Comprehensive phenotyping of neuropsychiatric traits in a multiplex 3q29 deletion family: a case report

Background 3q29 deletion syndrome is associated with a range of medical, neurodevelopmental, and psychiatric phenotypes. The deletion is usually de novo but cases have been reported where the deletion is inherited from apparently unaffected parents. The presence of these unaffected or mildly affected individuals suggests there may be an ascertainment bias for severely affected cases of 3q29 deletion syndrome, thus the more deleterious consequence of the 3q29 deletion may be overestimated. However, a substantial fraction of 3q29 deletion syndrome morbidity is due to psychiatric illness. In many case reports, probands and transmitting parents are not systematically evaluated for psychiatric traits. Here we report results from a systematic phenotyping protocol for neurodevelopmental and neuropsychiatric traits applied to all 3q29 deletion carriers in a multiplex family. Case presentation Through the 3q29 registry at Emory University, a multiplex family was identified where three offspring had a paternally inherited 3q29 deletion. We evaluated all 4 3q29 deletion family members using our previously described standardized, systematic phenotyping protocol. The transmitting parent reported no psychiatric history, however upon evaluation he was discovered to meet criteria for multiple psychiatric diagnoses including previously undiagnosed schizoaffective disorder. All four 3q29 deletion individuals in the pedigree had multiple psychiatric diagnoses that interfered with quality of life and prohibited successful academic and occupational functioning. Cognitive ability for all individuals was average or below average, but within the normal range. Conclusions This is the first case report of inherited 3q29 deletion syndrome where all affected individuals in the pedigree have been comprehensively and systematically evaluated for neurodevelopmental and psychiatric symptoms, using a standard battery of normed instruments administered by expert clinicians. Our investigation reveals that individuals with 3q29 deletion syndrome may have psychiatric morbidity that is debilitating, but only apparent through specialized evaluation by an expert. In the absence of appropriate evaluation, individuals with 3q29 deletion syndrome may suffer from psychiatric illness but lack avenues for access to care. The individuals evaluated here all have cognition in the normal range alongside multiple psychiatric diagnoses each, suggesting that cognitive ability alone is not a representative proxy for 3q29 deletion-associated disability. These results require replication in a larger cohort of individuals with 3q29 deletion syndrome.


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Conclusions: This is the first case report of inherited 3q29 deletion syndrome where all affected individuals in the pedigree have been comprehensively and systematically evaluated for neurodevelopmental and psychiatric symptoms, using a standard battery of normed instruments administered by expert clinicians. Our investigation reveals that individuals with 3q29 deletion syndrome may have psychiatric morbidity that is debilitating, but only apparent through specialized evaluation by an expert. In the absence of appropriate evaluation, individuals with 3q29 deletion syndrome may suffer from psychiatric illness but lack avenues for access to care. The individuals evaluated here all have cognition in the normal range alongside multiple psychiatric diagnoses each, suggesting that cognitive ability alone is not a representative proxy for 3q29 deletion-associated disability. These results require replication in a larger cohort of individuals with 3q29 deletion syndrome.
While the syndrome is most frequently de novo, 20-30% of cases are inherited [28]. At least eight multiplex families have been reported in the literature, and the parent from whom the deletion is inherited is most often described as "apparently unaffected" or "mildly affected" [1,13,[15][16][17][18][19]. These descriptions fuel suspicion that there are individuals in the general population who have the 3q29 deletion but manifest few of the phenotypic consequences. Mildly affected individuals are less likely to be referred for genetic testing, creating an ascertainment bias in favor of the most clinically overt cases rising to medical attention. This bias may result in overestimation of the disability associated with the 3q29 deletion phenotype.
However, in reports of multiplex families identifying these "mildly affected" or "apparently unaffected" individuals, the transmitting parent is typically evaluated with only a physical examination (Table 1) [1,13,[15][16][17][18][19]. Neurodevelopmental and neuropsychiatric phenotypes have not been directly evaluated. These data may be extrapolated from the medical history and usually focus on the sole dimension of cognitive ability. As our appreciation of the myriad neuropsychiatric manifestations associated with 3q29 deletion syndrome has increased over time, it is important to reevaluate whether these transmitting parents are truly unaffected.
Through the 3q29 registry, we ascertained a multiplex family with three children, all with a paternally inherited 3q29 deletion. We evaluated all four affected individuals for neuropsychiatric traits, using normed, gold-standard instruments as part of our previously described comprehensive phenotyping protocol [29]. These data show that even within a single family there can be wide-ranging heterogeneity of phenotypes, and these psychiatric phenotypes may go undetected without focused evaluation by appropriate diagnosticians. Our data also reveal that in 3q29 deletion syndrome, individuals may exhibit substantial neuropsychiatric morbidity even when cognitive function is preserved. We conclude that periodic screening for neuropsychiatric illness should be prioritized for all individuals with 3q29 deletion syndrome, without regard for the presence of intellectual disability.

