Autism Spectrum Disorders (ASD) via the NLGN4X Gene

  • Summary and Pricing
  • Clinical Features and Genetics
  • Citations
  • Methods
  • Ordering/Specimens
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Test Code Test Copy GenesPriceCPT Code Copy CPT Codes
2214 NLGN4X$910.00 81405 Add to Order
Targeted Testing

For ordering sequencing of targeted known variants, please proceed to our Targeted Variants landing page.

Turnaround Time

The great majority of tests are completed within 18 days.

Clinical Sensitivity

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 ( 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).

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Del/Dup via aCGH

Test Code Test Copy GenesPriceCPT Code Copy CPT Codes
600 NLGN4X$990.00 81404 Add to Order
Pricing Comments

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Over 100

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Turnaround Time

The great majority of tests are completed within 20 days.

Clinical Sensitivity

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).

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Clinical Features

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).

Testing Strategy

This test involves bidirectional Sanger sequencing using genomic DNA of all coding exons of the NLGN4X gene plus ~10 bp of flanking non-coding DNA on each side. We will also sequence any single exon (Test #100) or pair of exons (Test #200) in family members of patients with known mutations or to confirm research results.

Indications for Test

Individuals with family members having known NLGN4X variants and/or males displaying symptoms of autism, Asperger syndrome, or intellectual disabilities along with a family history of these diseases are candidates for this test.


Official Gene Symbol OMIM ID
NLGN4X 300427
Inheritance Abbreviation
Autosomal Dominant AD
Autosomal Recessive AR
X-Linked XL
Mitochondrial MT

Related Test

Autism Spectrum Disorders and Intellectual Disability (ASD-ID) Comprehensive Sequencing Panel with CNV Detection


Genetic Counselors
  • Bourgeron T. 2016. Comptes Rendus Biologies. 339: 300-7. PubMed ID: 27289453
  • Carrel L., Willard H.F. 2005. Nature. 434: 400-4. PubMed ID: 15772666
  • Center for Disease Control and Prevention. 2014. Morbidity and Mortality Weekly Report. Surveillance Summaries (Washington, D.C. : 2002). 63: 1-21. PubMed ID: 24670961
  • Guo H. et al. 2017. Scientific Reports. 7: 44155. PubMed ID: 28281572
  • Iossifov I. et al. 2014. Nature. 515: 216-21. PubMed ID: 25363768
  • Isrie M. et al. 2012. European Journal of Medical Genetics. 55: 577-85. PubMed ID: 22659343
  • Jamain S. et al. 2003. Nature Genetics. 34: 27-9. PubMed ID: 12669065
  • Karam S.M. et al. 2015. American Journal of Medical Genetics. Part A. 167: 1204-14. PubMed ID: 25728503
  • Krumm N. et al. 2015. Nature Genetics. 47: 582-8. PubMed ID: 25961944
  • Larsen E. et al. 2016. Molecular Autism. 7: 44. PubMed ID: 27790361
  • Laumonnier F. et al. 2004. American Journal of Human Genetics. 74: 552-7. PubMed ID: 14963808
  • Lawson-Yuen A. et al. 2008. European Journal of Human Genetics. 16: 614-8. PubMed ID: 18231125
  • Levy S.E. et al. 2009. Lancet. 374: 1627-38. PubMed ID: 19819542
  • Lim E.T. et al. 2013. Neuron. 77: 235-42. PubMed ID: 23352160
  • Marshall C.R. et al. 2008. American Journal of Human Genetics. 82: 477-88. PubMed ID: 18252227
  • McPartland J.C. et al. 2016. Encyclopedia of Mental Health. 2: 124-130.
  • Ross J.L. et al. 2015. Genes, Brain, and Behavior. 14: 137-44. PubMed ID: 25558953
  • Song J.Y. et al. 1999. Proceedings of the National Academy of Sciences of the United States of America. 96: 1100-5. PubMed ID: 9927700
  • Südhof T.C. 2008. Nature. 455: 903-11. PubMed ID: 18923512
  • Sztainberg Y., Zoghbi H.Y. 2016. Nature Neuroscience. 19: 1408-17. PubMed ID: 27786181
  • Talebizadeh Z. et al. 2006. Journal of Medical Genetics. 43: e21. PubMed ID: 16648374
  • Willemsen M.H. et al. 2012. European Journal of Medical Genetics. 55: 586-98. PubMed ID: 22796527
  • Yan J. et al. 2008. Psychiatric Genetics. 18: 204-7. PubMed ID: 18628683
  • Yu T.W. et al. 2013. Neuron. 77: 259-273. PubMed ID: 23352163
  • Zhang C. et al. 2009. The Journal of Neuroscience. 29: 10843-54. PubMed ID: 19726642
  • Zuo L. et al. 2013. Psychiatric Genetics. 23: 233-8. PubMed ID: 23907288
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Bi-Directional Sanger Sequencing

