Abstract

   Schizophrenia (SCZ) is a complex psychiatric disorder with high
   heritability; identifying risk genes is essential for deciphering the
   disorder’s pathogenesis and developing novel treatments. Using
   whole-exome sequencing, we screened for mutations within protein-coding
   sequences in a single family of patients with SCZ. In a pathway
   enrichment analysis, we found multiple transmitted variant genes
   associated with two KEGG pathways: herpes simplex virus 1 (HSV1)
   infection and the extracellular matrix (ECM)–receptor interaction. When
   searching for rare variants, six variants, SLC6A19^p.L541R,
   CYP2E1^p.T376S, NAT10^p.E811D, N4BP1^p.L7V, CBX2^p.S520C, and
   ZNF460^p.K190E, segregated with SCZ. A bioinformatic analysis showed
   that three of these mutated genes were associated with chromatin
   modulation. We found that HSV1 infection, ECM–receptor interaction
   pathways, and epigenetic mechanisms may contribute to the pathogenesis
   of SCZ in certain families. The identified polygenetic risk factors
   from the sample family provide distinctive underlying biological
   mechanisms of the pathophysiology of SCZ and may be useful in clinical
   practice and patient care.

   Keywords: schizophrenia, family, whole-exome sequencing, rare mutation,
   pathway analysis

1. Introduction

   Schizophrenia (SCZ) is a chronic debilitating mental disorder
   characterized by abnormal perception, thought disturbances, bizarre
   behavior, and cognitive deficits [[32]1]. This disorder affects
   approximately 1% of the general population worldwide. Genetic factors
   play a critical role in its etiology; the heritability of SCZ has been
   estimated at 70–80%, with numerous transmitted variants possibly
   involved [[33]2]. However, discovering genes directly responsible for
   SCZ is challenging because of its genetic complexity and the epigenetic
   mechanisms involved, which possibly act as downstream effectors of
   environmental signals [[34]3].

   The current hypotheses of the genetic basis for SCZ include rare
   deleterious mutations with large clinical penetrance in some patients,
   psychosis in families, or the addition of multiple common polymorphisms
   with minor effects [[35]4,[36]5]. Identifying common and rare variants
   associated with SCZ could increase our understanding of the
   neurobiology of psychiatric disorders. In this regard, a genome-wide
   association (GWAS) study identified 108 SCZ-associated genetic loci
   [[37]4]. Additionally, multiple rare or ultrarare variants, such as
   loss-of-function, missense, and chromosomal abnormalities, have been
   associated with SCZ [[38]5,[39]6]. These studies indicate that the
   genetic underpinnings of SCZ are very complex and heterogeneous. One
   strategy to better understand the etiology of this disorder is through
   a family-based analysis of the contribution of rare and common genetic
   variants to the disorder with whole-exome sequencing (WES)
   [[40]7,[41]8,[42]9].

   Here, we discovered variants in protein-coding sequences in a
   schizophrenic family with WES, which we confirmed with Sanger
   sequencing. The identified variants in the familial form of SCZ could
   provide a new landscape of the genetic architecture and alternative
   treatment targets for SCZ.

2. Materials and Methods

2.1. Sample Family Recruitment

   The sample family was a nuclear family, including an unaffected father
   and a mother, son, and daughter with SCZ. All subjects were Han Chinese
   from Taiwan. The unaffected father and the two children were recruited
   for the WES experiment. All affected family members were diagnosed
   according to the Diagnostic and Statistical Manual of Mental Disorders,
   Fifth Edition (DSM-V) criteria. The study was approved by the ethics
   committee of the Antai Tian-Sheng Memorial Hospital Institutional
   Review Board; written informed consent was obtained after all
   procedures were fully explained to the participants.

2.2. WES

   Genomic DNA was prepared from white blood cells (WBCs) using a Gentra
   Puregene Blood Kit (QIAGEN). The quality of the genomic DNA was
   assessed on a NanoDrop One spectrophotometer (Thermo Fisher Scientific,
   Waltham, MA, USA). The 260/280 ratio for genomic DNA was ~1.8. The DNA
   library was prepared using 1 μg of genomic DNA from each sample using
   an xGenTM Exome Research Panel V2 (IDT, San Diego, CA, USA) following
   the manufacturer’s protocols with added index codes to attribute
   sequences to each sample. The library quality was assessed on a Qubit
   4.0 fluorometer and Agilent bioanalyzer system. Sequencing was
   conducted on an Illumina NovaSeq 6000 platform to generate paired 150
   bp reads, and the read coverage depth was 22×. The paired-end reads
   were analyzed for mapping to the human reference genome using the
   Burrows–Wheeler Aligner (v2.2.1) with default settings. The aligned
   reads were subsequently processed with a Genome Analysis ToolKit (GATK)
   (v4.2.1.0), including MarkDuplicates and Base Quality Score
   Recalibration. Variant calling for single-nucleotide variants (SNVs)
   and small insertion/deletion mutations was performed using a
   HaplotypeCaller. High-confidence variants were then obtained with the
   standard GATK Variant Quality Score Recalibration tool and annotated
   with ANNOVAR (v2020), SnpEff (v4.3), and VEP (v100.4); the data were
   then integrated using an in-house algorithm. Copy-number variations and
   structural variations were called using Control-FREEC (v11.6) and Manta
   (v1.6.0); next, the variants were annotated with AnnotSV (v3.0.9).