Methods
A 4-person multiplex family ( Fig. 1) was ascertained through the 3q29 registry (3q29.org [3], housed and maintained at Emory University), and enrolled in our ongoing research study. This study was approved by Emory University's IRB (#IRB00088012). After an informed consent process with the parents was conducted by phone, we arranged the study visit. Medical records were obtained to confirm the genetic diagnosis. Informed consent was repeated in person at the beginning of the visit. Assent was also obtained from each of the children. Each family member was evaluated using our standardized and comprehensive phenotyping protocol with gold-standard instruments ( Table 2, [29]). Study subjects were evaluated for cognitive ability [40,41], anxiety [31,37], executive function [33,34], adaptive behavior [35], graphomotor ability [36], social disability [38,39], psychosis spectrum symptoms [31,32], and general psychopathology [30,31]. All study subjects also had a medical history interview and physical exam conducted by an experienced medical geneticist. Instruments were scored according to established guidelines. Diagnostic cutoffs were determined based on published criteria; expert clinicians administered and scored all instruments and interpreted the scores to arrive at clinician best-estimate diagnoses. Quantitative scores were also extracted from each instrument. At the study visit, we obtained a blood sample from each family member. All blood samples were processed with an optical mapping technology (Bionano Genomics, San Diego CA) to confirm coordinates of the 3q29 deletion. Each family member was confirmed to have the canonical 1.6 Mb 3q29 deletion with identical breakpoints chr3:195998740-197,667,295 (hg38).

Case presentations
Individual II-1 (Proband): 9 yo female History The proband was a term 3.18 kg infant delivered by Caesarean section (C-section) due to breech presentation. Pregnancy was otherwise uncomplicated. The child exhibited a delay in her gross motor milestones, starting to walk around 2 years of age. Other milestones were met as expected. At age 4, the child started school and some behavioral difficulties were noted. Also emerging at this time were the presence of focal seizures, café-au-lait macules, and high blood pressure secondary to renal artery stenosis (treated surgically with a stent). A clinical diagnosis of neurofibromatosis 1 (NF1) is documented in her medical records by her pediatrician at age 4 years 7 months. Existing behavioral abnormalities in the proband were atypical for NF1 prompting a chromosome microarray that detected a 1.6 Mb 3q29 deletion. The clinical diagnosis of NF1 was confirmed by our medical geneticist (MJG) upon physical examination; thus this individual has both NF1 and 3q29 deletion syndrome.  The child has poor weight gain due to food-related fussiness and retching. Current medications include two standard medications for blood pressure control, atenolol (beta blocker) and Lisinopril (ACE inhibitor); and carbamazepine, a standard anti-convulsant widely used for treating epilepsy. Of these medications only atenolol is not approved for use in children, though off-label use in pediatric cases is documented. At the time of our evaluation, the proband was receiving special education services for attention deficit hyperactivity disorder (ADHD).

Neuropsychiatric testing
This child was found to have a full scale IQ (FSIQ) of 82 (12th percentile), with verbal, nonverbal reasoning, and spatial reasoning subtest scores of 90, 74, and 91, respectively (noting a significantly lower nonverbal score). Her adaptive behavior (composite score = 65, 1st percentile) is lower than would be expected given her age and cognitive ability, indicating challenges with independent living skills. She also has clinically significant deficits in executive function (Tscore 86, > 99th percentile). Her visual-motor integration score (standard score 92, 30th percentile) was within the average range, but her motor coordination subtest score (standard score = 67) revealed motor control deficits compared to below average visual perception (standard score = 82). Upon evaluation with the Kiddie Schedule for Affective Disorders and Schizophrenia (K-SADS), the child was found to meet criteria for ADHD, combined type. She does not exhibit anxiety, ASD (although social vulnerabilities

