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NCAM2 deletion in a boy with macrocephaly and autism: Cause, association or predisposition?
European Journal of Medical Genetics 59 (2016) 493e498
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Original article
NCAM2 deletion in a boy with macrocephaly and autism: Cause, association or predisposition? €lzer a, Mandy Roy b, Caroline Scholz a, *, Doris Steinemann a, Madeleine Ma € rg Schmidtke a, Manfred Stuhrmann a Mine Arslan-Kirchner a, Thomas Illig a, c, Jo a b c
Institute of Human Genetics, Hannover Medical School, Hannover, Germany Psychiatric Clinic, Hannover Medical School, Hannover, Germany Hannover Unified Biobank, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 March 2016 Received in revised form 10 August 2016 Accepted 15 August 2016 Available online 2 September 2016
We report on an 8-year-old boy with autism spectrum disorder (ASD), speech delay, behavioural problems, disturbed sleep and macrosomia including macrocephaly carrying a microdeletion that contains the entire NCAM2 gene and no other functional genes. Other family members with the microdeletion show a large skull circumference but do not exhibit any symptoms of autism spectrum disorder. Among many ASD-candidate genes, NCAM2 has been assumed to play a pivotal role in the development of ASD because of its function in the outgrowth and bundling of neurites. Our reported case raises the questions whether the NCAM2-deletion is the true cause of the ASD or only a risk factor and whether there might be any connection in NCAM2 with skull-size Key words: autism spectrum disorder, macrocephaly, neural cell adhesion molecule 2 protein (NCAM2), array comparative genomic hybridization (microarray). © 2016 Elsevier Masson SAS. All rights reserved.
Keywords: Autism spectrum disorder Macrocephaly Neural cell adhesion Molecule 2 protein (NCAM2) Array comparative genomic hybridization (microarray)
1. Introduction Haldeman-Englert et al. have reported on an autistic male with a de novo deletion of 21q21.1-q21.3 as part of a complex rearrangement including the genes NCAM2 and GRIK1, amongst others. The authors concluded that their report underlines the former suggested role of these genes as possible candidate genes in autism and other neurobehavioral disorders (Haldeman-Englert et al., 2010). Additionally, Petit et al. have reported on three unrelated patients affected by global developmental delay, carrying a 21q21-deletion, which contained in two cases several additional genes, amongst them BTG3, and in one case no further coding genes in addition to NCAM2. The authors conclude that NCAM2 is a putative candidate gene for neurodevelopmental disorders (Petit et al., 2015). Copy number variations (CNV) and its affected genes are thought to be involved in several neurocognitive disorders. From the CNV morbidity map based on genomic data from more than 50.000 samples and controls, which has been recently published,
* Corresponding author. Institute of Human Genetics, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. E-mail address: [email protected] (C. Scholz). http://dx.doi.org/10.1016/j.ejmg.2016.08.006 1769-7212/© 2016 Elsevier Masson SAS. All rights reserved.
there is, however, no hint for a more frequent loss of NCAM2 in patients compared to controls (Coe et al., 2014). Autism is typically a multifactorial, multigenic disorder that affects the neurological development of patients. Autism spectrum disorder (ASD) encompasses different forms of autism with a broader phenotype. The prevalence of the whole autism spectrum is estimated at 1:167, with boys being four times more often affected than girls. In 10e25% of cases, genetic causes can be found (ORPHA106, OMIM, 209850). About one quarter of ASD patients appear to have a syndromic (complex) form (Miles, 2011). In 10e20% of non-syndromic patients, submicroscopic deletions or duplications can be found. Many different variants have been described, most of them with low frequency (Carter and Scherer, 2013). Here, we report on a boy with apparent non-syndromic ASD who presented with only minor dysmorphic features. As the cytogenetic analysis did not exhibit any pathologic findings, we performed microarray analysis and identified a 1.6-Mb deletion of 21q21.1-q21.2, containing the NCAM2 gene but no other functional gene.