2.3. Mutation Validation with PCR-Based Sequencing

   All PCR primer sequences were designed using the Primer3 website
   ([43]http://bioinfo.ut.ee/primer3-0.4.0/primer3/, accessed on 10 August
   2022), and the primer sequences, optimal annealing temperatures, and
   size of each amplicon are available on request. In a standard PCR
   reaction, genomic DNA (50 ng) was amplified in a reaction volume of 25
   μL containing 2× PCR buffer for KOD FX, 2 mM dNTPs, and 1.0 U/μL of KOD
   FX (TOYOBO Co., Ltd., Osaka-Shi, Japan). The PCR cycling condition
   consisted of an initial denaturation at 95 °C for 5 min, followed by 30
   cycles of 95 °C for 1 min, and an optimal annealing temperature of each
   target for 1 min and 72 °C for 1 min. For sequencing, aliquots of PCR
   products were purified using an Illustra™ ExoProStar™ 1-Step Kit (GE
   Healthcare Bio-Sciences, Piscataway, NJ, USA) and sequenced using a
   BigDye™ Terminator v3.1 Cycle Sequencing Kit on a SeqStudio DNA
   Analyzer (Thermo Fisher Scientific) according to the manufacturer’s
   protocols. The identified mutations were verified with repeated PCR and
   sequencing in both directions.

2.4. Bioinformatic Analysis

   The variant coding effect was checked using publicly available
   prediction programs: Polyphen-2
   ([44]http://genetics.bwh.harvard.edu/pph2/, accessed on 8 August 2022),
   SIFT ([45]http://sift.bii.a-star.edu.sg/, accessed on 8 August 2022),
   and CADD ([46]https://cadd.gs.washington.edu/, accessed on 8 August
   2022). We also determined whether the mutations were documented in the
   public databases gnomAD ([47]https://gnomad.broadinstitute.org/,
   accessed on 8 August 2022) and Taiwan BioBank
   ([48]https://taiwanview.twbiobank.org.tw, accessed on 8 August 2022).
   For the pathway analysis, genes with identified variants were uploaded
   to the database for annotation, visualization, and integrated discovery
   (DAVID v6.8, [49]https://david.ncifcrf.gov/tools.jsp, accessed on 1
   August 2022) using the official gene symbol method. Genes with
   identified variants associated with the Kyoto Encyclopedia of Genes and
   Genomes (KEGG) pathways were highlighted and annotated [[50]10].

3. Results

3.1. Clinical Manifestations of SCZ in the Study Family

   [51]Figure 1 shows the nuclear family with an unaffected father (I:1),
   a mother with SCZ (I:2), a son with SCZ (II:1), and a daughter with SCZ
   (II:2). The SCZ seemed to be transmitted following a dominant
   inheritance pattern. At the time of the study, the father (I-1) was 61
   years old and had no history of psychiatric disorders. The mother (I-2)
   died in an accident ten years ago. The elder son (II-1) was 30 years
   old and was born full-term without remarkable events. He began
   experiencing psychotic symptoms at the age of 14 years. His psychotic
   symptoms included auditory hallucinations, bizarre behavior, and
   aggressive behavior toward his family. He had no history of illicit
   drug abuse, head injury, or seizure attack. He had poor insight and
   poor drug adherence. As a result, he was hospitalized in psychiatric
   wards several times due to the relapse of his symptoms. He led a lazy
   lifestyle with poor socio-occupational functioning. Finally, he was
   admitted to a psychiatric day ward for long-term rehabilitation. The
   younger sister (II-2) was 29 years old and manifested auditory
   hallucinations, bizarre delusions (about aliens and gods), bizarre
   behaviors, persecutory delusion, and psychomotor agitation from the age
   of 20 years when she was diagnosed with SCZ. She had marked positive
   symptoms with multiple relapses, partly due to poor compliance with
   medication. Later, she did not respond very well to antipsychotic
   drugs; therefore, clozapine was prescribed in combination with other
   antipsychotics. She was finally hospitalized in a chronic psychiatric
   ward due to unremitted psychotic symptoms and poor family support. The
   clinical findings for the patients are shown in [52]Supplementary Table
   S1.

Figure 1.

   Figure 1
   [53]Open in a new tab

   Pedigree of the sample family. Circles and squares denote females and
   males, respectively. Black color indicates family members with SCZ. The
   arrow indicates the proband.

3.2. WES Analysis

   A WES analysis was conducted on peripheral WBC samples from the three
   family members (I:1, II:1, and II:2), and the reads were mapped against
   the hg38 human reference genome. The statistics of the sequencing reads
   and the number of identified variants after variant calling for SNVs
   are listed in [54]Supplementary Table S2. A list of complete variants
   in the study samples is available on request. Next, the identified
   variants were further filtered to exclude non-protein-modifying
   variants.

