarded patients without autism and in normal individuals. Eur J Med Genet. 2009;52(2-3):77–87.
Shinawi M, Liu P, Kang SH, et al. Recurrent reciprocal 16p11.2 rearrangements associated with global developmental delay, behavioural problems, dysmorphism, epilepsy, and abnormal head size. J Med Genet. 2010;47(5):332–41.
Marshall CR, Noor A, Vincent JB, et al. Structural variation of chromosomes in autism spectrum disorder. Am J Hum Genet. 2008;82(2):477–88.
Kumar RA, Marshall CR, Badner JA, et al. Association and mutation analyses of 16p11.2 autism candidate genes. PLoS One. 2009;4(2):e4582.
McCarthy SE, Makarov V, Kirov G, et al. Microduplications of 16p11.2 are associated with schizophrenia. Nat Genet. 2009;41(11):1223–7.
Fernandez BA, Roberts W, Chung B, et al. Phenotypic spectrum associated with de novo and inherited deletions and duplications at 16p11.2 in individuals ascertained for diagnosis of autism spectrum disorder. J Med Genet. 2010;47(3):195–203.
McDonald-McGinn DM, Fahiminiya S, Revil T, et al. Hemizygous mutations in SNAP29 unmask autosomal recessive conditions and contribute to atypical findings in patients with 22q11.2DS. J Med Genet. 2013;50(2):80–90.
Paciorkowski AR, Keppler-Noreuil K, Robinson L, et al. Deletion 16p13.11 uncovers NDE1 mutations on the non-deleted homolog and extends the spectrum of severe microcephaly to include fetal brain disruption. Am J Med Genet A. 2013;161A(7):1523–30.
Classen CF, Riehmer V, Landwehr C, et al. Dissecting the genotype in syndromic intellectual disability using whole exome sequencing in addition to genome-wide copy number analysis. Hum Genet. 2013;132(7):825–41.
Kolehmainen J, Black GC, Saarinen A, et al. Cohen syndrome is caused by mutations in a novel gene, COH1, encoding a transmembrane protein with a presumed role in vesicle-mediated sorting and intracellular protein transport. Am J Hum Genet. 2003;72(6):1359–69.
Seifert W, Kuhnisch J, Maritzen T, et al. Cohen syndrome-associated protein, COH1, is a novel, giant Golgi matrix protein required for Golgi integrity. J Biol Chem. 2011;286(43):37665–75.
Seifert W, Kuhnisch J, Maritzen T, et al. Cohen syndrome-associated protein COH1 physically and functionally interacts with the small GTPase RAB6 at the Golgi complex and directs neurite outgrowth. J Biol Chem. 2015;290(6):3349–58.
Chen X, Truong TT, Weaver J, et al. Intronic alterations in BRCA1 and BRCA2: effect on mRNA splicing fidelity and expression. Hum Mutat. 2006;27(5):427–35.
Seifert W, Holder-Espinasse M, Kuhnisch J, et al. Expanded mutational spectrum in Cohen syndrome, tissue expression, and transcript variants of COH1. Hum Mutat. 2009;30(2):E404–420.
El Chehadeh-Djebbar S, Blair E, Holder-Espinasse M, et al. Changing facial phenotype in Cohen syndrome: towards clues for an earlier diagnosis. Euro J Hum Genet. 2013;21(7):736–42.
Hurmerinta K, Pirinen S, Kovero O, et al. Craniofacial features in Cohen syndrome: an anthropometric and cephalometric analysis of 14 patients. Clin Genet. 2002;62(2):157–64.
Garcia-Ballesta C, Perez-Lajarin L, Lillo OC, et al. New oral findings in Cohen syndrome. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2003;95(6):681–7.
Kivitie-Kallio S, Norio R. Cohen syndrome: essential features, natural history, and heterogeneity. Am J Med Genet. 2001;102(2):125–35.
Howlin P, Karpf J, Turk J. Behavioural characteristics and autistic features in individuals with Cohen Syndrome. Eur Child Adoles Psychiatry. 2005;14(2):57–64.
Kivitie-Kallio S, Larsen A, Kajasto K, et al. Neurological and psychological findings in patients with Cohen syndrome: a study of 18 patients aged 11 months to 57 years. Neuropediatrics. 1999;30(4):181–9.
Yu TW, Chahrour MH, Coulter ME, et al. Using whole-exome sequencing to identify inherited causes of autism. Neuron. 2013;77 Suppl 2:259–73.
Douzgou S, Petersen MB. Clinical variability of genetic isolates of Cohen syndrome. Clin Genet. 2011;79(6):501–6.
Bedoyan JK, Kumar RA, Sudi J, et al. Duplication 16p11.2 in a child with infantile seizure disorder. Am J Med Genet A. 2010;152A(6):1567–74.
Schaaf CP, Goin-Kochel RP, Nowell KP, et al. Expanding the clinical spectrum of the 16p11.2 chromosomal rearrangements: three patients with syringomyelia. Eur J Hum Genet. 2011;19(2):152–6.
Filges I, Sparagana S, Sargent M, et al. Brain MRI abnormalities and spectrum of neurological and clinical findings in three patients with proximal 16p11.2 microduplication. Am J Med Genet A. 2014;164A(8):2003–12.
We would like to thank the families and the patients for their invaluable cooperation and participation.
This work was supported by funding from the Canadian Institutes for Health Research (CIHR) (Project Number: MOP-74502), BC Children’s Hospital Foundation (Project Number: KRZ75146) and BC Child and Family Research Institute (CFRI) (Project Number: 20R20410).
Availability of data and material
All relevant data are included in the manuscript and Additional file 1.
SML and ERS designed and initiated the study, monitored data collection and analysis for the study and revised the paper. JD contributed to the study design, analysed both clinical and WES data, implemented the technical methods, drafted and revised the paper. CC contributed to family recruitment, sample collection and genetic counselling. FT and YQ helped with utilizing Golden helix software. SM helped with technical parts. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Consent for publication of respective case presentations was obtained for each participant.
Ethics approval and consent to participate
The family was recruited through the B.C. Provincial Medical Genetics Program and Child & Family Research Institute of BC Children’s and Women’s Health Center. Ethics approval for clinical research involving human subjects was obtained through the Clinical Research Ethics Board of the University of British Columbia (Vancouver, B.C.). Samples from anonymized unaffected male and female individuals were used as controls for expression studies. Written informed consents were obtained from each participant.
Figure S1: Filtering strategies used for analysis of WES data; Additional material and methods. (DOCX 83 kb)