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2. Clinical report
3. Materials and methods
The patient presented in our genetics clinic at eight years of age with autism spectrum disorder, speech delay, behavioural problems, disturbed sleep and macrocephaly. He is the only child of non-consanguineous healthy parents of Russian origin. The pregnancy was complicated by hyperemesis gravidarum. There is no history of maternal diabetes, any other illness of the mother or intake of any medication, drugs or alcohol during pregnancy. Our patient was born at 42 weeks of gestational age to his 29year-old mother by caesarean section because of the absence of a spontaneous delivery. His postnatal measurements of length, weight and head circumference exceeded the 97th centile (birth weight 4.700 g > 97th centile, height 59.0 cm > 97th centile, OFC 39.0 cm > 97th centile). Prenatal measurements were within the normal range according to the mother, however, the exact data is not available. Early in infancy, the mother recognized her son's withdrawn behaviour. According to the mother, “he lives in his own world”. He has difficulties playing with other children, although he interacts with his mother and is able to maintain eye contact. He frequently has a fit of rage. The mother also reported that, at the age of one year, the patient was temporarily able to speak some words but only began to talk properly at the age of three and a half years. His motor development was unremarkable, apart from slight fine motor problems. Because of his disturbed sleep pattern, a sleep EEG was performed, revealing biparietal-occipital dysrhythmic abnormalities. There is no history of seizures. A mild ASD was diagnosed according to the Autism Diagnostic Observation Schedule (ADOS). The boy's intelligence was also tested, and was just within the normal range. Furthermore, enuresis and encopresis had been diagnosed without any obvious organic findings. On physical examination, the patient was capable of interacting with the examiner. He showed mild macrosomia including distinct macrocephaly. His height and weight were just above the 97th centile, his head circumference was 1 cm (according to Russian growth charts (Dr. Mila Tsepkova)) to 3 cm (according to Nellhaus and Tanner) above the 97th centile (height 140.5 cm, weight 38.5 kg, OFC 57.6 cm), (CDC growthcharts, Nellhaus and Tanner, Russian growth charts (Dr. Mila Tsepkova) (Nellhaus, 1968; Tanner, 1978)). His predicted genetic height according to the parental measurements is 180.5 cm, which is the 50th centile. As the patient grows along the 97th centile, so far he grows above his predicted centile. Unfortunately there is no data on bone age available. He showed only mild dysmorphic features including a high forehead, slightly protruding ears with horizontal ear lobes, genua valga and a wide space between the first and second toes (Fig. 1). Measurement of the head circumference of his accompanying mother and his maternal grandmother revealed a large head circumference in both according to centiles by Bushby et al. (mother: OFC 57.6 cm 2 mm < 97th centile, grandmother: OFC 56.8 cm 2 mm < 97th centile). For details of family measurements see Table 1 The patient's father and the father's niece and nephew were reported by the mother to show similar behavioural problems, but none of them was available for psychological testing. Three male maternal blood relatives were reported to have speech problems, but we were unable to examine them ourselves, because they live in Russia (see pedigree Fig. 2). After chromosomal analysis revealed a normal male karyotype 46,XY, and testing for Fragile X syndrome and Macrocephaly/ Autism-Syndrome (PTEN sequencing and MLPA analysis) did not reveal any abnormalities, no other specific diagnosis could be made and microarray analysis was performed.
3.1. Array-CGH Array-CGH was performed using the Agilent Human Genome Microarray Kits 2 400 K (Agilent Technologies, Santa Clara, CA, USA), a high resolution 60-mer oligonucleotide-based microarray with median overall probe spacing of about 5.3 kb. Labeling and hybridization of genomic DNA was performed according to the protocol provided by Agilent, using the Agilent Genomic DNA Labeling Kit Plus. Purified labeled and mixed products were hybridized to Agilent's 2 400 K Human Genome CGH microarray at 65 C with 20 rpm rotation for 24 h. Washing steps were performed according to the Agilent protocol. Microarray slides were scanned immediately using an Agilent microarray scanner G2505B at a resolution of 2 mm. For image analysis, default CGH settings of Feature Extraction Software (Agilent Technologies, Waldbronn, Germany) were applied. Output files from Feature Extraction were subsequently imported into Agilent's CGH data analysis software, Genomic Workbench. The aberration algorithm ADM2 was applied and aberration filters were set to: threshold 5.0, at least four probes with mean log2 ratio of 0.5 leading to a resolution of approx. 20 kb. 3.2. Multiplex ligation-dependent probe amplification (MLPA) MLPA analysis was performed using the SALSA MLPA P095-A3 Aneuploidy probe mix (MRC-Holland, Amsterdam, Netherlands) according to the manufacturer's MLPA General Protocol. For data analysis, we focused on the SALSA MLPA probe 00813-L00636, which is located just inside the deleted region 21q21.1. Samples from our index patient, both parents, and six maternal relatives were analysed (Fig. 2). The father and an unrelated person, both tested negative for the deletion by microarray analysis, were used as negative controls. Our patient and his mother were considered positive controls. 3.3. Sequencing analysis The patient's second NCAM2 allele was amplified and all coding regions and adjacent intronic regions were sequenced using standard methods (NCBI RefSeq NM_004540.3). Sequencing was performed using the ABI PRISM 3130xl Genetic Analyzer (Life Technologies GmbH, Darmstadt, Germany). For data analysis, Sequence Pilot (JSI medical systems GmbH, Kippenheim, Germany) was used. The used NCAM2 primers are available upon request. 3.4. Psychological testing ASD in adulthood was tested by using a semi-structured interview that thoroughly explores the DSM-IV criteria of autism (Diagnostic and Statistical Manual of Mental Disorders (1994)). The interviews were conducted by an experienced investigator. An inhouse interpreter was present for the interview with the Russian grandmother. 4. Results 4.1. Microarray analysis and sequencing analysis Array-CGH analysis was performed on the patient, his mother and his father. A heterozygous 1.6 Mb deletion on 21q21.1-q21.2 (22444986-24047363) was detected in our patient (listed in Decipher #231386). This deleted region ranged from 22.444 to 24.047 Mb and contained the entire NCAM2 gene and two long
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Fig. 1. Patient at the age of eight years and four months. The patient was of tall stature and heavy weight. He showed only mild dysmorphic features including a macrocephaly, a high forehead, slightly protruding ears with horizontal ear lobes, genua valga and a wide space between the first and second toes.