3.3. Autosomal Dominant Analysis and Rare Mutation Identification

   An autosomal dominant transmission analysis identified 951
   protein-altering variants (within 736 genes) in the two affected
   children (II:1 and II:2) absent in the unaffected father (I:1)
   ([55]Supplementary Table S3). Furthermore, 736 genes were uploaded to
   the DAVID database for pathway analysis. According to the DAVID
   analysis (after multiple tests), multiple genes were associated with
   the following two KEGG pathways: herpes simplex virus 1 (HSV1)
   infection (hsa05168) and the extracellular matrix (ECM)–receptor
   interaction (hsa04512) ([56]Table 1). The detailed genetic and
   bioinformatic information of the variants identified in these two KEGG
   pathways is listed in [57]Table 1.

Table 1.

   Protein-altering variants in the HSV1 infection pathway and
   ECM–receptor interactions observed in the sample family under the
   autosomal dominant inheritance model.
   Gene Coding: Amino Acid Change rs Number Public Database MAF In Silico
   Analysis
   gnomAD Taiwan BioBank SIFT Polyphen-2 CADD^raw/phred
   Herpes Simplex Virus 1 Infection Pathway ^#
   ZNF573:[58]NM_001172692 c.A1303G:p.M435V rs3095726 0.831 0.3895 T B
   −0.073/0.943
   ZNF177:[59]NM_001172651 c.C335T:p.T112M rs2217652 0.61 0.408 T B
   −0.13/0.718
   ZNF177:[60]NM_001172651 c.A1363T:p.I455F rs2230752 0.566 0.4 D B
   0.744/8.855
   ZNF132:[61]NM_003433 c.C755T:p.P252L rs1465789 0.547 0.296 T B
   0.265/3.882
   ZNF132:[62]NM_003433 c.G608A:p.G203D rs1122955 0.174 0.0615 T B
   −1.234/0.002
   ZNF891:[63]NM_001277291 c.G1375C:p.V459L rs2173970 0.189 0.1555 T B
   1.398/15.07
   ZNF571:[64]NM_001321272 c.A1777G:p.K593E rs16973890 0.0148 0.062 T P
   2.087/19.92
   SP100:[65]NM_001080391 c.T2477C:p.M826T rs836237 0.797 0.4545 T B
   −1.434/0.001
   ZNF790:[66]NM_001242800 c.A902G:p.Q301R rs3745775 0.273 0.245 D B
   0.255/3.759
   ZNF44:[67]NM_001353551 c.A394G:p.T132A rs11879168 0.181 0.301 D B
   0.955/11.05
   ZNF529:[68]NM_001145650 c.T337G:p.L113V rs2912444 0.694 0.489 T B
   −0.538/0.099
   ZNF527:[69]NM_032453 c.A638G:p.H213R rs4452075 0.272 0.304 D B
   −0.35/0.245
   ZNF229:NM_00127851 c.G1966A:p.G656R rs1434579 0.27 0.3265 D P
   2.495/22.4
   ZNF229:NM_00127851 c.C991T:p.R331C rs12151338 0.274 0.3365 T P
   1.057/12.32
   ZNF568:[70]NM_001204839 c.G1696C:p.G566R rs1363753 0.57 0.3325 T B
   −0.68/0.048
   ZNF302:[71]NM_001012320 c.A266G:p.Y89C rs2290652 0.303 0.234 T P
   2.082/19.88
   ZNF565:[72]NM_001366190 c.G140T:p.G47V rs75766177 0.00356 0.102 T B
   −0.092/0.863
   ZNF543:[73]NM_213598 c.C163G:p.P55A rs6510057 0.528 0.295 T B
   0.407/5.505
   ZNF268:[74]NM_001165887 c.A220T:p.M74L rs61960670 0.182 0.137 T B
   −0.68/0.049
   ZNF268:[75]NM_001165881 c.G2579C:p.R860T rs62644541 0.182 0.137 D B
   2.755/22.9
   ZNF300:[76]NM_052860 c.5_7del:p.M2del rs72332188 0.027317 0.038 NA NA
   N/A
   ZIM3:[77]NM_052882 c.A1135G:p.I379V rs4801433 0.451 0.363 T P
   1.731/17.24
   ZIM3:[78]NM_052882 c.T205A:p.L69M rs4801200 0.45 0.363 T P 0.283/4.096
   ZIM3:[79]NM_052882 c.G82A:p.E28K rs2370134 0.143 0.2665 T P 0.174/2.83
   ZNF584:[80]NM_001318002 c.C298T:p.R100C rs200779956 0.000105 0.0615 T B
   0.202/3.141
   ZNF584:[81]NM_001363680 c.C289T:p.P97S rs11668789 0.178 0.061 T B
   −0.063/0.991
   ZNF484:[82]NM_001007101 c.G1397A:p.G466D rs3739602 0.0239 0.2785 D P
   2.451/22.3
   ZNF440:[83]NM_152357 c.A371G:p.N124S rs427880 0.515 0.3965 D B
   0.11/2.181
   ZNF440:[84]NM_152357 c.G1561A:p.G521R rs117998813 0.00385 0.0755 D B
   1.105/12.82
   ZNF285:[85]NM_001291491 c.G158A:p.S53N rs2571089 0.192 0.362 T B