Neuropsychiatric testing
This child was found to have a FSIQ of 79 (8th percentile), with verbal, nonverbal reasoning, and spatial reasoning subtest scores of 86, 80, and 79, respectively. His adaptive behavior score of 59 falls below the 1st percentile for his age and denotes significant delays in his adaptive functioning. He has clinically significant deficits in executive function (T-score = 88, > 99th percentile). Graphomotor weakness is present, indicated by a Visualmotor integration (VMI) standard score of 77, which falls at the 6th percentile for his age. On the VMI, he also showed significant motor coordination deficits (standard score = 45, < 1st percentile) compared to below average visual perception (standard score = 80, 6th percentile). Upon evaluation with the K-SADS, this child was found to qualify for diagnoses of ADHD, combined type; disruptive mood dysregulation disorder; and conduct disorder with childhood onset.

Neuropsychiatric testing
FSIQ was measured at 96 (39th percentile), with verbal, nonverbal reasoning, and spatial reasoning subtest scores of 103, 98, and 91, respectively. His adaptive behavior score of 68 (2nd percentile) indicates significant delays in his adaptive functioning given his age and cognitive ability. Due to young age, executive function, visualmotor integration, prodrome/psychosis, and anxiety were not evaluated (instruments are not normed at age 4). Symptoms endorsed by his mother using the KSADS parent interview revealed that this child was found to qualify for diagnoses of ADHD, hyperactive type. No evidence of ASD was present.

Individual I-1: 39 yo male History
The father's genetic testing was completed when he was 34 years of age, after his daughter (the proband) was diagnosed with 3q29 deletion syndrome. He reports that he had early learning challenges. After graduating high school, he completed 3 years of postsecondary education, but did not complete his degree. He has held several jobs in the past but is currently unemployed and living on governmental assistance. He is working on obtaining a driver's license. His father and paternal grandmother are both reported to have schizophrenia; these individuals were not available for genetic testing nor direct evaluation by our team. , and an independent qualitative evaluation by our team psychiatrist (JFC), the study subject endorsed multiple symptoms consistent with psychosis, including bizarre beliefs and auditory and visual hallucinations. Based on the study subjects' report, his unusual sensory experiences most often coincided with mood disturbances and was therefore consistent with a diagnosis of schizoaffective disorder.

