Novel Hemizygous Missense Variation in AFF2 Gene Underlies Fragile XE Syndrome

Background: Fragile XE (FRAXE) is an X-linked recessive condition of intellectual disability affecting 1 in 50,000 new born male. FRAXE is characterized by mild ID, cognitive impairment, speech delay and some cases patients display Autism Spectrum disorder (ASD) like phenotypes. . Method: In this study, we investigated a family with two male siblings with neurodevelopmental delay Whole exome sequencing analysis (WES) was employed to identify the pathogenic variant. Co-segregation analysis was performed through Sanger sequencing in affected and normal family members. Results: Two affected Proband of family were diagnosed with intellectual disability. A novel hemizygous variant, c.3348G>T; p.Asp1150Tyr, in AFF2 gene was identied as the pathogenic cause in affected individuals. It is rst novel variant report in AFF2 gene within Pakistani population. Conclusion: In this study, novel hemizygous variant, c.3348G>T; p.Asp1150Tyr, in AFF2 gene was identied. The ndings broaden the clinical and genetic spectrum of rare X-linked recessive disorders causing ID.


Background
Cognition is the result of multiple molecular, cellular and biological events in nervous system, and a minor defect in any of the event ultimately leads to cognitive impairment or intellectual disability (ID) [1,2].
Moreover, it is also believed that developmental delay or intellectual disability is consequence of a defect in synapse formation and plasticity; therefore it is also termed as neurodevelopmental disorder [3,4].
Prevalence of intellectual disability (ID) in general population is 2-3% [5,6]. Clinical and genetic heterogeneity of ID pose a great challenge for clinicians and scientists involved in this research [7,8].
The syndromic intellectual disability has several types e.g. Joubert syndrome, Kahrizi syndrome, and the majority of them have limited knowledge. So far, non-syndromic intellectual disability (NSID) is least studied and hence only 47 loci and 26 genes have been identi ed. Comparison between X linked genes and autosomes (chromosomes other than sex chromosomes), there is a large number of genes that control brain and its function are still unidenti ed. Even in case of non-syndromic intellectual disability, still twenty one loci are identi ed and their causative genes are remained to be recognized. FRAXE syndrome is a condition in which cognitive function, learning and thinking ability has been affected. Poor writing skills, delayed speech, hyperactivity and short attention span are the common symptoms of people affected with FRAXE syndrome. Autistic behaviour such as intense interest in a particular subject, repetitive behavior, hand apping are displayed by some affected individuals. In FRAXE syndrome unlike in some other types of ID, cognitive function remains steady and did not decrease with age. [11].

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The study was approved by the Board of Advanced Study Research (BASR) and ethical committee of the International Islamic University Islamabad. DNA was isolated from the collected blood samples using standard protocol. Microarray genotyping was performed using Illumina cytoSNP-12v2.0 arrays in both probands to identify the known genes in family.

Whole Exome Sequence Analysis
Whole exome sequencing was performed commercially on Illumina platform (Macrogen, Seoul, South Korea). WES was performed on one sample in family by using the Agilent SureSelect human all exon V4 exome enrichment kit. Sequence reads aligned to the reference genome UCSC: hg19/ GRCH37 NCBI.
GATK tool was used to perform the sequence realignment and base quality recalibration. Read depth of the sequences were determined by using BedTools method [12]. Single nucleotide variations (SNVs), insertion and deletions were detected by using SamTool software [13]. Data annotation was performed with wANNOVAR [14]. Allele speci c primer was designed to amplify the target region of DNA using primer3plus tool [15].

Sanger Sequencing
Polymerase chain reaction (PCR) was performed using genomic DNA of the proband and accessible family members for the con rmation of the veracity of the likely causative variant and to assess segregation within the family. The PCR product was puri ed using Exo-Sap at 37 o c for 15 minutes, followed by heating temperature 80 o c for 15 minutes. Sequencing reaction was performed using BigDye terminator kit (Applied Biosystems). The puri ed sequencing reaction product sequencing was performed on ABI 3730xl. Sequences were manually reviewed and compared to reference sequence NM 004595.4 of SMS gene using Codon Code Aligner software.
Predicted models reliability was checked using RAMPAGE [19]. Models were visualized via UCSF Chimera [20]. CASTp server were used to identi ed proteins pockets on 3D-structure of AFF2 protein [21,22]. Meta SNP [23], I-mutant 2.0 [24] and PREDICT SNP [25] used to check the impact of mutation on protein stability. Multiple in-silico tools were used to check the pathogenicity of the mutation. Multiple sequence alignment was performed using Clustal Omega.