   −0.604/ 0.071
   ZNF460:[86]NM_001330622 c.A568G:p.K190E ND ND ND D P 3/23.4
   ZNF284:[87]NM_001037813 c.369_371del:p.S124del rs139900131 0.473322
   0.3493 NA NA N/A
   ZNF382:[88]NM_001256838 c.C1637T:p.T546M rs61732180 0.162 0.188 T B
   0.854/9.938
   ZNF180:[89]NM_001278508 c.C740G:p.S247C rs1897820 0.532 0.59 D B
   1.601/16.38
   ZNF180:[90]NM_001278508 c.C192G:p.C64W rs2253563 0.517 0.5765 D P
   1.158/13.3
   ZNF180:[91]NM_001278508 c.T122C:p.V41A rs2571108 0.692 0.697 T B
   0.209/3.23
   ZNF57:[92]NM_001319083 c.C572A:p.T191N rs2288958 0.418 0.6085 T B
   −1.136/0.003
   ZNF816:[93]NM_001202456 c.C485T:p.S162L rs11084210 0.0929 0.2365 T B
   −0.468/0.138
   ZNF30:[94]NM_001099437 c.A1202G:p.Y401C rs765746 0.188 0.403 D P
   1.728/17.22
   ZNF439:[95]NM_001348724 c.C1013G:p.A338G rs72994214 0.023 0.08 D B
   0.772/9.12
   ZNF317:NM_00119079 c.G57T:p.Q19H rs3752199 0.154 0.117 T B 0.43/5.76
   ZNF416:[96]NM_001353405 c.C284T:p.S95L rs151324898 0.000952 0.035 T P
   0.947/10.97
   ZNF735:[97]NM_001159524 c.C670A:p.H224N rs4320434 0.224 0.2575 T B
   −1.599/0.001
   ZNF875:[98]NM_001329773 c.G1358T:p.S453I rs3745765 0.278 0.318 T B
   −0.005/1.302
   ZNF460:[99]NM_001330622 c.A568G:p.K190E ND ND ND D P 3/23.4
   ZNF333:[100]NM_001352243 c.C917T:p.A306V rs3764626 0.357 0.5945 T B
   0.635/7.848
   TLR3:NM_00326 c.C1234T:p.L412F rs3775291 0.236 0.361 D P 2.844/23.1
   IFNAR1:[101]NM_000629 c.G502C:p.V168 rs2257167 0.156081 0.354 T P
   1.153/13.25
   ECM–receptor interaction pathway ^$
   LAMA2:[102]NM_000426 c.G1856A:p.R619H rs3816665 0.257227 0.0725 T B
   −1.085/0.004
   LAMA2:NM_00107982 c.C7748T:p.A2583V rs2229848 0.370535 0.587 T P
   4.105/27.8
   VWF:[103]NM_000552 c.G6104A:p.G2035D rs186806674 0.000157 0.0045 T B
   0.811/9.502
   ITGA4:[104]NM_000885 c.G2633A:p.R878Q rs1143676 0.288230 0.8475 T B
   −0.899/0.014
   LAMC3:[105]NM_006059 c.C1564T:p.P522S rs869457 0.282728 0.2955 T B
   0.839/9.786
   LAMC3:[106]NM_006059 c.A1631G:p.E544G rs10901333 0.477113 0.471 T B
   2.173/20.7
   LAMC3:[107]NM_006059 c.C2308G:p.R770G rs3739510 0.845 0.6075 T B
   −0.095/0.851
   LAMC3:[108]NM_006059 c.A3244G:p.S1082G rs2275140 0.243787 0.663 T B
   0.421/5.661
   LAMA4:[109]NM_001105206 c.G5443A:p.V1815I rs3734292 0.001740 0.0755 D P
   3.647/25.2
   DMP1:[110]NM_001079911 c.A157T:p.S53C rs10019009 0.281 0.4205 D P
   3.056/23.5
   HMMR:[111]NM_001142557 c.G737A:p.R246H rs2303078 0.037807 0.04 T B
   0.075/1.873
   FRAS1:[112]NM_001166133 c.T1396A:p.L466I rs12504081 0.350818 0.3855 T P
   2.658/22.7
   FRAS1:[113]NM_001166133 c.A2060G:p.D687G rs345513 0.425 0.4485 T B
   0.327/4.597
   FRAS1:[114]NM_001166133 c.C2450T:p.A817V rs6835769 0.433817 0.197 T B
   0.351/4.87
   FRAS1:[115]NM_001166133 c.G3406A:p.E1136K rs12512164 0.242134 0.207 T P
   1.844/18.06
   TNN:[116]NM_022093 c.C3467T:p.A1156V rs2072036 0.114564 0.178 D B
   0.296/4.24
   COL6A2:[117]NM_001849 c.G2039A:p.R680H rs1042917 0.478396 0.43 D P
   3.684/25.3
   COL9A1:[118]NM_001377290 c.A1133G:p.Q378R rs1135056 0.382833 0.2785 T B
   0.922/10.68
   TNR:[119]NM_003285 c.G382T:p.A128S rs2239819 0.241553 0.514 T B
   1.383/14.97
   COL6A6:[120]NM_001102608 c.G5216A:p.R1739Q rs16830494 0.095544 0.1835 D
   B 2.068/19.77
   [121]Open in a new tab

   ^# Fold enrichment: 2.0736; Bonferroni: 0.0101; Benjamini: 0.0093; FDR:
   0.0093. ^$ Fold enrichment: 4.0982; Bonferroni: 0.0184; Benjamini:
   0.0093; FDR: 0.0093; MAF: minor allele frequency; ND: not documented;
   NA: not applicable; T: tolerated; D: damaging; B: benign; P: probably
   or possibly damaging.

