Autism Spectrum Disorders (ASD) via the NLGN4X Gene

Autism Spectrum Disorders (ASD) encompasses several neurodevelopmental disorders characterized by varying degrees of social impairment, communication ability, and propensity for restricted interests and repetitive behavior(s) which usually present by age 3. Diagnosis is based on the degree and severity of symptoms and behaviors (Diagnostic and Statistical Manual of Mental Disorders (DSM-5); Levy et al. 2009; McPartland et al. 2016). Comorbidities occur in more than 70% of cases and include intellectual disability, epilepsy, language deficits, and gastrointestinal problems (Sztainberg and Zoghbi 2016).

NLGN4X (neuroligin 4X) is a member of the neuroligin gene family encoding a neuronal trans-synaptic adhesion molecule (neuroligin 4X) that interacts with the pre-synaptic cell surface receptor neurexin. Together, these molecules play an important role in formation, organization, and remodeling of synapses, maintenance of synapse function, and circuit-specific traits (Südhof et al. 2008; Zhang et al. 2009). Variants in NLGN4X have been associated with autism, Asperger syndrome, and intellectual disability, however presentation is variable even among individuals inheriting the same variant (Jamain et al. 2003).

Genetic aberrations are reported to be responsible for 50-90% of ASD cases (Larsen et al. 2016; Karam et al. 2015). Incidence of ASD is approximately 1 in 68 individuals with a male-to-female ratio of 4:1 (Center for Disease Control 2014). De novo missense and likely gene disrupting variants are 15% and 75% more frequent in ASD patients than unaffected controls, respectively (Iossifov et al. 2014).

In the case of NLGN4X, inheritance is thought to occur in an X-linked recessive manner. Individuals with NLGN4X point variants or small indels have to date been exclusively male (Zhang et al. 2009; Yu et al. 2013; Jamain et al. 2003; Laumonnier et al. 2004). CNVs disrupting NLGN4X are most frequent in males (Willemsen et al. 2012; Isrie et al. 2012). However, there is at least one report of an affected female with one gross deletion encompassing NLGN4X and no second candidate (Willemsen et al. 2012). Duplications of NLGN4X are not sex-biased and have been reported in both affected males and females (Willemsen et al. 2012; Isrie et al. 2012).

NLGN4X is located in the proximal region of p.22.33 within the pseudoautosomal region of the X chromosome which escapes X-inactivation in females. In males, a homolog on the Y chromosome (NLGN4Y) is believed to pair with NLGN4X during meiosis. However, NLGN4X and NLGN4Y are more dissimilar at the amino acid (97.5% identity) and nucleotide (<96% identity) level than other genes within the pseudoautosomal region (>99% identity) (Jamain et al. 2003; Zuo et al. 2013; Carrel and Willard 2005). Therefore, in males two distinct neuroligin 4 genes are expressed (NLGN4X and NLGN4Y) while in females only one is expressed (NLGN4X). A subtle difference in protein function between these genes has been proposed, but not proven (Ross et al. 2015). One missense variant from a single family has been reported in NLGN4Y to contribute to ASD (Yan et al. 2008).

The NLGN4X gene consists of 5 coding exons with up to 2 non-coding exons in the 5'UTR region depending on the transcript. The NLGN4X protein contains a noncatalytic acetylcholinesterase domain necessary for pre-synapatic neurexin binding, extracellular, transmembrane, and intracellular domains, and a PDZ-binding domain important for specific targeting to excitatory synapses (Laumonnier et al. 2004; Song et al. 1999­).

Missense and loss of function variants (including nonsense and frameshift variants) within NLGN4X have been documented in individuals with variable phenotypes, including autism, Asperger syndrome, and intellectual disability (Lamonnier et al. 2004; Jamain et al. 2003; Yu et al. 2013). The majority of affected individuals (male) inherit a NLGN4X variant (predominantly before the transmembrane domain) from their unaffected carrier mother (SFARI Database; Yu et al. 2013; Laumonnier et al. 2004; Jamain et al. 2003), although apparent de novo variants have been reported (Zhang et al. 2009). Internal NLGN4X deletions encompassing up to several exons as well as larger multi-gene deletions have also been reported in individuals with ASD phenotypes (Lawson-Yuen et al. 2008; Talebizadeh et al. 2006; Willemsen et al. 2012; Isrie et al. 2012).

Currently, the contribution of de novo and inherited factors to Autism Spectrum Disorders (ASD) risk is estimated to be approximately 50-60% (Krumm et al. 2015). NLGN4X is classified in the Simons Foundation Autism Research Initiative (SFARI) Database as a gene with ‘suggestive evidence’ regarding ASD risk (https://gene.sfari.org/GeneDetail/NLGN4X). However, more than 700 genes have been associated with ASD features (Bourgeron 2016). Rare complete knockouts of genes on the X chromosome (such as copy number variants encompassing NLGN4X) are estimated to contribute 2% to ASD risk (Lim et al. 2013).

Overall, de novo copy number variants (CNVs) are estimated to account for approximately 6% of ASD risk, while rare complete knockouts (de novo or inherited) of genes on the X chromosome are estimated to account for 2% of overall risk (Lim et al. 2013). The contribution of NLGN4X CNVs specifically is unclear; however the chromosomal region Xp22 in which NLGN4X is located has been implicated with ASD in numerous reports (Guo et al. 2017; Lawson-Yuen et al. 2008; Marshall et al. 2008; Talebizadeh et al. 2006; Isrie et al. 2012; Willemsen et al. 2012).

This test provides full coverage of all coding exons of the NLGN4X gene plus 10 bases of flanking noncoding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define full coverage as >20X NGS reads or Sanger sequencing. PGnome panels typically provide slightly increased coverage over the PGxome equivalent. PGnome sequencing panels have the added benefit of additional analysis and reporting of deep intronic regions (where applicable).

Dependent on the sequencing backbone selected for this testing, discounted reflex testing to any other similar backbone-based test is available (i.e., PGxome panel to whole PGxome; PGnome panel to whole PGnome).