Clinical details
The family comprise of six members with two Proband i.e. IV: 2 and IV: 3 (Fig. 1A).
Patient Iv: 2 The Proband (IV: 2) was an 8 year and 3 month old male. He was born at normal gestational age and his Birth weight, birth length and OFC was 2.2 kg, 41.6 cm and 31 cm, respectively at birth. Developmental mile stones were observed at early age. At the age of 14 months, he started walking but unable to speak single word. Proband exhibited clinical manifestations of intellectual disability, memory loss, as well as learning disability. In addition, dysmorphic facial features including prominent nasal tip, large ears was noted.
Patient Iv: 3 The second Proband i.e. IV: 3 was 6 years old male at the time of study. He was born after an uneventful pregnancy and his weight and OFC were 3 kg and 30 cm respectively at birth. Clinical symptoms of both Proband were similar.

Genetic Analysis
The variant NM_002025.4: c.3448G > T; p.Asp1150Tyr; Chr X: 148059506G > T (GRCh37) was identi ed in the AFF2 gene (MIM 309548) in the index patient (IV: 2), through whole-exome sequencing analysis. WES raw data was ltered through bioinformatics tools with minor allelic frequency (MAF) less (1%).This variant was then con rmed by Sanger sequencing. Co-segregation analysis of candidate variant in AFF2 was performed in both affected and normal members. The pathogenic variant, c.3448G > T; p. Asp1150Tyr in AFF2 was found associated with disease phenotype. Two affected males (IV: 2 and IV: 3) were hemizygous for mutant allele in coding exon 17 of AFF2 gene where the normal mother III: 1 was heterozygous for the allele and the remaining members IV: 1 and IV: 5 were of wild type (Fig. 1B). This variant leads to a substitution of aspartic acid by tyrosine and at the evolutionary conserved amino acid position (p.Asp1150Tyr) according to the UCSC Human Genome (GRCh37/hg19). The variant is listed in the Genome Aggregation Database (gnomAD v2.1.1) (https://gnomad.broadinstitute.org/) in heterozygous form in two females from European (non-Finnish) population and is not reported in males. The variant is absent in south Asian population of gnomAD database.
The pathogenicity of the variant predicated to damaging by FATHMM-MKL with score of (0.88), damaging by DEOGEN2, damaging by PROVEAN with score (-2.75), DANN and GERP score were (0.99), (5.98) respectively. In-silico results strongly indicate that variant (p.Asp1150Tyr) is likely to be disease causing and deleterious to the AFF2 protein and responsible for disease in family.

Effect Of Mutation On Protein
To study the pathogenicity of p.Asp1150Tyr mutation, structure of AFF2 protein was predicted by using I-TASSER Tool. The mutated model created by using template 6R80 [26] (Fig. 1C). The result showed that template trigger release of polymerase-II enzyme from promoter proximal pausing site. Paused polymerase-II was playing role in the maintenances of promoter region structure. Affected model shows loss of structure of protein but no change in protein length observed. Loss of function of protein involved in the altering conformation of protein, it also shows that loss of c-terminal helices disturb the function of paused polymerase-II. Therefore the reported mutation p.Asp1150tyr is probably pathogenic for this family.
Multiple sequence alignment revealed conservation of mutated amino acid (p.Asp1150Try) and nearby amino acids in 7 different species. Mutation site denoted with red arrow (Fig. 1D).

Discussion
Fragile X E (FRAXE) is an X-linked syndrome characterized by cognitive impairment, Speech delay and autistic behavior. AFF2 gene is reported to be involved in the syndrome. The AFF2 gene is responsible for making a protein which is present in the cell's nucleus but its function is not well known. It has been reported that this protein serves as a transcription factor, which binds to speci c regions of DNA and helps to control the activity of some other genes, though these genes are unknown [27]. Current investigation involves a family diagnosed with ID in two members and resulted in identi cation of novel missense mutation, c.3448G > T; p.Asp1150Tyr, in the coding exon 17 of AFF2 gene.
Three dimensional protein models revealed conformation changes of amino acids due to mutation. Point mutation altered AFF2 protein structure although it was a single base change. Figure 1C

Consent for Publication
Written informed consent for publication of this research work was obtained from the research participant and child's < 16 years of age written informed consent was obtained from their parents for the publication of their clinical and genetic data.

Availability of Data
Variant has been submitted to the NCBI database ClinVar, available online by using following Accession number: SCV001423533. All data related to paper available on request from corresponding authors

Competing Interests
Authors declare they have no competing interests.

Funding
The study was supported by higher education commission (HEC) of Pakistan under student scholarship program (award No: 1-8/HEC/HRD/2018/8590, NRPU7028). Microarray/exome sequencing, Sanger sequencing was carried out at RILD Wellcome Wolfson Center, Royal Devon, University of Exeter.