Keywords autism - neuroligin 4Y - India - novel missense mutation - male predominance
Introduction
Autism [ASD] [MIM 299850] is a heterogeneous neurodevelopmental disorder. Autism is
not characterized based on a single symptom. It is usually characterized by the triad
of symptoms viz lack of social interaction, abnormal verbal and nonverbal communications
and stereotyped or repetitive behaviors.[1 ] Autism is classified as syndromic and non-syndromic autism. Syndromic autism is
one in which patients who have pre-existing neurological disorders, example, a subset
of patients with fragile x syndrome, tubers sclerosis, Rett syndrome displays phenotypes
which are attributed to ASD. Non-syndromic autism accounts for autism cases that are
not linked to any neurological disorders.[2 ] Neuroligin is trans-synaptic cell adhesion molecule present post-synaptically and
plays a very important role in synaptogenesis with presynaptic neurexin.[3 ] Humans have neuroligin 4X [NLGN4X ] on the X chromosome and neuroligin 4Y [NLGN4Y ] on the Y chromosome. NLGN4X and NLGN4Y genes share 97% sequence identify.[4 ] The male bias seen from NLGN4X mutations is unclear since NLGN4Y plays a function similar to NLGN4X and should be sufficient to reimburse for NLGN4X ASD-related mutations. This lack of compensation in males suggested that NLGN4Y may have an uncharacterized distinct function that needs to be explored. Several
studies are reported that synaptic cell adhesion molecules have been strongly involved
in autism. Neuroligin has an important role in the maturation and functions of synapses.[5 ]
[6 ] The mechanism of Y chromosome contribution on to neurodevelopmental disorders is
still not known very well. Originally, it was thought that Y chromosome contains only
a few genes that are primarily involved in sex determination and testicular functions
but now it is known to contain numerous genes with diverse functions.[7 ] Several shreds of evidence strongly suggested that NLGN4X deficiencies can cause autism and still there is no clear understanding of sex bias
in autism.
We hypothesize that male individuals have both X and Y chromosome so analysis of sequence
variants in NLGN4Y gene may be associated with sex bias in male autistic individuals. To address this
objective, we sequenced all the exonic regions of NLGN4Y gene in 85 male autistic children from north Karnataka region of India.
Methods
Sample Collection
One-hundred fifty autistic children were identified after screening a large mentally
ill population of 1,870 children from the entire North Karnataka region of India (n
male = 117, n
female = 33 mean age = ± 11.5]. Eighty-five male autistic children were included for the
genetic analysis of NLGN4Y gene. Screening of autistic children was performed using
Diagnostic and Statistical Manual of Mental Disorders-V (DSM-V, American Psychiatric
Association, 2000) (https://www.psychiatry.org/psychiatrists/practice/dsm ) and/or International Classification of Diseases-10 (ICD-10, WHO) (https://www.who.int/ classifications/ icd/ icdonlineversions/en/). Child with associated neurological
disorders and other comorbid diseases was excluded from the study. Around 1 to 2 mL
of peripheral blood was collected in EDTA-coated vacutainers and stored in −20°C until
further analysis.
DNA Isolation and PCR Amplification
Genomic DNA was isolated from peripheral blood using blood and tissue DNA isolation
kit (QIAGEN, Germany) as per manufactures guidelines. Quality and quantity of isolated
genomic DNA were checked using agarose gel electrophoresis and nanodrop UV spectrophotometer
(Quawell, Q3000 UV spectrophotometer). Amplification of NLGN4Y gene was carried with designed primers using standard PCR reagents (New England Bio
Labs, United States). Quality and quantity of PCR product were analyzed.
Sequencing
PCR products of NLGN4Y gene was sequenced using Sanger sequencing kit v3.1 on ABI 3500 Sanger sequencer
platform. Sequence data were analyzed with ABI sequence analysis Software v5.4 (Applied
Biosystem, United States).
Bioinformatics Analysis
Pathogenic effect of missense mutation was analyzed using Insilco tools like PROVEAN,
SNAP2, polyphen2, SNP&GO, and CADD. Conservation status of amino acid residues of
NLGN4Y protein at 163 position was checked using the ConSurf Server (https://consurf.tau.ac.il/ ).[8 ] Three-dimensional structure of wild type and mutant protein was developed using
Swiss-model and structures were visualized and analyzed using UCSF Chimera tool.
Results
Detailed screening of 1,870 mentally ill children below 18 years of age from North
Karnataka population of India revealed 150 autistic children [n
male = 117, n
female = 33 mean age = ± 11.5] which accounts for 8.02% of autism in North Karnataka region
of India. Sanger Sequence analysis of neuroligin 4Y gene from 85 male autistic children
revealed the nine variants, which include one missense and eight synonymous variants.
Four variants which were recorded in our study cohort are not previously recorded
in any in house human SNP databases viz dbSNP, 1000 genomes, ExAc and ClinVar shown
in [Table 1 ]. Novel missense, p.N163K mutation was recorded in three autistic children and clinical
features of those autistic children with missense mutation are shown in [Table 2 ]. Pathogenicity prediction of missense variants was analyzed using Insilco tools
viz PROVEAN, POLYPHEN2, SNAP2, SNP&GO, and CADD. Only Missense variant, p.N163K was
found to be harmless on the functions of NLGN4Y protein by PROVEAN, POLYPHEN2, SNAP2, SNP&GO, and CADD shown in [Table 3 ]. Conservation status analysis of NLGN4Y protein sequences shows that amino acid
residue at 163 position is not conserved; it is variable and exposed residue according
to the neural network algorithm shown in [Fig. 1 ].