Table 1 Family measurements. Height, head circumference (OFC) and individual centiles of the OFC for all adults are listed according to the centiles by Bushby et al. (Bushby et al., 1992). N.a. (not available). Measurements for probands III:2, I:2 and II:6 were carried out by the patients themselves. Person
Height [cm]
OFCa
Centile OFC
NCAM2 deletion
IV:5 III:3 III:2 II:4 I:2 II:6
140.5 164.0 185.0 154.4 147.0 151.0
57.6 57.6 n.a.b 56.8 53.0 55.0
1 cm > 97th 2 mm < 97th
Yes Yes No Yes No No
a b
2 mm < 97th 2 mm > 25th 1 mm < 75th
Head circumference. Not available.
intergenic non-protein coding RNAs (LINC00317 and LINC00308). The patient's mother, but not his father, also carried this microdeletion, proving that this was a maternally derived deletion (Fig. 3). Sequencing of the entire NCAM2 allele showed no mutations leading to the exclusion of compound heterozygosity of our patient.
4.2. MLPA analysis As expected, the NCAM2 deletion was verified by MLPA analysis in the patient and his mother, indicated by a reduced peak solely of the respective probe but not of the other 35 probes. Using MLPA, the deletion was also observed in the maternal grandmother of our patient. The other tested maternal relatives, namely the greatgrandmother, the sister of the grandmother, her two daughters and the son of one of the daughters did not carry the deletion, according to MLPA analysis (Fig. 2).
4.3. Psychological testing Since the mother and maternal grandmother of our patient also carried the NCAM2 deletion, we initiated a psychological test to rule out minor symptoms of autism spectrum disorder in both of them. The patient's mother and maternal grandmother showed no autistic symptoms. They had no problems in social interaction and communication and had no special interests or stereotype behaviour. We, therefore, excluded an Asperger syndrome in the patient's mother and maternal grandmother. 5. Discussion Autism spectrum disorder (ASD, OMIM 209850) encompasses different forms of autism with a broader phenotype. Among the genes involved, NCAM2 has been assumed to play a role in the development of ASD because of its function in the outgrowth and bundling of neurites. NCAM2 (Neural cell adhesion molecule 2) also known as OCAM (olfactory cell adhesion molecule) is a member of the immunoglobulin superfamily. Through homophilic transinteractions NCAM2 mediates cell-cell adhesion, and probably also mediates cell-cell repulsion via unknown heterophilic interactions and modulates intracellular signaling cascades through cis activation (Winther et al., 2012). Ncam2 (Ocam)-knockout-mice do not show any evidence of behavioural abnormalities and their sense of olfactory acuity is increased. Furthermore, Ncam2 (Ocam)knockout mice reproduce normally and are indistinguishable from their wild-type littermates (Walz et al., 2006). There is a second member of the NCAM family, the paralog (possibly ohnolog) NCAM1. Ncam1-deficient mice do show impairment of learning abilities and exploratory behaviour (Cremer et al., 1994). Unlike in mice, in humans there is evidence of a causal connection between the NCAM2 region and neurobehavioral disorders such as autism (Hussman et al., 2011). A linkage to 21q in
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Fig. 2. Pedigree diagram depicting the patient and other affected family members. Pedigree of the patient's family, arrow indicates patient.
Fig. 3. Array-CGH analysis showing NCAM2-microdeletion. A: Results of the array-CGH analysis: Arr 21q21.1-q21.2(22,444,986-24,047,363)x1 mat. Genomic profiles of chromosome 21 are shown below the ideogram of chromosome 21: patient (lower profile), mother (middle profile) and father (upper profile); B: zoom of deleted region to gene view (according to hg19); C: Copy number variations according to DGV with losses in red, gains in blue.
children with ASD and a history of developmental regression has been reported previously (Molloy et al., 2005). There has been a case report of an autistic boy with an 8.8 Mb microdeletion involving 19 genes, including NCAM2 and another autism-related candidate gene, GRIK1 (Haldeman-Englert et al., 2010). A second report on three unrelated patients affected by global developmental delay has also detailed 21q21-deletions containing NCAM2 (Petit et al., 2015). In two of the reported cases BTG3, which has been reported to play a role in neurogenesis in the central nervous system, has been amongst several additional genes included in the
deletion (Tirone, 2001). In addition, Decipher and dbVar list six cases with smaller deletions containing NCAM2 (#289444, #289445, #nsv531520 and the three cases reported by Petit et al., #254181, #274603 and # 276325); These cases all share a global developmental delay or intellectual disability. Macrocephaly has not been reported in these cases, but short stature was noticed in one case (Haldeman-Englert et al., 2010; Bacrot et al., 2015). Other reported patients carried even larger deletions (Ahlbom et al., 1996; Korenberg et al., 1991; Takhar et al., 2002; Wakui et al., 2002). In fact, there has been only one other report on a patient with a
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small deletion exclusively containing the NCAM2 gene as the sole coding gene, as seen in our patient. This patient, reported by Petit et al., (Petit et al., 2015)., and our patient share developmental delay and impaired social interaction. Furthermore, our patient and the patient reported by Haldeman-Englert et al. share many features such as ASD, speech defect, behavioural problems and a large head circumference. Which role the two intergenic long non-coding RNAs contained in the deletion of our patient may play in the development of the symptoms is unclear to date. Our case seems to support the assumption made by other authors that similar to other cell adhesion protein defects NCAM2 deficiency plays a role in the development of neurobehavioral disorders such as autism and speech defects (Lopez-Hernandez et al., 2011). However, our patient's mother, who also carries the deletion, did not show any symptoms of ASD. Her mother was also a carrier of the deletion and was likewise not affected by ASD. Moreover, the maternal second degree cousin, who was