   Among the 951 protein-altering variants segregated with SCZ, 55 had
   MAFs < 0.5% in the gnomAD and Taiwan BioBank databases, of which six
   variants (SLC6A19^p.L541R, CYP2E1^p.T376S, NAT10^p.E811D, N4BP1^p.L7V,
   CBX2^p.S520C, and ZNF460^p.K190E) were not reported in either of the
   two databases ([122]Table 2). Sanger sequencing confirmed the presence
   of these six rarely transmitted protein-altering variants in the sample
   family ([123]Figure 2).

Table 2.

   Protein-altering variants with minor allele frequency (MAF) < 0.5%
   observed in the sample family under the autosomal dominant inheritance
   model.
   Gene Coding: Amino Acid Change rs Number Public Database MAF In Silico
   Analysis
   gnomAD Taiwan BioBank SIFT Polyphen-2 CADD^raw/phred
   SSX2IP:[124]NM_001166294 c.G1492C:p.G498R rs191448293 0.0002900
   0.001648 D P 1.85/18.1
   SPAG17:[125]NM_206996 c.A4837G:p.N1613D rs200562117 0.00008007 0.000660
   T B 0.468/6.162
   STYXL2:[126]NM_001080426 c.C91T:p.R31X rs377245710 0.00005664 0.001318
   NA NA 7.113/37
   ZNF648:[127]NM_001009992 c.T1105C:p.C369R rs765064028 0.00005709
   0.000668 D P 3.636/25.2
   RGSL1:[128]NM_001137669 c.T226C:p.F76L rs1403931074 0.00004728 ND D P
   3.83/26
   EMILIN1:[129]NM_007046 c.G2366A:p.R789Q rs566620372 0.00007649 ND T P
   3.552/24.9
   ARHGEF33:[130]NM_001145451 c.C484G:p.P162A rs190587324 0.0001200
   0.000989 T P 2.677/22.8
   USP34:[131]NM_014709 c.A3444G:p.I1148M rs769808228 0.00007653 0.002307
   T B 1.537/15.97
   CLEC4F:[132]NM_001258027 c.1421delA:p.K474Sfs*44 rs781870818 0.00003535
   0.000330 NA NA NA
   STEAP3:[133]NM_001008410 c.G982A:p.A328T rs376635282 0.00002651 ND T P
   0.49/6.395
   MYO7B:[134]NM_001080527 c.A694C:p.I232L rs559170047 0.0001176 0.001979
   D P 3.607/25.1
   USF3:[135]NM_001009899 c.A934G:p.T312A rs192213473 0.0001033 0.000659 T
   B 0.336/4.707
   FIP1L1:[136]NM_001134938 c.1225_1226del:p.R413Gfs*3 rs143671659
   0.000160 ND NA NA NA
   NDST3:[137]NM_004784 c.A2027T:p.E676V rs757955201 0.00003188 0.000330 T
   B 2.585/22.6
   UCP1:[138]NM_021833 c.G242A:p.G81E rs555800104 0.0001272 0.000659 D P
   3.426/24.5
   SLC6A19:[139]NM_001003841 c.T1622G:p.L541R rs1176698743 ND ND D P
   3.562/24.9
   CENPK:[140]NM_001349368 c.G487A:p.V163I rs769017086 0.00008767 0.001649
   T P 2.989/23.4
   DMGDH:[141]NM_013391 c.T2324G:p.L775R rs201416183 0.000003992 ND D P
   4.262/29.2
   ZFYVE16:[142]NM_001284237 c.C2189T:p.T730I rs140823634 0.00006367 ND T
   B 1.397/15.06
   SLC22A4:[143]NM_003059 c.442_443del:p.L148Vfs*131 rs765247850
   0.00001988 0.000330 NA NA NA
   TCOF1:[144]NM_000356 c.C122T:p.A41V rs56180593 0.002966 0.003 D P 1.682
   CRYBG1:[145]NM_001624 c.G166C:p.E56Q rs201789082 0.0005189 0.002444 D P
   3.799/25.8
   COL10A1:[146]NM_000493 c.T788C:p.I263T rs200461789 0.00008876 0.000989
   T B 0.282/4.078
   CREB5:[147]NM_001011666 c.G671T:p.G224V rs142716067 0.0002903 0.006338
   D P 4.015/27.1
   SEMA3E:[148]NM_001178129 c.G1552A:p.V518I rs200779956 0.0001667
   0.003626 T B 1.179/13.47
   ATP6V1B2:[149]NM_001693 c.C7A:p.L3M rs201057159 0.0001301 0.001252 T P
   2.71/22.8
   MTSS1:[150]NM_001363300 c.G1181A:p.R394Q rs780673847 0.00001990
   0.000330 D P 3.739/25.6
   EXD3:[151]NM_017820 c.C707T:p.A236V rs370748438 0.0001587 0.000660 T B
   −0.38/0.212
   KIAA1217:[152]NM_001282769 c.T1169G:p.V390G rs780371689 0.00006011
   0.000330 D P 4.244/29.1
   SORBS1:[153]NM_001290296 c.C649G:p.R217G rs371784791 0.0002812 0.001995
   D B 0.928/10.74
   CYP2E1:[154]NM_000773 c.C1127G:p.T376S ND ND ND D B 1.605/16.41
   NAT10:[155]NM_001144030 c.G2433T:p.E811D ND ND ND D P 3.284/24.1
   PACSIN3:[156]NM_001184974 c.C82T:p.R28W rs185936979 0.0004014 0.002970
   D P 4.48/32
   OR8K5:[157]NM_001004058 c.C821A:p.A274D rs201996541 0.00005177 0.000989
   D P 1.472/15.55
   VWF:[158]NM_000552 c.G6104A:p.G2035D rs186806674 0.0004034 0.003626 T B
   0.811/9.502
   SLC39A5:[159]NM_001135195 c.C1517T:p.T506M rs2293511 0.0007447 0.004293
   T B −0.791/0.027
   DNAH3:NM_00134788 c.G10796A:p.R3599H rs750487156 0.0001485 0.001320 D P
   2.868/23.1
   PRSS53:[160]NM_001039503 c.C1004T:p.A335V rs188342896 0.0005128
   0.003955 T B 0.433/5.786
   N4BP1:[161]NM_153029 c.C19G:p.L7V ND ND ND T P 2.527/22.5
   CBX2:[162]NM_005189 c.C1559G:p.S520C ND ND ND D P 4.002/27
   GDF15:[163]NM_004864 c.G763A:p.A255T rs779865510 0.000008450 ND D P
   2.955/23.3
   ZNF784:[164]NM_203374 c.C298A:p.P100T rs558625495 0.0002610 0.004529 D
   B 1.118/12.94
   NLRP13:[165]NM_001321057 c.C2764G:p.P922A rs181274636 0.0001805
   0.004285 T B 0.173/2.827
   PEG3:[166]NM_001369735 c.G1736A:p.S579N rs532173382 0.00009551 0.000989
   T P 3.266/24
   ZNF460:[167]NM_001330622 c.A568G:p.K190E ND ND ND D P 3/23.4
   C20orf96:[168]NM_080571 c.C58G:p.Q20E rs756843278 0.00002503 0.001978 D
   P 1.474/15.57
   SALL4:[169]NM_001318031 c.T1262G:p.L421W rs758373411 0.0001061 ND D P
   3.88/26.2
   ZNF831:[170]NM_178457 c.G3106A:p.A1036T rs749543585 0.0001320 0.000330
   T B 0.618/7.683
   MCM3AP:[171]NM_003906 c.T5555C:p.M1852T rs182565117 0.0002757 0.004614
   T B 0.393/5.345
   IL17RA:[172]NM_001289905 c.G1817A:p.G606E rs761619526 0.0001468
   0.003067 T P −0.335/0.264
   AIFM3:[173]NM_001018060 c.A151C:p.T51P rs753362705 0.00009996 0.003014
   T B 0.363/5.011
   MYO18B:[174]NM_001318245 c.G5399A:p.R1800Q rs371283219 0.00004649
   0.000659 D P 4.151/28.2
   ELFN2:[175]NM_052906 c.G62A:p.R21H rs368781861 0.0001428 0.002125 T B
   0.686/8.33
   SCUBE1:[176]NM_173050 c.G2146A:p.G716S rs550015784 0.0001239 0.000342 T
   B −0.44/0.159
   SCUBE1:[177]NM_173050 c.G2141A:p.R714H rs535650437 0.00003703 0.000341
   D P 3.831/26
   [178]Open in a new tab

