Derivation of four iPSC lines from a male ASD patient carrying a deletion in the middle coding region of NRXN1 α gene (NUIGi039-A and NUIGi039-B) and a male sibling control (NUIGi040-A and NUIGi040-B)

NRXN1 deletions are commonly found in autism spectrum disorder (ASD) and other neurodevelopmental/neuropsychiatric disorders. Derivation of induced pluripotent stem cells (iPSCs) from different diseases involving different deletion regions are essential, as NRXN1 may produce thousands of splicing variants. We report here the derivation of iPSCs from a sibling control and an ASD proband carrying de novo heterozygous deletions in the middle region of NRXN1 , using a non- integrating Sendai viral kit. The genotype and karyotype of the iPSCs were validated by whole genome SNP array. All iPSC lines highly expressed pluripotency markers and could be differentiated into three germ layers

NRXN1 deletions are commonly found in autism spectrum disorder (ASD) and other neurodevelopmental/neuropsychiatric disorders. Derivation of induced pluripotent stem cells (iPSCs) from different diseases involving different deletion regions are essential, as NRXN1 may produce thousands of splicing variants. We report here the derivation of iPSCs from a sibling control and an ASD proband carrying de novo heterozygous deletions in the middle region of NRXN1, using a nonintegrating Sendai viral kit. The genotype and karyotype of the iPSCs were validated by whole genome SNP array. All iPSC lines highly expressed pluripotency markers and could be differentiated into three germ layers.
Resource Table: Unique stem cell lines identifier

Resource utility
The ND1 iPSCs from an ASD patient at the severe end of ASD spectrum may add to resources for coupling NRXN1 regions with the severity of clinical and cellular phenotype. The sibling control iPSC lines may minimize genetic background effects and help creation of closely related isogenic lines for phenotypic validation.

Resource details
Autism spectrum disorder (ASD) is a common neurodevelopmental disorder associated with a spectrum of core clinical symptoms and an array of central and peripheral comorbidities, however, different individuals may have different types and/or severity of clinical presentations (Reilly et al., 2017). It affects patients from early childhood with no cure. The drug development is complicated by the fact that hundreds of rare genetic risk factors are involved. The deletions of NRXN1 encoding presynaptic Neurexins are known as one of the strongest rare risk factors (Pinto et al., 2014;Al Shehhi et al., 2019), however, NRXN1 deletions/mutations are also found in other brain disorders including schizophrenia, intellectual disability, epilepsy and developmental delay (Grayton et al., 2012). Human stem cell modelling including 3-D organoids may help understanding how NRXN1 lesions in different individuals lead to different clinical symptoms, and if different NRXN1 regions are associated with specific NRXN1 function and/or clinical severity. A differential second hit of risk factors may also be present in different family background which may involve in expressing distinct clinical symptoms, therefore, the availability of sibling control cells are important from this point of view to minimize the genetic background effect and to create closely related isogenic cells to understand regional function of the NRXN1 gene.
We report the generation of four iPSC lines in this study using Sendai virus vectors expressing four Yamanaka reprogramming factors. The iPSCs were derived from skin biopsy of a 4-year male sibling and a nonverbal 8-year-old ASD male with severe intellectual disability, infant seizures, developmental delay, self-injurious and aggressive behaviour. The proband carries 328,067 bp heterozygous deletion (de novo) from intron 5 to intron 15 of NRXN1α +/− gene (chr2:50485874-50813940, Hg38, Table 1). All the derived iPSC lines showed a large nucleus/ cytoplasm ratio, small cell body and grew in tightly packed colonies with no obvious cell boundary ( Fig. 1A). High alkaline phosphatase activity were confirmed by AP staining (Fig. 1B). Strong anti-OCT4, SSEA4, SOX2 and NANOG staining as the pluripotency markers were revealed by immunocytochemistry (Fig. 1C). Abundant mRNA expression of endogenous OCT4, SOX2 and NANOG genes were demonstrated by qPCR for cells at P15 (Fig. 1G). All iPSC lines could form embryoid bodies and spontaneously differentiated into three embryonic germ layers with positive staining for endoderm marker α-fetoprotein (AFP), mesoderm maker α-smooth muscle actin (α-SMA), and ectodermal marker βIII-tubulin (TUJ1) (Fig. 1D).
Whole genome SNP array was done on all lines at P5-8. An internal control was created from 83 samples which filtered out non-specific CNVs. One copy deletion at chr2:50485874-50813940 (Hg38, NRXN1α +/− ) was revealed by IGV software in the proband iPSCs ( Fig. 1E, Supplementary Fig. 1), with no other consistent/specific CNVs among the family members (Table 1, Supplementary Fig. 1). The iPSCs were shown free of transgene integration at P15 (Fig. 1H). All lines were PCR-negative for mycoplasma contamination at P14, with an internal PCR control (500 bp) in all reactions and 330 bp band resulted from the commercial mycoplasma template (Fig. 1F). The finger printing of iPSCs and fibroblasts were performed both commercially and by PCR in own laboratory. The data will be archived with the journal as per the instructions. The STR profiles of the two siblings are identical except for one locus (D21S2055), which demonstrates the limits of DNA identification when siblings are involved (Zaken et al., 2013).
The full characterization is summarized in Table 2 and Fig. 1. These NRXN1α +/− ASD iPSC lines and sibling control lines will become valuable resources for investigating ASD molecular mechanisms and NRXN1 regional function.
Transgene-free confirmation: Total cellular RNA were extracted by RNeasy Mini Kit (Qiagen), reversely transcribed to cDNA with  (Table 3). DNA fingerprinting analysis: The iPSC lines were confirmed by PCR with primers of eight STR loci (Table 3).
Pluripotency validation: Alkaline Phosphatase Staining Kit II (Stemgent) was used to demonstarte the Alkaline phosphatase activity. The iPSCs were fixed in 4% PFA for 20 min, permeabilized with 0.1% Triton X-100 (Sigma) for 15 min, and blocked for 1 h in 1% BSA-DPBS, and incubated with anti-OCT4, SSEA4, SOX2 or NANOG (Table 2) at 4 • C overnight, then with secondary antibodies and Hoechst 33,342 (Life Technologies) before imaging under Olympus FluoView 1000 system. Expression of endogenous pluripotency genes were detected by qRT-PCR with Fast SYBR™ Green Master Mix (Applied Biosystems) and specific primers listed in Table 3. The OCT4, SOX2 and NANOG expression was adjusted with GAPDH and converted to log2 fold of expression over fibroblast mRNA (negative control).
Three germ layer differentiation: Detached iPSC colonies were shaken at 50 rpm on an orbital shaker inside a 37 • C incubator for 5 days in DMEM/F12 medium supplemented with 20% FBS, 1% MEM NEAA, 1% L-Glutamine (200 mM), 0.2% β-mercaptoethanol and 1% penicillin-streptomycin to form embryonic bodies (EB). EBs were then cultures on Geltrex-coated 8-well chambers (iBiDi) for 3-4 weeks. Sspontaneously differentiated cells were fixed and tested by immunocytochemical staining for expression of AFP, α-SMA and TUJ1 (Table 3).  Karyotyping: The molecular karyotype was analyzed with 990 k SNP array by Beijing Hyslar Biotech Limited Corporation (Beijing, China). SNP data was analyzed by Axiom Analysis software (ThermoFisher, USA). LogR ratio and B allele plots were generated using an internal control created from 83 samples. The molecular karyotyping of fibroblasts and iPSC lines were visualized with IGV software.
Mycoplasma detection: The free of mycoplasma contamination was tested using the MycoSensor PCR Assay Kit (Agilent), following manufacturer's instructions.