Table 1
Showing list of mutations recorded in our study cohort
Variation
N. change
A.A Change
SNP id
Frequency of mutation
Missense
g. 205526 C > A
p.N163K
Not recorded
3 (3.5%)
Synonymous
g.312652 T > C
p. H447=
rs777234513
4 homozygous (4.7%)
2 heterozygous (2.3%)
Synonymous
g.312781C > T
p.G490=
rs767447455
3 (3.5%)
Synonymous
g.312787 A > G
p.E492=
rs750273940
3 homozygous (3.5%)
2 heterozygous (2.3%)
Synonymous
g.312826 A > C
p.T505=
Not recorded
1 (1.2%)
Synonymous
g.312844 T > C
p.N512=
Not recorded
2 (2.3%)
Synonymous
g. 312847 C > T
p.F513=
Not recorded
1 (1.2%)
Synonymous
g. 312871 T > C
p.S520=
rs1423308667
1 (1.2%)
Synonymous
g. 312880 G > C
p.V523=
rs753006927
3 (3.5%)
Table 2
Clinical features of autism children with missense mutation, p.N163K of NLGN4Y gene
Demographic character
Child 1
Child 2
Child 3
Ethnic origin
Indian
Indian
Indian
Sex
Male
Male
Male
Age of father at child's birth
38
29
35
Age of mother at child's birth
33
19
34
Consanguineous marriage
No
Yes
No
Prenatal damage
preeclampsia
None
None
Postnatal damage
None
None
Birth asphyxia
IQ
25
30
28
CARS Score and Severity
40; Severe
44; Severe
52 ; Severe
Comorbidity
None
None
None
Table 3
Showing pathogenicity prediction of a missense variant
Variant
PROVEAN
SNP&GO
PolyPhen2
SNAP2
CADD score
p.N163K
Neutral
Score: −0.992
Neutral
0.381
Benign
Score: 0.023
Neutral
Score: −79
19.34
[Raw score 2.010689]
Fig. 1 Conservation status of p.N163K mutation, amino acid residue at 163 position is not
conserved; it is variable and exposed residue.
Three-dimensional protein modeling analysis of NLGN 4Y protein revealed that mutant
residue is bigger than wild type residue and it possesses a positive charge whereas
wild type protein possesses neutral charge. The wild type of residue is predicted
to be located in its preferred secondary structure, a turn but the mutant residue
prefers to be in another secondary structure; therefore, the local conformation will
be slightly destabilized shown in [Fig. 2A ] and [Fig. 2B ]. The mutated residue is located in a domain that is important for binding of other
molecules. Mutation of the residue might disturb this function of NLGN4Y protein.
Fig. 2 (A ) Three-dimensional model of wild type protein; wild type amino acid residue is small
and neutrally charged. (B ) Three-dimensional model of mutant, p.N163K protein. Mutant residue is bigger than
wild type residue and it is positively charged.
Discussion
Autism [MIM 299850] is a complex neurological condition which is characterized by
abnormal social interaction, verbal and nonverbal communication and impaired behaviors.
“Autism,” the term was first used by Ukrainian-Austrian-American psychiatrist, Leo
Kenner in 1943.[9 ] Rates of autism cases are increasing globally over the period of time when it comes
to the Indian perspectives, cases are increasing dramatically, and it may be due to
increased scientific knowledge and awareness or it may be an improper diagnosis. In
recent days, neuroligin gene is the most targeted gene for the molecular studies on
neurological disorders like autism, anxiety, attention deficit hyperactivity disorder
and intellectual disability due to its active role in synaptogenesis.[10 ]
[11 ]
[12 ]
[13 ]
The Simons Foundation Autism Research Initiative (SFARI) [geneSFARI.org] lists four
genes of Y chromosome associated with autism viz NLGN4Y, ASMT, USP9Y, and SHOX .[14 ] Only a few studies have been undertaken till now to study the role of NLGN4Y gene in autism. Studies conducted in 2005 and 2006, failed to identify the variants
in the NLGN4Y gene in autistic patients.[15 ]
[16 ] But later several studies, record polymorphisms of NLGN4Y gene involved in neurodevelopmental disorder and synaptic functions have been associated
with autism.[17 ]
[18 ] Peripheral blood NLGN4Y gene expression showed an increased risk of autism in children with XYY symptoms.[7 ]
In the present study, for the first time in Indian autistic population, we analyzed
a cohort of 85 male autistic children under the age of 18 years after screening large
neurological disorders population. Our study population records 7.4% of autism over
different neurological disorders. Sequencing analysis of the entire exonic region
revealed 09 mutations. One is novel missense mutation and reaming 08 (88.9%) are synonymous
variants. Out of eight synonymous variants, five (62.5%) were already reported in
in-house human SNP databases. Insilco functional effect prediction of a novel missense
mutation, p.N163K by PROVEAN, POLYPHEN2, SNAP2, SNP&GO, and CADD shows nonpathogenic
effects on the functions of neuroligin 4Y protein. But structural analysis of p.N163K
mutant protein shows slight destabilization in the local configuration by positioning
on different secondary structure, a turn and it might cause bumps in protein structure
due to differences in the size of amino acid. Meanwhile, the mutation introduces a
charge; this can cause repulsion of ligands or other residues with the same charge.
Structural and functional prediction of novel missense mutation indicates slight changes
related only to the structure of neuroligin 4Y protein, and not to the functions of
the protein. Absence of functional analysis of gene was the important limitation of
our study.
Conclusion
Mutation in NLGN4Y may be an uncommon cause of autism in Indian autistic population. But further detailed
functional investigation of neuroligin 4Y gene in autism is important to understand
the male predominance of autism and increased rate of autism in males in India autistic
population.