   MAF: minor allele frequency; ND: not documented; NA: not applicable; T:
   tolerated; D: damaging; B: benign; P: probably or possibly damaging.

Figure 2.

   [179]Figure 2
   [180]Open in a new tab

   Results of Sanger sequencing of the six ultrarare variants
   (SLC6A19^p.L541R, CYP2E1^p.T376S, NAT10^p.E811D, N4BP1^p.L7V,
   CBX2^p.S520C, and ZNF460^p.K190E) identified in this study. The blue
   box indicates the position of the identified missense mutations.

4. Discussion

   Identifying the specific genetic susceptibility and polygenetic risk
   factors for a family with SCZ is essential for establishing the
   molecular diagnosis, providing insight into the pathogenesis, and
   guiding the personalized treatment for each affected patient [[181]11].
   In this case, to search for the genetic underpinnings, we conducted a
   WES analysis of a nuclear family with an unaffected father, a son with
   SCZ, and a daughter with SCZ. Because the mother with SCZ died in an
   accident ten years ago, we did not collect her sample for the WES
   analysis. Given that this sample family is a classical SCZ family with
   a dominant inheritance pattern, we conducted an autosomal dominant
   transmission analysis to identify protein-altering variants in the two
   affected children but absent in the unaffected father. After the
   autosomal dominant analysis, we found multiple transmitted variant
   genes associated with two KEGG pathways: HSV1 infection and the
   ECM–receptor interaction. In addition, we detected six variants
   (SLC6A19^p.L541R, CYP2E1^p.T376S, NAT10^p.E811D, N4BP1^p.L7V,
   CBX2^p.S520C, and ZNF460^p.K190E) absent in the gnomAD and Taiwan
   BioBank databases, which indicated that these coding variants may be
   ultrarare and confer a risk for SCZ in this sample family. Among these
   ultrarare variants, three (CYP2E1^p.T376S, NAT10^p.E811D, and
   CBX2^p.S520C) may be associated with epigenetic mechanisms in the
   pathogenesis of SCZ. The above polygenetic risk factors we identified
   from the sample family provide distinctive underlying biological
   mechanisms for the pathophysiology of SCZ and may be helpful in
   clinical practice and patient care.

