Clinical characterization of patients with schizophrenia and 16p13.11 duplication: A case series

Abstract Background Chromosome 16p13.11 duplication is a well‐known genetic risk factor for schizophrenia (SCZ) (odds ratio = 1.84). However, no case reports focusing on patients with SCZ and 16p13.11 duplication have been published. Therefore, here, we report the detailed clinical cases of four patients with SCZ and 16p13.11 duplication who were identified in our previous whole‐genome copy number variant (CNV) study. Case Presentation In the four patients with SCZ and 16p13.11 duplication detected by array comparative genomic hybridization, one patient was found to have treatment‐resistant SCZ and an additional pathogenic rare CNV. Two of the four patients in this study had environmental risk factors that may have been involved in the development of SCZ. Conclusions The results of this case series suggest that a genetic cohort study would be useful for evaluating which genetic and environmental risk factors could better explain the variable expressivity of 16p13.11 duplication. Furthermore, this work could be useful for elucidating the pathophysiology of SCZ.


| INTRODUC TI ON
Hundreds of genetic variants with a high effect size for psychiatric disorders have recently been identified, and genetic counseling based on these high-effect variants has been performed. However, the detailed clinical course of carriers with these high-risk variants remains unclear. Therefore, it is important to accumulate knowledge about carriers with high-risk variants for psychiatric disorders to improve future genetic counseling.
Chromosome 16p13.11 duplication is one of the most wellknown risk factors for schizophrenia (SCZ) 1 (odds ratio [OR] = 1.84). 2 The pathophysiology of 16p13.11 duplication in SCZ is suggested to be derived from the genes related to the neurodevelopment in this region, such as nuclear distribution E homolog 1 (NDE1). 3 However, to our knowledge, no case reports focusing on patients with SCZ and 16p13.11 duplication have been reported. Therefore, here, we report detailed clinical cases of four patients with SCZ and 16p13.11 duplication who were identified in our previous copy number variant (CNV) study. 4

| Participants and phenotype evaluation
All patients were of Japanese ancestry and had been diagnosed with SCZ according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition. 5 We retrospectively collected clinical data from the medical records of four patients with SCZ and 16p13.11 duplication. The data evaluated included developmental history, family history, academic career, professional career, medical history, psychiatric symptoms, age at onset of SCZ, history of hospitalization, medications, response to pharmacotherapy, comorbidity of physical disease, and other clinical manifestations. Treatment-resistant schizophrenia (TRS) 6 is defined as the persistence of symptoms despite receiving ≥2 antipsychotic medications of an adequate dose (≥600 mg/day chlorpromazine equivalent 7 ) and duration (≥4 weeks at a therapeutic dosage) with documented adherence.
Controls used for the expression analysis were selected from the general population and had no history of mental disorders based on responses to questionnaires or self-reporting.

| Genetic analysis
Genomic DNA was extracted from blood samples. CNVs in the 16p13.11 region were identified in four patients with SCZ using two types of array comparative genomic hybridization: NimbleGen 720 k Whole-Genome Tiling Arrays (Roche NimbleGen) and Agilent SurePrint G3 Human CGH 400 K (Agilent Technologies). 4 We generated CNV calls using Nexus Copy Number software, v9.0 (BioDiscovery). All genomic locations are given in hg18 coordinates.

| Expression analysis of the NDE1 gene in LCLs
To investigate the effect of 16p13.11 duplication on NDE1 mRNA transcription, we compared the relative expression of a patient (Patient 2) and 16p13.11 duplication with 55 healthy controls (40.5 ± 11.8) using lymphoblastoid cell lines (LCLs). LCLs were established by Epstein-Barr virus transformation of lymphocytes and cultured in RPMI-1460 medium containing 20% fetal bovine serum, penicillin, and streptomycin. Total RNA was extracted from LCLs using the RNAqueous Kit (Invitrogen) and treated with DNase using the TURBO DNA-free™ Kit (Invitrogen), then reverse-transcribed to cDNA using the high-capacity RNA-to-cDNA Kit (Invitrogen). Two housekeeping genes, hypoxanthineguanine phosphoribosyltransferase (HPRT) and glucuronidase-beta Measurement of the cycle threshold was implemented in duplicate.
The data, including the amplifying efficiency and relative expression on quantification, were analyzed using the 2 -ΔΔ C T method. Patient 1 was a 79-year-old female with hypertension and hyperlipidemia. At age 7 years, her father passed away from pneumonia. Her grades at junior high school were above average. After graduating from junior high school, she was employed as an office worker. At age 22 years, she developed delusions and hallucinations with soliloquy and excitation. Subsequently, she was diagnosed with SCZ and hospitalized repeatedly. At the time of this study, she had been hospitalized for 30 years from age 49 years. Due to her negative symptoms, such as abulia and social withdrawal, she did not engage with other patients in the hospital. At study recruitment, she was prescribed risperidone (2 mg/day) and estazolam (1 mg/day).