Test Procedure

Nomenclature for sequence variants was from the Human Genome Variation Society (  As required, DNA is extracted from the patient specimen.  PCR is used to amplify the indicated exons plus additional flanking non-coding sequence.  After cleaning of the PCR products, cycle sequencing is carried out using the ABI Big Dye Terminator v.3.0 kit.  Products are resolved by electrophoresis on an ABI 3730xl capillary sequencer.  In most cases, sequencing is performed in both forward and reverse directions; in some cases, sequencing is performed twice in either the forward or reverse directions.  In nearly all cases, the full coding region of each exon as well as 10 bases of non-coding DNA flanking the exon are sequenced.

Analytical Validity

As of February 2018, we compared 26.8 Mb of Sanger DNA sequence generated at PreventionGenetics to NextGen sequence generated in other labs. We detected only 4 errors in our Sanger sequences, and these were all due to allele dropout during PCR. For Proficiency Testing, both external and internal, in the 14 years of our lab operation we have Sanger sequenced roughly 14,300 PCR amplicons. Only one error has been identified, and this was an error in analysis of sequence data.

Our Sanger sequencing is capable of detecting virtually all nucleotide substitutions within the PCR amplicons. Similarly, we detect essentially all heterozygous or homozygous deletions within the amplicons. Homozygous deletions which overlap one or more PCR primer annealing sites are detectable as PCR failure. Heterozygous deletions which overlap one or more PCR primer annealing sites are usually not detected (see Analytical Limitations). All heterozygous insertions within the amplicons up to about 100 nucleotides in length appear to be detectable. Larger heterozygous insertions may not be detected. All homozygous insertions within the amplicons up to about 300 nucleotides in length appear to be detectable. Larger homozygous insertions may masquerade as homozygous deletions (PCR failure).

Analytical Limitations

In exons where our sequencing did not reveal any variation between the two alleles, we cannot be certain that we were able to PCR amplify both of the patient’s alleles. Occasionally, a patient may carry an allele which does not amplify, due for example to a deletion or a large insertion. In these cases, the report contains no information about the second allele.

Similarly, our sequencing tests have almost no power to detect duplications, triplications, etc. of the gene sequences.

In most cases, only the indicated exons and roughly 10 bp of flanking non-coding sequence on each side are analyzed. Test reports contain little or no information about other portions of the gene, including many regulatory regions.

In nearly all cases, we are unable to determine the phase of sequence variants. In particular, when we find two likely causative mutations for recessive disorders, we cannot be certain that the mutations are on different alleles.

Our ability to detect minor sequence variants, due for example to somatic mosaicism is limited. Sequence variants that are present in less than 50% of the patient’s nucleated cells may not be detected.

Runs of mononucleotide repeats (eg (A)n or (T)n) with n >8 in the reference sequence are generally not analyzed because of strand slippage during PCR and cycle sequencing.

Unless otherwise indicated, the sequence data that we report are based on DNA isolated from a specific tissue (usually leukocytes). Test reports contain no information about gene sequences in other tissues.

