Deletions of NRXN1 (neurexin-1) predispose to a wide spectrum of developmental disorders

Structure and Function of NRXN1

NRXN1, located on chromosome 2p16.3, is one of the largest known human genes (1.1 Mb with 24 exons) [Tabuchi and Sudhof 2002]. It is subject to relatively frequent disruption including missense changes, translocation, whole gene deletion and intragenic copy number alterations [Feng et al. 2006; Glessner et al. 2009; International Schizophrenia Consortium 2008; Kim et al. 2008; Kirov et al. 2008; Marshall et al. 2008; Morrow et al. 2008; Rujescu et al. 2009; Szatmari et al. 2007; Yan et al. 2008; Zahir et al. 2008].

The longer transcript, NRXN1-α, encodes an N-terminal signal peptide with three repeats of two laminin/neurexin/sex hormone-binding globulin (LNS) domains separated by an EGF-like sequence (Figure 1). Following these repeats, there is an O-glycosylation sequence, a transmembrane domain, and a cytoplasmic tail of 55 amino acids.

Neurexin 1-α has been shown to interact with certain neuroligin isoforms and neurexin-binding proteins known as neurexophilins. This pre-synaptic molecule is also required for calcium-triggered neurotransmitter release and the function of voltage-gated calcium channels in the synapses of the brainstem and neocortex [Dudanova et al. 2006; Missler et al. 2003; Zhang et al. 2005]. Mouse knockouts of all three α-neurexin genes do not demonstrate major abnormalities of axonal pathfinding during development [Dudanova et al. 2007], although synaptic function is severely impaired. Mice with knockouts of individual α-neurexin genes have modestly decreased post-natal viability, while double knockout mice have greatly decreased postnatal survival. Triple knockout mice do not survive past the first day of life [Missler et al. 2003].

Neurexin1-β is much shorter than Neurexin 1-α, as five of the six LNS domains and the intervening EGF sequences are replaced with a short β-neurexin specific sequence (Figure 1) [Missler and Sudhof 1998]. Neurexin 1-β has been shown to interact with the post-synaptic neuroligin family of cell adhesion molecules and dystroglycans [Arac et al. 2007; Chen et al. 2008; Comoletti et al. 2007; Ichtchenko et al. 1995; Sugita et al. 2001]. No mouse models with knockouts of NRXN1-β, alone or in combination with NRXN1-α, have yet been analyzed [Sudhof 2008]. For each of Neurexin 1-α and Neurexin 1-β, multiple protein coding isoforms of NRXN1 have been identified, whose structure and functions are not well understood.

NRXN1 Mutations in Humans

There is increasing evidence that NRXN1 disruptions [Kim et al. 2008], point mutations [Feng et al. 2006; Yan et al. 2008] and deletions [Glessner et al. 2009; Marshall et al. 2008; Morrow et al. 2008; Szatmari et al. 2007] are associated with autism spectrum disorders. NRXN1 has also been found to be associated with autism in a large genome-wide single nucleotide polymorphism association study [Wang et al. 2009].

NRXN1 deletions have also been associated with a variety of other conditions including schizophrenia [International Schizophrenia Consortium 2008; Kirov et al. 2008; Need et al. 2009; Rujescu et al. 2009; Vrijenhoek et al. 2008; Walsh et al. 2008], nicotine dependence [Bierut et al. 2007; Nussbaum et al. 2008] and other physical manifestations such as vertebral anomalies [Zahir et al. 2008].

Prior reports of abnormalities in NRXN1 have focused on populations with specific diagnoses (e.g., autism, schizophrenia). However, the clinical significance of copy number variants (CNV), such as deletion involving one or more exons of NRXN1, and the range of phenotypic manifestations of subjects with NRXN1 deletion CNV remains unclear. We describe here a group of subjects with NRXN1 deletions who demonstrate a wide range of physical and developmental phenotypes.

Clinical Cohort Record Review

From March 2007 to January 2009, a total of 3540 subjects at Children’s Hospital Boston were evaluated for genomic imbalance (deletion and duplication) using the Agilent 244K human genome oligonucleotide comparative genomic hybridization (CGH) microarrays (G4411B, Agilent Technologies, Palo Alto, CA) according to the manufacturer’s instructions [Oligonucleotide Array-Based CGH for Genomic DNA Analysis protocol version 3 (Agilent Technologies, Palo Alto, CA, USA)]. The majority of the referrals were for clinical features of developmental disorders (developmental delay, autism spectrum disorders, mental retardation) or multiple congenital malformations as determined by specialists in Clinical Genetics, Neurology and Developmental Medicine.