   Accumulating evidence from genetic and epidemiologic studies
   demonstrates that the link between immunological processes and
   neuropsychiatric disorders has increased [[182]12,[183]13,[184]14]. For
   example, an inflammatory state in the brain plays a role in developing
   neuropsychiatric disorders [[185]12]. A meta-analysis observed a
   positive association between non-neurological autoimmune disorders and
   psychosis [[186]15]. Furthermore, previous research highlighted a
   potential role for the immune system in autism spectrum disorder
   [[187]14]. Thus, it seems there is a role of immune systems in
   neuropsychiatric disorders, and understanding how they interact can
   improve our understanding of neuropsychiatric disorders and give rise
   to alternative treatments in psychiatry. Serological evidence has
   demonstrated that exposure to the neurotropic virus HSV1 is associated
   with cognitive deficits in individuals with SCZ [[188]16,[189]17]. In
   addition, neuroanatomic studies have revealed reduced brain gray matter
   volumes among patients with SCZ exposed to HSV1 compared with healthy
   subjects [[190]18,[191]19,[192]20]. Moreover, cognitive impairment in
   patients with SCZ could be associated with HSV1 infection due to the
   ability of HSV1 to infect neurons in brain areas involved in working
   memory and executive functions [[193]21]. Thus, HSV1 could contribute
   to the psychotic symptoms, behavioral abnormalities, and cognitive
   impairment that characterize SCZ. However, given the high worldwide
   prevalence of HSV1 infection, it may act additively or interact with
   other factors, such as genetic variation [[194]22]. For example, Prasad
   et al. observed that differences in gray matter variations in the
   prefrontal cortex among HSV1-exposed patients with SCZ and healthy
   controls were related to an exonic polymorphism of the MHC class I
   polypeptide-related sequence B gene [[195]23]. In the present findings,
   multiple missense variants segregated with SCZ were associated with the
   HSV1 infection pathway. Taken together, we speculate that a specific
   genetic susceptibility to HSV1 infection may contribute to the
   development of SCZ. However, the associations between these gene
   variants and HSV1 infection are complex. Further research is needed to
   fully understand the relationships between these gene variants and HSV1
   infection.

   ECM molecules, derived from neurons and glial cells, are located in
   extracellular space, and brain-ECM-interaction molecules are involved
   in synaptogenesis and GABAergic, glutamatergic, and dopaminergic
   neurotransmission [[196]24]. The brain ECM plays multiple roles in
   brain development, shaping synaptic plasticity, stability, cognitive
   flexibility, context discrimination, as well as the physiology and
   pathology of the central nervous system (CNS) [[197]25]. For example,
   research has demonstrated the contribution of extracellular glycans and
   glycoconjugates, major constituents of the neural ECM, to the etiology
   and pathogenesis of idiopathic autism spectrum disorders and other
   pervasive neurodevelopmental disorders [[198]26]. Several lines of
   evidence point to the involvement of the ECM in the pathophysiology of
   neuropsychiatric disorders such as autism, SCZ, and Alzheimer’s
   disease, suggesting possible etiological mechanisms linking the ECM and
   neuropsychiatric disorders [[199]26,[200]27,[201]28]. The
   pathophysiology of SCZ is characterized by substantial alterations and
   a functional impact of the ECM’s composition and integrity
   [[202]25,[203]27,[204]28]. Studies have demonstrated some ECM-related
   genes potentially associated with SCZ in animal models
   [[205]6,[206]29,[207]30,[208]31]. For example, a study demonstrated
   that patients with SCZ presented significantly lower levels of four
   brain ECM proteins (Lumican, a secreted acidic protein rich in
   cysteine, Nidogen-1, and Fibronectin) than healthy volunteers,
   suggesting that the brain ECM and its components are potential
   pharmacological targets to treat SCZ [[209]29]. Fraser extracellular
   matrix complex subunit 1 (FRAS1) encodes an ECM protein that appears to
   regulate epidermal–basement membrane adhesion and organogenesis during
   development [[210]30]. The ECM was disorganized in cortical and
   subcortical areas in Fras1 knockout mice, which exhibited many
   behavioral defects, including impaired egocentric spatial memory and
   aberrant olfactory learning and memory [[211]31]. De novo mutations in
   an ECM gene, LAMA2, were described in patients with SCZ [[212]6]. In
   addition, our previous study identified that the genetic deletion of an
   SCZ-susceptible gene, an activity-regulated cytoskeleton-associated
   protein, disturbed multiple genes involved in the ECM [[213]32]. Here,
   we identified multiple missense variants within ECM-related genes
   transmitted by a mother with SCZ. These findings point toward
   associations between SCZ and multiple missense mutations within
   ECM-related genes, consistent with previous reports implicating a
   pathophysiological dysregulation of the brain ECM proteins in SCZ.