tested by MLPA and who was reported to be affected by a speech defect, was not a carrier of the deletion. Hence, there are at least two family members who did not develop any signs of ASD although carrying the deletion. Also, a link between the origins of our patient's symptoms with the origins of his second degree cousin's symptoms cannot be made. We must, therefore, question whether the NCAM2deletion in our case (and other cases reported) is really causative of the ASD or whether it only functions as a risk factor in the sense that there must be additional genetic and/or non-genetic factors that lead to the presentation of clinical symptoms in our patient. Possible explanations include variable expression or decreased penetrance of the NCAM2 deletion. ASD and the other features of our patient, could even be unlinked to NCAM2, and could represent, for example, an X-linked disorder, a de novo mutation or a paternally derived alteration. In the literature, many non-syndromic ASD patients with parentally derived microdeletions in different loci have been described. In many of these cases, the carrier parent has not been reported to show any clinical signs of autism (Carter and Scherer, 2013). Together with our case, these findings provide evidence for a multigene-multifactorial genesis of ASD, with NCAM2 being a predisposing or associated factor (Muhle et al., 2004). There is, however, no proof yet for a true causality of an NCAM2-deletion and ASD. Our patient (see pedigree IV:5), his mother (see pedigree III:3), his maternal grandmother (see pedigree II:4) and the previously published patient shared a large head circumference as a common symptom. On our request, the great-grandmother (see pedigree I:2) and the great-aunt (see pedigree II:6), mother and sister of the maternal grandmother, measured their head circumferences as well. As they live in Russia, we were unable to confirm their measurements. They both did not carry the microdeletion and they both, according to their latest own measurements, did not exhibit a large head circumference (Centile OFC great-grandmother 2 mm > 25th, Centile OFC aunt 1 mm < 75th, see Table 1 and Fig. 2). We cannot be sure, whether there is a causality of the macrocephaly of our patient and the large head circumferences measured in other family members and the NCAM2 deletion. The macrocephaly could be part of a constitutional macrosomia of our patient, although there is only mild overgrowth for height and weight whereas there is a distinct overgrowth of the head. Furthermore a large OFC might segregate in this family independently, or our patient might suffer from a complete different disease causing macrocephaly and ASD. Particularly because of non-confirmed measurements of the Russian family members and because of the missing co-incidence of macrocephaly and ASD in all family members carrying the NCAM2-deletion we cannot exclude these possibilities. However, a link between a large sized head circumference and the NCAM2 deletion cannot entirely be ruled out, as all
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family members containing the deletion and one previously published patient have a rather large head circumference. NCAM2 is a cell adhesion molecule and is assumed to play a role in the outgrowth of neurites. There is evidence that the dysregulation of the cellular growth of neuronal elements leads to megalencephaly, which is a possible cause of clinical macrocephaly (Mirzaa and Poduri, 2014). A different cell adhesion molecule, GlialCAM, has previously been described as a cause of macrocephaly. Lopez-Hernandez et al. have described dominant GlialCAM mutations in patients with benign familial macrocephaly and other specific mutations in a clinical syndrome of macrocephaly and mental retardation with or without autism (Lopez-Hernandez et al., 2011; Favre-Kontula et al., 2008). Since both GlialCAM and NCAM2 are cell adhesion molecules and are assumed to both play a role in the outgrowth of neurites, there might be overlapping functions, potentially associated with the development of macrocephaly. In a large genome-wide association study in children there has been no evidence that would support a role of NCAM2 in the normal variation of head circumference (Taal et al., 2012). This adds weight to our assumption that a potential NCAM2-associated macrocephaly could be caused by a specific pathomechanism. However, not all reported patients with NCAM2-deletions show macrocephaly. In conclusion, our reported case provides additional evidence for the association of NCAM2 and the development of ASD. However, we must question whether the NCAM2-deletion in our case (and other cases reported) is really causative of the ASD or whether it only reveals a predisposing or risk factor for the ASD. In addition, our case raises the question whether there might be a connection in NCAM2 with skull-size In order to gain more knowledge about the clinical relevance of (NCAM2-containing) copy number alterations in ASD it is important to perform further comprehensive molecular and phenotypical characterization, including thorough measurements of patients and families. As is probably true for many other cases, the interpretation of the genetic test results appeared to be rather complex in our patient. Our family exemplifies the challenges of relating genetic findings to phenotypic features and vice versa. Therefore, one should conclude advisedly on microarray-results concerning the causality for any clinical symptoms found. Conflict of interest The authors declare no conflict of interest. Web resources Orphanet www.orpha.net. Decipher https://decipher.sanger.ac.uk/ dbVar http://www.ncbi.nlm.nih.gov/dbvar/variants/ DGV http://projects.tcag.ca/variation/ CDC http://www.cdc.gov/growthcharts/clinical_charts.htm. Russian Growthcharts, Dr. Mila Tsepkova, found at http:// adoption.squarespace.com/topics/growth-charts.html. Acknowledgments The authors would like to thank the patient and his family for their participation and Claudia Davenport for helping to prepare the manuscript. References Ahlbom, B.E., Sidenvall, R., Anneren, G., 1996. Deletion of chromosome 21 in a girl with congenital hypothyroidism and mild mental retardation. Am. J. Med.