One hundred thirty probes cover the 1.12Mb region of the NRXN1 gene on the Agilent 244K CGH array. The average interprobe space within the NRXN1 gene is 8.6Kb. This permits the reliable detection of small intragenic deletions down to 43Kb in size. Images were captured by Agilent scanner and quantified using Feature Extraction software v9.0 (Agilent Technologies, Palo Alto, CA, USA). CGH analytics software v3.4 (Agilent Technologies, Palo Alto, CA, USA) was subsequently used for data normalization, quality evaluation and data visualization. Copy number aberration was indicated using the ADM-2 (Aberration Detection Method 2) algorithm. Deletions involving 5 or more consecutive probes were considered as true CNV.

For two larger deletions, fluorescent in situ hybridization (FISH) testing using probe RP11-800C7 was carried out for deletion confirmation and parental testing. The smaller deletions were confirmed by PCR-based breakpoint mapping methods. The primers used for each case are listed in the supplementary material.

Subjects with deletions involving exonic sequence of NRXN1 were included in our review. Two developmental behavioral pediatricians (RHN, MSC), a clinical geneticist (WHT), and a pediatric neurologist (SSJ) reviewed each of the medical records. The clinical history, physical examination, laboratory data, and radiological reports of each subject were reviewed.

Additional Report of Cases with NRXN1 Deletion and Autism

Cases with exonic and intragenic NRXN1 deletions were also contributed from the Homozygosity Mapping Collaborative for Autism (HMCA) which utilized the Affymetrix GeneChip Human Mapping 500K Array Set using CNV detection methods previously described [Morrow et al. 2008].

This work was approved by the Institutional Review Boards at the corresponding hospitals.

Clinical Cohort Record Review

We identified 12 subjects through Children’s Hospital Boston with deletions involving exonic sequences of NRXN1 (Table I and Figure 1). The deletions reported here range from 65Kb to 5Mb and most of these cases are predicted to affect the initial structural domains of the protein (Figure 1).

Of these 12 deletions, 4 were de novo CNV not identified in either parent, 3 were maternally inherited, 2 were paternally inherited, and the parental samples for one (subject 9) were not available. In another two (subjects 1 and 7), paternal samples were not available but the deletion was not identified in maternal testing.

In subjects 1-9, the deletions involved at least 2 exons of NRXN1-α, while in subjects 10-12, the deletions involved only an exon of a minor expressed NRXN1 isoform. The genomic imbalances involving NRXN1 are summarized in Table I and the clinical manifestations are summarized in Tables II and III. Further clinical data are available in the Supplementary Material.

Detailed clinical records were available from geneticists in 9 out of 12 subjects, developmental-behavioral pediatricians in 6/12, psychologists in 6/12 and neurologists in 4/12. Four of the twelve subjects (4, 6, 7, and 10) were diagnosed with autism spectrum disorders; in each positive case, this diagnosis was supported by the Autism Diagnostic Observation Schedule. Another subject (2) was suspected of having autism but the evaluation was not available for review; he also had global developmental delays. Two subjects had mental retardation without a diagnosis of an autism spectrum disorder (1 and 8). Subject 1, in addition, had absence seizures and an EEG consistent with a primary generalized epilepsy. One subject (3) was too young to ascertain for an autism spectrum disorder or cognitive delays. Nine subjects had clinical documentation of expressive or receptive language delays.

Mild dysmorphic features were present in seven subjects (2, 3, 4, 7, 8, 9, 12); three subjects had hemangiomas (2, 6, 10). Hypotonia was present in four subjects (3, 4, 9, 11). Two subjects (5, 12) had ventricular septal defects.

Medical record review also revealed the following characteristics in the 6 parents from whom the NRXN1 deletion was inherited. Subject 4, who had Pervasive Developmental Disorder, Not Otherwise Specified (PDD-NOS) and hypotonia, inherited his deletion from his father who is also reported to be socially awkward. Subject 6, who had PDD-NOS and coordination issues, inherited the deletion from a mother with a history of language delay and social skill difficulties. Subject 11, who has hypotonia, weakness and Poland anomaly, inherited the deletion from a mother who has a history of joint hypermobility, osteoarthritis, mitral valve prolapse, severe migraines and severe breast asymmetry. The father of subject 3 (hypotonia, gross motor delay), the father of subject 7 (autism, mental retardation) and the mother of subject 12 (poor weight gain, craniofacial dysmorphism) are reported to be healthy without developmental or medical concerns.

Additional Report of Cases with NRXN1 Deletion and Autism

In addition to the Children’s Hospital Boston cases, we report here three cases from two families ascertained through the Homozygosity Mapping Collaborative for Autism [Morrow et al. 2008]. The NRXN1 deletions in each were discovered to segregate with IQ below 70 in these pedigrees (Figure 2). All three affected children were carriers and unaffected children were not. The deletions were inherited from fathers who were found to have ASD symptoms and IQ between 60 and 70, while non-carrier mothers were not on the autism spectrum and with IQs in the normal range. The deletion for the subject in the first family is exonic and intragenic, while the deletion for the two siblings in the second family is upstream and may affect gene expression. Further investigation is necessary to substantiate this as a functional deletion, even though it segregates with disease.