| Case presentations
However, her response to these psychotropic drugs was poor. The results of expression analysis of NDE1, which is located in the 16p13.11 region. We compared the relative expression of a patient with SCZ and 16p13.11 duplication (Patient 2) with 55 healthy controls using lymphoblastoid cell lines. Box plot: the box represents the middle 50% of observations. The middle bold line represents the median gene expression. Whiskers represent the minimum and maximum observations. Each dot represents the relative expression of each sample calculated by the 2 -ΔΔ C T method. The orange dots represent the relative expression of controls (CON). The blue dot represents the relative expression of the carrier (Patient 2) with 16p13.11 duplication, and was higher than the first quartile of healthy controls. The relative quantity of 16p13.11 dup (n = 1) was 1.98, and the median (interquartile range) of CON (n = 55) was 1.00 (0.16). hyperthymia and unrestrained behavior, followed by a manic state with verbosity, hyperactivity, extravagant behavior, and violence, leading to hospitalization. At study recruitment, she was prescribed levomepromazine (50 mg/day), chlorpromazine (200 mg/day), and carbamazepine (400 mg/day); this pharmacotherapy was effective for her psychiatric symptoms.

| DISCUSS ION
To our knowledge, this is the first case series focusing on patients with SCZ and 16p13.11 duplication.
Although 16p13.11 duplication is a well-known risk factor for SCZ (OR = 1.84), 2 it is not sufficient to cause SCZ. In other words, additional genetic or environmental factors may be involved in the development and severity of SCZ as a second hit. 9 Patient 2 with TRS had 16p13.11 duplication and VPS13B deletion. Patients with SCZ with two pathogenic CNVs are more likely to show severe clinical manifestations. 10 Considering that patient 2 had TRS, antipsychotic treatment with clozapine was chosen. NDE1 mRNA in the LCLs from patient 2 was high compared with the control samples. Regarding the effects of 16p13.11 duplication on the phenotypes of patient 2, we speculate that alterations in NDE1 dosage may affect brain development, as argued by Houlihan and Feng. 11 In patients 1 and 3, environmental risk factors may have been involved in the development of SCZ. These patients experienced childhood adversities such as parental loss or separation. Such adversities are associated with a more than threefold increased risk of SCZ. 12 Although 16p13.11 duplication has been reported to be associated with neurodevelopmental disorders (e.g., intellectual disability, autism spectrum disorder) and cardiovascular disease, 13 no clinical information on these disorders was identified in this case series. This may be due in part to our limited access to health records. Moreover, the patients in this series may not be representative of individuals with 16p13.11 duplication because we only reported four patients with 16p13.11 duplication and SCZ.
Therefore, a genetic cohort study would be useful for evaluating which genetic and environmental risk factors could better explain the variable expressivity of 16p13.11 duplication. 14 Furthermore, to elucidate the molecular pathophysiology and drug development for SCZ, developing disease models such as induced pluripotent stem cells derived from carriers with 16p13.11 duplication would be useful. 15 We believe that this work could be useful for elucidating the pathophysiology of SCZ.

ACK N OWLED G M ENTS
We thank all the patients and their families for participating in this study. We also thank Mami Yoshida, Kiyori Monta, and Yukari Mitsui for their technical assistance.

DATA AVA I L A B I L I T Y S TAT E M E N T
In light of the ethical concerns surrounding the inclusion of detailed clinical information in this article, we make the raw data for CNVs identified in four patients available upon reasonable request to the corresponding author.

E TH I C A L A PPROVA L
This study was approved by the ethics committee of Nagoya