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European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Original article
NCAM2 deletion in a boy with macrocephaly and autism: Cause, association or predisposition? €lzer a, Mandy Roy b, Caroline Scholz a, *, Doris Steinemann a, Madeleine Ma € rg Schmidtke a, Manfred Stuhrmann a Mine Arslan-Kirchner a, Thomas Illig a, c, Jo a b c
Institute of Human Genetics, Hannover Medical School, Hannover, Germany Psychiatric Clinic, Hannover Medical School, Hannover, Germany Hannover Unified Biobank, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 March 2016 Received in revised form 10 August 2016 Accepted 15 August 2016 Available online 2 September 2016
We report on an 8-year-old boy with autism spectrum disorder (ASD), speech delay, behavioural problems, disturbed sleep and macrosomia including macrocephaly carrying a microdeletion that contains the entire NCAM2 gene and no other functional genes. Other family members with the microdeletion show a large skull circumference but do not exhibit any symptoms of autism spectrum disorder. Among many ASD-candidate genes, NCAM2 has been assumed to play a pivotal role in the development of ASD because of its function in the outgrowth and bundling of neurites. Our reported case raises the questions whether the NCAM2-deletion is the true cause of the ASD or only a risk factor and whether there might be any connection in NCAM2 with skull-size Key words: autism spectrum disorder, macrocephaly, neural cell adhesion molecule 2 protein (NCAM2), array comparative genomic hybridization (microarray). © 2016 Elsevier Masson SAS. All rights reserved.
Keywords: Autism spectrum disorder Macrocephaly Neural cell adhesion Molecule 2 protein (NCAM2) Array comparative genomic hybridization (microarray)
1. Introduction Haldeman-Englert et al. have reported on an autistic male with a de novo deletion of 21q21.1-q21.3 as part of a complex rearrangement including the genes NCAM2 and GRIK1, amongst others. The authors concluded that their report underlines the former suggested role of these genes as possible candidate genes in autism and other neurobehavioral disorders (Haldeman-Englert et al., 2010). Additionally, Petit et al. have reported on three unrelated patients affected by global developmental delay, carrying a 21q21-deletion, which contained in two cases several additional genes, amongst them BTG3, and in one case no further coding genes in addition to NCAM2. The authors conclude that NCAM2 is a putative candidate gene for neurodevelopmental disorders (Petit et al., 2015). Copy number variations (CNV) and its affected genes are thought to be involved in several neurocognitive disorders. From the CNV morbidity map based on genomic data from more than 50.000 samples and controls, which has been recently published,
* Corresponding author. Institute of Human Genetics, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. E-mail address: [email protected] (C. Scholz). http://dx.doi.org/10.1016/j.ejmg.2016.08.006 1769-7212/© 2016 Elsevier Masson SAS. All rights reserved.
there is, however, no hint for a more frequent loss of NCAM2 in patients compared to controls (Coe et al., 2014). Autism is typically a multifactorial, multigenic disorder that affects the neurological development of patients. Autism spectrum disorder (ASD) encompasses different forms of autism with a broader phenotype. The prevalence of the whole autism spectrum is estimated at 1:167, with boys being four times more often affected than girls. In 10e25% of cases, genetic causes can be found (ORPHA106, OMIM, 209850). About one quarter of ASD patients appear to have a syndromic (complex) form (Miles, 2011). In 10e20% of non-syndromic patients, submicroscopic deletions or duplications can be found. Many different variants have been described, most of them with low frequency (Carter and Scherer, 2013). Here, we report on a boy with apparent non-syndromic ASD who presented with only minor dysmorphic features. As the cytogenetic analysis did not exhibit any pathologic findings, we performed microarray analysis and identified a 1.6-Mb deletion of 21q21.1-q21.2, containing the NCAM2 gene but no other functional gene.