Discussion and conclusions
Our team has performed direct, systematic evaluation of physical, neurodevelopmental, and psychiatric phenotypes using gold-standard instruments in members of a multiplex family where four related individuals have the 3q29 deletion. Physical manifestations are moderate and include subtle facial dysmorphology, an inability to gain weight, sleep abnormalities, and mild musculoskeletal abnormalities (Table 3). In contrast, the burden of psychiatric illness among the 3q29 deletion carriers in the pedigree is substantial (Table 4). All members of the pedigree qualify for a diagnosis of ADHD and have significant delays in adaptive behavior that are far greater than expected given their level of cognitive functioning. In fact, individual I-1's (proband's father) adaptive deficits were evident in his inability to sustain a job and function independently in many aspects of his life. Three individuals who were evaluated have clinically significant deficits in executive function, and two children have deficits in motor coordination. In two individuals, anxiety disorders were present, and in these same two individuals, evidence of a psychotic disorder is present. Individual II-1 (the proband) had a prior diagnosis of ADHD but no other neurodevelopmental or psychiatric diagnoses were reported in any member of the family. These data provide further evidence that the disability associated with 3q29 deletion syndrome is predominantly in the neuropsychiatric domain. The high burden of mental illness associated with the 3q29 deletion is an impediment to quality of life and independent functional living, but may be undetected without deliberate and focused evaluation.
Prior reports of multiplex families where transmitting parents are reported as "unaffected" or "mildly affected" have had an outsized effect on our interpretation of the 3q29 deletion phenotypic spectrum, introducing skepticism about the burden of illness associated with the deletion [1,13,[15][16][17][18][19]. The existence of unaffected transmitting parents implies that there are individuals with the 3q29 deletion in the general population who are functioning within the average or above average range. These higher functioning individuals are not typically ascertained through genetic testing, thus it has been suggested that only the most severe cases of 3q29 deletion syndrome are described in the literature [10]. The implication of prior multiplex case reports is that the severity of the 3q29 deletion phenotype is overestimated. However, a reexamination of these multiplex reports reveals that phenotypic evaluation of transmitting parents has been limited to physical traits only, with no formal evaluation of cognitive ability or psychiatric illness. Many of these multiplex reports date back 10 years or more, when the association between the 3q29 deletion and psychiatric illness was not appreciated. In light of new findings that the 3q29 deletion is associated with generalized anxiety disorder, social disability, and increased risk for both autism and schizophrenia [2][3][4][5][6], it is appropriate to reconsider prior multiplex reports. Without direct evaluation and deliberate solicitation of psychiatric phenotypes, it is not known whether the transmitting parents reported in past case reports are truly unaffected. We note that in the multiplex pedigree we report on here, if criteria from prior case reports were used, the transmitting father would also have been judged to be only mildly affected. It was only through direct evaluation with appropriate instruments, and consideration of his psychosocial functioning and history, that his degree of disability emerged. Our data suggest a competing hypothesis: the adverse manifestations of the 3q29 deletion phenotype may be underestimated, as many prior case reports may not have ascertained the full spectrum of neurodevelopmental and psychiatric phenotypes in individuals with the 3q29 deletion.
In early descriptions of 3q29 deletion syndrome, subjects were noted to have "mild to moderate intellectual disability." Subsequent case reports have therefore typically focused on the dimension of cognitive ability. However, in at least one prior report there is a hint that 3q29 comorbidity may be uncoupled from intellectual function. Cobb et al. reported on a 6.75 year old male with a de novo 3q29 deletion, with full-scale IQ measured at 84, well within the normal range [10]. However, this child was noted to have features consistent with autism and ADHD, inattentive type. The phenotypic features of Cobb et al's patient are consistent with the individuals reported here, where cognitive measures for all 3q29 deletion individuals in the pedigree are within the normal range, yet multiple comorbid psychiatric diagnoses are present in each individual. Taken together, these data imply that the psychiatric phenotypes observed in 3q29 deletion syndrome are partially independent of intellectual function, and further suggest that the measure of cognitive ability is not a useful proxy for overall behavioral disability in individuals with 3q29 deletion syndrome. A larger sample of well-phenotyped 3q29 deletion study subjects is required to confirm these findings.
3q29 deletion syndrome has a heterogeneous presentation with variable penetrance of medical, neurodevelopmental and neuropsychiatric phenotypes [2,3,7]. This has been seen in other genomic disorders, such as 22q11.2 deletion syndrome [42] and 16p11.2 deletion and duplication [43,44]. Various hypotheses have been invoked to explain this heterogeneity, including the presence of individual rare or common genetic variants that may act as modifier loci [45], genetic background effects [46], and/or polygenic risk scores [47,48]. In the family evaluated in the current study, there is concordance among multiple neurodevelopmental and neuropsychiatric phenotypes that are exhibited (ADHD, executive function, adaptive behavior, visual-motor deficits are present in all evaluated) or not present (no member of the pedigree was diagnosed with autism spectrum disorder), suggesting that either these phenotypes are less sensitive to modifying influences, or the possible modifiers (whether genetic or environmental) are present in all family members. In contrast medical phenotypes are more variable, as are measures of intellectual function. FSIQ ranges from low (79) to average (96). In addition, two members of the pedigree were found to have a psychotic disorder. Individual III-3 was only 4 years of age at the time of evaluation; at this age psychotic phenotypes cannot be reliably assessed. The proband (Individual II-1) does not exhibit psychotic features at this time, but has not moved through the age at risk. Future follow up of this family would be useful to assess concordance of psychotic phenotypes. The current data suggest that medical phenotypes, intellectual function, and psychotic manifestations may be more susceptible to modifying influences. A larger sample of well-phenotyped individuals with 3q29 deletion syndrome will be required to support or refute these observations.
In conclusion, we report a multiplex family where multiple individuals have the 3q29 deletion syndrome and varied neurodevelopmental and psychiatric manifestations. These data serve to expand upon the 3q29 deletion phenotype and suggest that disability associated with the syndrome is not restricted to or solely a consequence of intellectual deficit. Our data further suggest that care and management of individuals with the 3q29 deletion should include screening for neurodevelopmental and psychiatric traits at multiple timepoints across the lifespan. Ethics approval and consent to participate This study was reviewed and approved by Emory University's Institutional Review Board (IRB00088012). All participating study subjects gave informed consent. Individuals younger than age 16 gave assent to participate.

Consent for publication
A copy of this manuscript was provided to the subjects participating in this study; written and verbal consent for publication of the data herein was obtained from participating study subjects.