Deletion/Duplication Testing via Array Comparative Genomic Hybridization

Test Procedure

Equal amounts of genomic DNA from the patient and a gender matched reference sample are amplified and labeled with Cy3 and Cy5 dyes, respectively. To prevent any sample cross contamination, a unique sample tracking control is added into each patient sample. Each labeled patient product is then purified, quantified, and combined with the same amount of reference product. The combined sample is loaded onto the designed array and hybridized for at least 22-42 hours at 65°C. Arrays are then washed and scanned immediately with 2.5 µM resolution. Only data for the gene(s) of interest for each patient are extracted and analyzed.

Analytical Validity

PreventionGenetics' high density gene-centric custom designed aCGH enables the detection of relatively small deletions and duplications within a single exon of a given gene or deletions and duplications encompassing the entire gene. PreventionGenetics has established and verified this test's accuracy and precision.

Analytical Limitations

Our dense probe coverage may allow detection of deletions/duplications down to 100 bp; however due to limitations and probe spacing this cannot be guaranteed across all exons of all genes. Therefore, some copy number changes smaller than 100-300 bp within a targeted large exon may not be detected by our array.

This array may not detect deletions and duplications present at low levels of mosaicism or those present in genes that have pseudogene copies or repeats elsewhere in the genome.

aCGH will not detect balanced translocations, inversions, or point mutations that may be responsible for the clinical phenotype.

Breakpoints, if occurring outside the targeted gene, may be hard to define.

The sensitivity of this assay may be reduced when DNA is extracted by an outside laboratory.

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Ordering Options

myPrevent - Online Ordering
  • The test can be added to your online orders in the Summary and Pricing section.
  • Once the test has been added log in to myPrevent to fill out an online requisition form.
  • A completed requisition form must accompany all specimens.
  • Billing information along with specimen and shipping instructions are within the requisition form.
  • All testing must be ordered by a qualified healthcare provider.


(Delivery accepted Monday - Saturday)

  • Collect 3 ml -5 ml (5 ml preferred) of whole blood in EDTA (purple top tube) or ACD (yellow top tube). For Test #500-DNA Banking only, collect 10 ml -20 ml of whole blood.
  • For small babies, we require a minimum of 1 ml of blood.
  • Only one blood tube is required for multiple tests.
  • Ship blood tubes at room temperature in an insulated container. Do not freeze blood.
  • During hot weather, include a frozen ice pack in the shipping container. Place a paper towel or other thin material between the ice pack and the blood tube.
  • In cold weather, include an unfrozen ice pack in the shipping container as insulation.
  • At room temperature, blood specimen is stable for up to 48 hours.
  • If refrigerated, blood specimen is stable for up to one week.
  • Label the tube with the patient name, date of birth and/or ID number.


(Delivery accepted Monday - Saturday)

  • Send in screw cap tube at least 5 µg -10 µg of purified DNA at a concentration of at least 20 µg/ml for NGS and Sanger tests and at least 5 µg of purified DNA at a concentration of at least 100 µg/ml for gene-centric aCGH, MLPA, and CMA tests, minimum 2 µg for limited specimens.
  • For requests requiring more than one test, send an additional 5 µg DNA per test ordered when possible.
  • DNA may be shipped at room temperature.
  • Label the tube with the composition of the solute, DNA concentration as well as the patient’s name, date of birth, and/or ID number.
  • We only accept genomic DNA for testing. We do NOT accept products of whole genome amplification reactions or other amplification reactions.


(Delivery preferred Monday - Thursday)

  • PreventionGenetics should be notified in advance of arrival of a cell culture.
  • Culture and send at least two T25 flasks of confluent cells.
  • Some panels may require additional flasks (dependent on size of genes, amount of Sanger sequencing required, etc.). Multiple test requests may also require additional flasks. Please contact us for details.
  • Send specimens in insulated, shatterproof container overnight.
  • Cell cultures may be shipped at room temperature or refrigerated.
  • Label the flasks with the patient name, date of birth, and/or ID number.
  • We strongly recommend maintaining a local back-up culture. We do not culture cells.
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