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2. Clinical report
3. Materials and methods
The patient presented in our genetics clinic at eight years of age with autism spectrum disorder, speech delay, behavioural problems, disturbed sleep and macrocephaly. He is the only child of non-consanguineous healthy parents of Russian origin. The pregnancy was complicated by hyperemesis gravidarum. There is no history of maternal diabetes, any other illness of the mother or intake of any medication, drugs or alcohol during pregnancy. Our patient was born at 42 weeks of gestational age to his 29year-old mother by caesarean section because of the absence of a spontaneous delivery. His postnatal measurements of length, weight and head circumference exceeded the 97th centile (birth weight 4.700 g > 97th centile, height 59.0 cm > 97th centile, OFC 39.0 cm > 97th centile). Prenatal measurements were within the normal range according to the mother, however, the exact data is not available. Early in infancy, the mother recognized her son's withdrawn behaviour. According to the mother, “he lives in his own world”. He has difficulties playing with other children, although he interacts with his mother and is able to maintain eye contact. He frequently has a fit of rage. The mother also reported that, at the age of one year, the patient was temporarily able to speak some words but only began to talk properly at the age of three and a half years. His motor development was unremarkable, apart from slight fine motor problems. Because of his disturbed sleep pattern, a sleep EEG was performed, revealing biparietal-occipital dysrhythmic abnormalities. There is no history of seizures. A mild ASD was diagnosed according to the Autism Diagnostic Observation Schedule (ADOS). The boy's intelligence was also tested, and was just within the normal range. Furthermore, enuresis and encopresis had been diagnosed without any obvious organic findings. On physical examination, the patient was capable of interacting with the examiner. He showed mild macrosomia including distinct macrocephaly. His height and weight were just above the 97th centile, his head circumference was 1 cm (according to Russian growth charts (Dr. Mila Tsepkova)) to 3 cm (according to Nellhaus and Tanner) above the 97th centile (height 140.5 cm, weight 38.5 kg, OFC 57.6 cm), (CDC growthcharts, Nellhaus and Tanner, Russian growth charts (Dr. Mila Tsepkova) (Nellhaus, 1968; Tanner, 1978)). His predicted genetic height according to the parental measurements is 180.5 cm, which is the 50th centile. As the patient grows along the 97th centile, so far he grows above his predicted centile. Unfortunately there is no data on bone age available. He showed only mild dysmorphic features including a high forehead, slightly protruding ears with horizontal ear lobes, genua valga and a wide space between the first and second toes (Fig. 1). Measurement of the head circumference of his accompanying mother and his maternal grandmother revealed a large head circumference in both according to centiles by Bushby et al. (mother: OFC 57.6 cm 2 mm < 97th centile, grandmother: OFC 56.8 cm 2 mm < 97th centile). For details of family measurements see Table 1 The patient's father and the father's niece and nephew were reported by the mother to show similar behavioural problems, but none of them was available for psychological testing. Three male maternal blood relatives were reported to have speech problems, but we were unable to examine them ourselves, because they live in Russia (see pedigree Fig. 2). After chromosomal analysis revealed a normal male karyotype 46,XY, and testing for Fragile X syndrome and Macrocephaly/ Autism-Syndrome (PTEN sequencing and MLPA analysis) did not reveal any abnormalities, no other specific diagnosis could be made and microarray analysis was performed.
3.1. Array-CGH Array-CGH was performed using the Agilent Human Genome Microarray Kits 2 400 K (Agilent Technologies, Santa Clara, CA, USA), a high resolution 60-mer oligonucleotide-based microarray with median overall probe spacing of about 5.3 kb. Labeling and hybridization of genomic DNA was performed according to the protocol provided by Agilent, using the Agilent Genomic DNA Labeling Kit Plus. Purified labeled and mixed products were hybridized to Agilent's 2 400 K Human Genome CGH microarray at 65 C with 20 rpm rotation for 24 h. Washing steps were performed according to the Agilent protocol. Microarray slides were scanned immediately using an Agilent microarray scanner G2505B at a resolution of 2 mm. For image analysis, default CGH settings of Feature Extraction Software (Agilent Technologies, Waldbronn, Germany) were applied. Output files from Feature Extraction were subsequently imported into Agilent's CGH data analysis software, Genomic Workbench. The aberration algorithm ADM2 was applied and aberration filters were set to: threshold 5.0, at least four probes with mean log2 ratio of 0.5 leading to a resolution of approx. 20 kb. 3.2. Multiplex ligation-dependent probe amplification (MLPA) MLPA analysis was performed using the SALSA MLPA P095-A3 Aneuploidy probe mix (MRC-Holland, Amsterdam, Netherlands) according to the manufacturer's MLPA General Protocol. For data analysis, we focused on the SALSA MLPA probe 00813-L00636, which is located just inside the deleted region 21q21.1. Samples from our index patient, both parents, and six maternal relatives were analysed (Fig. 2). The father and an unrelated person, both tested negative for the deletion by microarray analysis, were used as negative controls. Our patient and his mother were considered positive controls. 3.3. Sequencing analysis The patient's second NCAM2 allele was amplified and all coding regions and adjacent intronic regions were sequenced using standard methods (NCBI RefSeq NM_004540.3). Sequencing was performed using the ABI PRISM 3130xl Genetic Analyzer (Life Technologies GmbH, Darmstadt, Germany). For data analysis, Sequence Pilot (JSI medical systems GmbH, Kippenheim, Germany) was used. The used NCAM2 primers are available upon request. 3.4. Psychological testing ASD in adulthood was tested by using a semi-structured interview that thoroughly explores the DSM-IV criteria of autism (Diagnostic and Statistical Manual of Mental Disorders (1994)). The interviews were conducted by an experienced investigator. An inhouse interpreter was present for the interview with the Russian grandmother. 4. Results 4.1. Microarray analysis and sequencing analysis Array-CGH analysis was performed on the patient, his mother and his father. A heterozygous 1.6 Mb deletion on 21q21.1-q21.2 (22444986-24047363) was detected in our patient (listed in Decipher #231386). This deleted region ranged from 22.444 to 24.047 Mb and contained the entire NCAM2 gene and two long
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Fig. 1. Patient at the age of eight years and four months. The patient was of tall stature and heavy weight. He showed only mild dysmorphic features including a macrocephaly, a high forehead, slightly protruding ears with horizontal ear lobes, genua valga and a wide space between the first and second toes.