   Although twin and adoption studies indicate that SZ has a genetic
   component with heritability [[214]33], environmental influences are an
   alternative explanation for the non-hereditary portion of schizophrenia
   [[215]3,[216]34]. Epigenetic modifications, which possibly act as
   downstream effectors of environmental signals, not only play a
   regulatory role in cell differentiation and the development of the
   brain but also as a mechanism underlying behavioral changes [[217]35].
   Research has demonstrated that epigenetic alterations, such as altered
   DNA methylation, histone modifications, and RNA interference, may
   provide an alternative explanation for the pathogenesis of SCZ
   [[218]36]. In the present study, we identified three ultrarare variants
   in three epigenetic-associated genes (CYP2E1, NAT10, and CBX2) that may
   be associated with the pathogenesis of SCZ. The cytochrome P450 family
   2 subfamily E member 1 (CYP2E1) gene encodes a member of the cytochrome
   P450 superfamily, being considered an oxidative-stress-related gene
   [[219]37]. CYP2E1 enzyme expression is differently influenced by
   epigenetic variation such as DNA methylation [[220]38]. DNA methylation
   levels in the promoter of the CYP2E1 gene have been associated with SCZ
   and tardive dyskinesia [[221]39]. In fact, a Chinese case–control study
   revealed that two CYP2E1 SNPs were associated with SCZ [[222]40]. Thus,
   it should be noted that (epi)genetic variations of the CYP2E1 gene play
   a role in SCZ. Further, N-acetyltransferase 10 (NAT10) is involved in
   epigenetic events, including histone acetylation and mRNA modification
   [[223]41,[224]42]. Recently, Guo and colleagues found that NAT10
   elevation in hippocampal neurons was related to anxiety- and
   depression-like behavior [[225]43]. Finally, Chromobox 2 (CBX2) encodes
   a component of the polycomb multiprotein complex, which is required to
   maintain the transcriptionally repressive state of many genes
   throughout development via chromatin remodeling and histone
   modification [[226]44]. Gu and colleagues reported that CBX2 inhibits
   neurite development by interacting with neuro-associated genes
   [[227]45]. Thus, we suggest that aberrant chromatin modulation and
   histone modifications due to CYP2E1, NAT10, and CBX2 mutations may have
   contributed to the pathogenesis of SCZ in this family, but an
   additional functional study is required.

   This study had the following limitations. First, we performed a
   whole-exome analysis in only one family. The present data may represent
   a specific genetic background in the sample family. Second, because we
   did not investigate whole-genome sequencing, several potentially
   valuable regions, such as non-protein-coding regions, were not
   sequenced.

5. Conclusions

   We hypothesize that multiple coding variants in two KEGG pathways (HSV1
   infection and the ECM–receptor interaction) indirectly affect the
   etiology of SCZ. With respect to rare variants, we hypothesize that
   damaging coding variants associated with epigenetic mechanisms are
   associated with SCZ, followed by ultra-rarely transmitted variants,
   given the evidence that newly arising mutations are enriched for more
   harmful mutation types. This present study provides insights with
   potential benefits for treating SCZ.

Supplementary Materials

   The following supporting information can be downloaded at
   [228]https://www.mdpi.com/article/10.3390/jpm13091392/s1, Table S1:
   Clinical findings of the sample family; Table S2: Summary of
   whole-exome sequencing statistics and variants found in study samples;
   Table S3: Identified protein-altering variants in two affected children
   (II:1 and II:2) absent in the unaffected father (I:1).
   [229]Click here for additional data file.^ (157.1KB, zip)

Author Contributions

   Conceptualization, Y.-C.H., L.-Y.P. and M.-C.C.; methodology, S.-H.H.,
   H.-Y.T. and M.-C.C.; formal analysis, S.-H.H., H.-Y.T. and M.-C.C.;
   investigation, Y.-C.H., L.-Y.P., S.-H.H., H.-Y.T. and M.-C.C.;
   resources, L.-Y.P. and M.-C.C.; writing—original draft preparation,
   Y.-C.H. and M.-C.C.; writing—review and editing, M.-C.C.; funding
   acquisition, M.-C.C. All authors have read and agreed to the published
   version of the manuscript.

Institutional Review Board Statement

   The study was conducted in accordance with the Declaration of Helsinki
   and approved by the Institutional Review Board of the Antai Medical
   Care Cooperation, Antai Tian-Sheng Memorial Hospital (approval number:
   18-144-A).

Informed Consent Statement

   Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

   The raw data are available upon the request of the corresponding
   author.

Conflicts of Interest

   The authors declare no conflict of interest. The funders had no role in
   the design of the study; in the collection, analyses, or interpretation
   of data; in the writing of the manuscript; or in the decision to
   publish the results.

Funding Statement

   This research was funded by the Yuli Branch, Taipei Veterans General
   Hospital, Taiwan, grant number VHYL108-008.

Footnotes

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References