Significance Test

To establish the relevance of these CNV, we compared the frequency of deletions involving NRXN1-α exons in our Children’s Hospital Boston population, in whom CGH testing was considered to be clinically indicated, to the frequency of similar deletions detected by array genomic profiling of equivalent resolution in normal populations. Itsara et al. [2009] detected 3 deletions involving NRXN1-α exons in 2493 normal individuals. The International Schizophrenia Consortium [2008] reported 2 exonic deletion cases in 3181 normal controls. Another large scale schizophrenia study identified 5 deletion cases among 33,746 normal controls [Rujescu et al. 2009]. Recently, Glessner et al. [2009] reported no deletion CNV involving NRXN1-α among 1409 ACC (Autism Case-Control) control samples and 1110 AGRE (Autism Genetic Resource Exchange) controls. Collectively, the frequency of exonic deletion of NRXN1-α in control populations is 10/51939 (0.019%); this differs significantly from the frequency of exonic deletion CNV we observed in our clinically referred population (9/3540) (0.25%; p=8.9 × 10⁻⁹, two-tailed Fisher’s exact test). There are no available data on the frequency of minor isoform exonic deletions in control populations and thus these subjects (n=3) were excluded from the significance test.

NRXN1 and Synapse Function

Prior studies have functionally linked other molecules that are associated with NRXN1 to a range of neuropsychiatric disorders including autism. These include neuroligins 3 and 4 (NLGN3, NLGN4) and SH3 and multiple ankyrin repeat domains 3 (SHANK3) [Durand et al. 2007; Jamain et al. 2002; Laumonnier et al. 2004; Lawson-Yuen et al. 2008; Moessner et al. 2007]. In addition, CNTNAP2 (contactin associated protein-like 2) [Alarcon et al. 2008; Arking et al. 2008; Bakkaloglu et al. 2008] and cadherin 10 (CDH10) and 9 (CDH9) have been also associated with autism spectrum disorders [Wang et al. 2009]. Our finding that NRXN1 is also associated with autism and developmental disorders adds further evidence to the importance of this molecular family to the development of neurodevelopmental disorders.

The function of NRXN1 in facilitating synaptic transmission suggests that mutations in this gene may predispose to a neurologic disconnection syndrome. Long range disconnections between neural networks have been hypothesized to be causative in some populations with autism [Barnea-Goraly et al. 2004; Frith 2004; Geschwind and Levitt 2007; Just et al. 2004]. The effects of NRXN1 on language development and hypotonia may likewise be related to long range connectivity within the brain.

Phenotypic Variation

Phenotypic variations may reflect the highly pleiotropic effects observed for specific CNVs such as associated with NRXN1. In addition, a number of our subjects inherited NRXN1 deletions from their parents. The detailed phenotype of these parents were not described in the medical records except in the family history, but the parents were ostensibly less affected than their children. This suggests that deletion in the NRXN1 gene may not be fully penetrant, or interacts with other genes resulting in the variable phenotype. Further research efforts to investigate such variable phenotypes associated with this unstable genomic region will provide further insight into the role of NRXN1 in the development of language delays, autism spectrum disorders and physical features.

Limitations

The accuracy and completeness of the clinical phenotype identified in this study is entirely dependent on the clinical information that was documented in the medical records of these subjects, often before the NRXN1 deletions were identified in them. Because of the clinical variability exhibited in our cohort, the subjects were seen by a variety of specialists, which affected the completeness of data.

In addition, the parents were not formally assessed to ascertain their cognitive, physical and behavioral phenotypes. As noted above, review of family history suggests that some parents may have shared similar phenotypes to their children. We are conducting further testing on both the subjects and their parents to better clarify developmental and/or social cognition issues in subjects and their parents.

For the deletion CNV significance test, we used the normal control data generated by different genomic profiling array platforms as reference. Knowing that the sensitivity and specificity differ from one array platform to another, this may not be an optimal comparison. However, the effort was made to minimize the detection bias between different array platforms. Here we have only chosen recent studies using array platform of similar resolution as ours. All these published papers reported the detection of smaller CNV, suggesting that technically all these array platforms were able to detect any CNV identified in this study. Thus this comparison, although an approximation, is on the conservative side.

Finally we acknowledge that while our clinically ascertained subjects were not drawn from a cohort with a single diagnosis such as autism or schizophrenia, they were ascertained from a heterogeneously affected group in whom genetic testing was considered clinically relevant. As a result, there is ascertainment bias and our findings may not reflect the true distribution of physical and developmental findings in the NRXN1 deletion phenotype. Nevertheless, we have demonstrated that there are a number of other phenotypic features present in this clinical population beyond what has previously been identified in the literature.