Table 1 Family measurements. Height, head circumference (OFC) and individual centiles of the OFC for all adults are listed according to the centiles by Bushby et al. (Bushby et al., 1992). N.a. (not available). Measurements for probands III:2, I:2 and II:6 were carried out by the patients themselves. Person
Height [cm]
OFCa
Centile OFC
NCAM2 deletion
IV:5 III:3 III:2 II:4 I:2 II:6
140.5 164.0 185.0 154.4 147.0 151.0
57.6 57.6 n.a.b 56.8 53.0 55.0
1 cm > 97th 2 mm < 97th
Yes Yes No Yes No No
a b
2 mm < 97th 2 mm > 25th 1 mm < 75th
Head circumference. Not available.
intergenic non-protein coding RNAs (LINC00317 and LINC00308). The patient's mother, but not his father, also carried this microdeletion, proving that this was a maternally derived deletion (Fig. 3). Sequencing of the entire NCAM2 allele showed no mutations leading to the exclusion of compound heterozygosity of our patient.
4.2. MLPA analysis As expected, the NCAM2 deletion was verified by MLPA analysis in the patient and his mother, indicated by a reduced peak solely of the respective probe but not of the other 35 probes. Using MLPA, the deletion was also observed in the maternal grandmother of our patient. The other tested maternal relatives, namely the greatgrandmother, the sister of the grandmother, her two daughters and the son of one of the daughters did not carry the deletion, according to MLPA analysis (Fig. 2).
4.3. Psychological testing Since the mother and maternal grandmother of our patient also carried the NCAM2 deletion, we initiated a psychological test to rule out minor symptoms of autism spectrum disorder in both of them. The patient's mother and maternal grandmother showed no autistic symptoms. They had no problems in social interaction and communication and had no special interests or stereotype behaviour. We, therefore, excluded an Asperger syndrome in the patient's mother and maternal grandmother. 5. Discussion Autism spectrum disorder (ASD, OMIM 209850) encompasses different forms of autism with a broader phenotype. Among the genes involved, NCAM2 has been assumed to play a role in the development of ASD because of its function in the outgrowth and bundling of neurites. NCAM2 (Neural cell adhesion molecule 2) also known as OCAM (olfactory cell adhesion molecule) is a member of the immunoglobulin superfamily. Through homophilic transinteractions NCAM2 mediates cell-cell adhesion, and probably also mediates cell-cell repulsion via unknown heterophilic interactions and modulates intracellular signaling cascades through cis activation (Winther et al., 2012). Ncam2 (Ocam)-knockout-mice do not show any evidence of behavioural abnormalities and their sense of olfactory acuity is increased. Furthermore, Ncam2 (Ocam)knockout mice reproduce normally and are indistinguishable from their wild-type littermates (Walz et al., 2006). There is a second member of the NCAM family, the paralog (possibly ohnolog) NCAM1. Ncam1-deficient mice do show impairment of learning abilities and exploratory behaviour (Cremer et al., 1994). Unlike in mice, in humans there is evidence of a causal connection between the NCAM2 region and neurobehavioral disorders such as autism (Hussman et al., 2011). A linkage to 21q in
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Fig. 2. Pedigree diagram depicting the patient and other affected family members. Pedigree of the patient's family, arrow indicates patient.
Fig. 3. Array-CGH analysis showing NCAM2-microdeletion. A: Results of the array-CGH analysis: Arr 21q21.1-q21.2(22,444,986-24,047,363)x1 mat. Genomic profiles of chromosome 21 are shown below the ideogram of chromosome 21: patient (lower profile), mother (middle profile) and father (upper profile); B: zoom of deleted region to gene view (according to hg19); C: Copy number variations according to DGV with losses in red, gains in blue.
children with ASD and a history of developmental regression has been reported previously (Molloy et al., 2005). There has been a case report of an autistic boy with an 8.8 Mb microdeletion involving 19 genes, including NCAM2 and another autism-related candidate gene, GRIK1 (Haldeman-Englert et al., 2010). A second report on three unrelated patients affected by global developmental delay has also detailed 21q21-deletions containing NCAM2 (Petit et al., 2015). In two of the reported cases BTG3, which has been reported to play a role in neurogenesis in the central nervous system, has been amongst several additional genes included in the
deletion (Tirone, 2001). In addition, Decipher and dbVar list six cases with smaller deletions containing NCAM2 (#289444, #289445, #nsv531520 and the three cases reported by Petit et al., #254181, #274603 and # 276325); These cases all share a global developmental delay or intellectual disability. Macrocephaly has not been reported in these cases, but short stature was noticed in one case (Haldeman-Englert et al., 2010; Bacrot et al., 2015). Other reported patients carried even larger deletions (Ahlbom et al., 1996; Korenberg et al., 1991; Takhar et al., 2002; Wakui et al., 2002). In fact, there has been only one other report on a patient with a
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small deletion exclusively containing the NCAM2 gene as the sole coding gene, as seen in our patient. This patient, reported by Petit et al., (Petit et al., 2015)., and our patient share developmental delay and impaired social interaction. Furthermore, our patient and the patient reported by Haldeman-Englert et al. share many features such as ASD, speech defect, behavioural problems and a large head circumference. Which role the two intergenic long non-coding RNAs contained in the deletion of our patient may play in the development of the symptoms is unclear to date. Our case seems to support the assumption made by other authors that similar to other cell adhesion protein defects NCAM2 deficiency plays a role in the development of neurobehavioral disorders such as autism and speech defects (Lopez-Hernandez et al., 2011). However, our patient's mother, who also carries the deletion, did not show any symptoms of ASD. Her mother was also a carrier of the deletion and was likewise not affected by ASD. Moreover, the maternal second degree cousin, who was tested by MLPA and who was reported to be affected by a speech defect, was not a carrier of the deletion. Hence, there are at least two family members who did not develop any signs of ASD although carrying the deletion. Also, a link between the origins of our patient's symptoms with the origins of his second degree cousin's symptoms cannot be made. We must, therefore, question whether the NCAM2deletion in our case (and other cases reported) is really causative of the ASD or whether it only functions as a risk factor in the sense that there must be additional genetic and/or non-genetic factors that lead to the presentation of clinical symptoms in our patient. Possible explanations include variable expression or decreased penetrance of the NCAM2 deletion. ASD and the other features of our patient, could even be unlinked to NCAM2, and could represent, for example, an X-linked disorder, a de novo mutation or a paternally derived alteration. In the literature, many non-syndromic ASD patients with parentally derived microdeletions in different loci have been described. In many of these cases, the carrier parent has not been reported to show any clinical signs of autism (Carter and Scherer, 2013). Together with our case, these findings provide evidence for a multigene-multifactorial genesis of ASD, with NCAM2 being a predisposing or associated factor (Muhle et al., 2004). There is, however, no proof yet for a true causality of an NCAM2-deletion and ASD. Our patient (see pedigree IV:5), his mother (see pedigree III:3), his maternal grandmother (see pedigree II:4) and the previously published patient shared a large head circumference as a common symptom. On our request, the great-grandmother (see pedigree I:2) and the great-aunt (see pedigree II:6), mother and sister of the maternal grandmother, measured their head circumferences as well. As they live in Russia, we were unable to confirm their measurements. They both did not carry the microdeletion and they both, according to their latest own measurements, did not exhibit a large head circumference (Centile OFC great-grandmother 2 mm > 25th, Centile OFC aunt 1 mm < 75th, see Table 1 and Fig. 2). We cannot be sure, whether there is a causality of the macrocephaly of our patient and the large head circumferences measured in other family members and the NCAM2 deletion. The macrocephaly could be part of a constitutional macrosomia of our patient, although there is only mild overgrowth for height and weight whereas there is a distinct overgrowth of the head. Furthermore a large OFC might segregate in this family independently, or our patient might suffer from a complete different disease causing macrocephaly and ASD. Particularly because of non-confirmed measurements of the Russian family members and because of the missing co-incidence of macrocephaly and ASD in all family members carrying the NCAM2-deletion we cannot exclude these possibilities. However, a link between a large sized head circumference and the NCAM2 deletion cannot entirely be ruled out, as all
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family members containing the deletion and one previously published patient have a rather large head circumference. NCAM2 is a cell adhesion molecule and is assumed to play a role in the outgrowth of neurites. There is evidence that the dysregulation of the cellular growth of neuronal elements leads to megalencephaly, which is a possible cause of clinical macrocephaly (Mirzaa and Poduri, 2014). A different cell adhesion molecule, GlialCAM, has previously been described as a cause of macrocephaly. Lopez-Hernandez et al. have described dominant GlialCAM mutations in patients with benign familial macrocephaly and other specific mutations in a clinical syndrome of macrocephaly and mental retardation with or without autism (Lopez-Hernandez et al., 2011; Favre-Kontula et al., 2008). Since both GlialCAM and NCAM2 are cell adhesion molecules and are assumed to both play a role in the outgrowth of neurites, there might be overlapping functions, potentially associated with the development of macrocephaly. In a large genome-wide association study in children there has been no evidence that would support a role of NCAM2 in the normal variation of head circumference (Taal et al., 2012). This adds weight to our assumption that a potential NCAM2-associated macrocephaly could be caused by a specific pathomechanism. However, not all reported patients with NCAM2-deletions show macrocephaly. In conclusion, our reported case provides additional evidence for the association of NCAM2 and the development of ASD. However, we must question whether the NCAM2-deletion in our case (and other cases reported) is really causative of the ASD or whether it only reveals a predisposing or risk factor for the ASD. In addition, our case raises the question whether there might be a connection in NCAM2 with skull-size In order to gain more knowledge about the clinical relevance of (NCAM2-containing) copy number alterations in ASD it is important to perform further comprehensive molecular and phenotypical characterization, including thorough measurements of patients and families. As is probably true for many other cases, the interpretation of the genetic test results appeared to be rather complex in our patient. Our family exemplifies the challenges of relating genetic findings to phenotypic features and vice versa. Therefore, one should conclude advisedly on microarray-results concerning the causality for any clinical symptoms found. Conflict of interest The authors declare no conflict of interest. Web resources Orphanet www.orpha.net. Decipher https://decipher.sanger.ac.uk/ dbVar http://www.ncbi.nlm.nih.gov/dbvar/variants/ DGV http://projects.tcag.ca/variation/ CDC http://www.cdc.gov/growthcharts/clinical_charts.htm. Russian Growthcharts, Dr. Mila Tsepkova, found at http:// adoption.squarespace.com/topics/growth-charts.html. Acknowledgments The authors would like to thank the patient and his family for their participation and Claudia Davenport for helping to prepare the manuscript. References Ahlbom, B.E., Sidenvall, R., Anneren, G., 1996. Deletion of chromosome 21 in a girl with congenital hypothyroidism and mild mental retardation. Am. J. Med.
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