The role of the TCF4 in the phenotype of individuals with 18q segmental deletions

Minire Hasi, Bridgette Soileau, Courtney Sebold, Annice Hill, Daniel E. Hale, Louise O’Donnell & Jannine D. Cody

Human Genetics

ISSN 0340-6717

Hum Genet DOI 10.1007/s00439-011-1020- y

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Hum Genet DOI 10.1007/s00439-011-1020-y

ORIGINAL INVESTIGATION

The role of the TCF4 gene in the phenotype of individuals with 18q segmental deletions

Minire Hasi • Bridgette Soileau • Courtney Sebold • Annice Hill • Daniel E. Hale • Louise O’Donnell • Jannine D. Cody

Received: 15 February 2011 / Accepted: 25 May 2011 Ó Springer-Verlag 2011

Abstract The goal of this study is to define the effects individuals failed to reach developmental milestones of TCF4 hemizygosity in the context of a larger seg- beyond those typically acquired by 12 months of age. mental deletion of 18q. Our cohort included TCF4 hemizygosity also conferred an increased risk of 37 individuals with deletions of 18q. Twenty-seven had early death principally due to aspiration-related compli- deletions including TCF4 (TCF4?/-); nine had deletions cations. Hemizygosity for TCF4 confers a significant that did not include TCF4 (TCF4?/?); and one individual impact primarily with regard to cognitive and motor had a microdeletion that included only the TCF4 gene. development, resulting in a very different prognosis for We compared phenotypic data from the participants’ individuals hemizygous for TCF4 when compared to medical records, survey responses, and in-person evalu- individuals hemizygous for other regions of distal 18q. ations. Features unique to the TCF4?/- individuals included abnormal corpus callosum, short neck, small penis, accessory and wide-spaced nipples, broad or Introduction clubbed fingers, and sacral dimple. The developmental data revealed that TCF4?/? individuals were only mod- It has recently been shown that hemizygosity of the TCF4 erately developmentally delayed while TCF4?/- gene causes Pitt–Hopkins syndrome (MIM ID #610042) through a haploinsufficiency mechanism (Zweier et al. 2007). The TCF4 gene is located at 18q21.1 and is, therefore, also hemizygous in some individuals with larger segmental deletions of 18q. The goal of this study is to Web Resources Online Mendelian Inheritance in Man (OMIM), define the effect of TCF4 hemizygosity in individuals with http://www.ncbi.nlm.nih.gov/Omim/. 18q deletions. M. Hasi Á B. Soileau Á C. Sebold Á A. Hill Á The constellation of phenotypic features known as D. E. Hale Á L. O’Donnell Á J. D. Cody (&) Pitt–Hopkins syndrome is characterized by severe intel- Department of Pediatrics, UT Health Science Center, lectual disability, wide mouth with fleshy lips, a beaked 7703 Floyd Curl Dive, San Antonio, TX 78229, USA nose, and intermittent hyperventilation followed by apnea e-mail: [email protected] (Pitt and Hopkins 1978). Since it was first described, the D. E. Hale Á J. D. Cody phenotype has been shown to have three different CHRISTUS Santa Rosa Children’s Hospital, genetic causes. It can be caused dominantly by hemi- San Antonio, TX, USA zygosity of or inactivating mutations in the TCF4 gene, L. O’Donnell or recessively by mutations in the CNTNAP2 gene on Department of Psychiatry, UT Health Science Center chromosome 7q35 or the NRXN1 gene on chromosome at San Antonio, San Antonio, USA 2p16.3 (Zweier et al. 2009). Because this phenotype has multiple underlying molecular mechanisms, the phrase J. D. Cody The Registry and Research Society, ‘‘Pitt–Hopkins’’ refers only to the clinically-defined San Antonio, TX, USA syndrome. 123 Author's personal copy

Hum Genet

The identification of the genetic bases of Pitt–Hopkins One of the overall goals of our research group is to has allowed a fuller appreciation of the range of phenotypic determine which on 18q contribute to the 18q- features associated with mutations or deletions of these phenotype through haploinsufficiency. This information genes. It has recently been realized that 2% of individuals will eventually allow the molecular karyotype to be pre- with phenotypic Angelman syndrome actually had TCF4 dictive with regard to the phenotype. The aim of this study aberrations (Takano et al. 2010). In addition, Rosenfeld was to determine the extent to which hemizygosity of et al. (2009) identified seven cases with TCF4 deletions TCF4 contributes to the physical and behavioral phenotype which were referred for chromosomal microarray analysis in people with segmental 18q deletions. due to intellectual disability. In this genotypically ascer- tained group, a reevaluation of the phenotypic effects of TCF4 hemizygosity was undertaken. Of these patients, Methods only 3 of 7 had a breathing abnormality and none had seizures, indicating that the penetrance of these features is Subject recruitment significantly less than 100%. In their review of published cases likely caused by haploinsufficiency of TCF4 Potential participant families learn about the research study (N = 36), they found that the Pitt–Hopkins facial appear- from a variety of sources. Primarily, however, families are ance was present in 91% of cases. Other features seen in referred to the Research Center from the Chromosome 18 more than half of the subjects included: severe psycho- Registry & Research Society. Eligibility for the study motor delay (97%), hypotonia (88%), happy disposition requires a cytogenetic or molecular diagnosis of an 18q (85%) (though no elaboration of this behavior was pro- deletion. This study was approved by the Institutional vided), single palmar crease (69%), microcephaly (64%), Review Board of the University of Texas Health Science constipation (58%), and brain abnormalities (56%). Center at San Antonio. All families were and continue to Interestingly, variations in all three genes responsible be involved in the informed consent process, which is for Pitt–Hopkins syndrome are associated with schizo- appropriately documented. phrenia and autism. Hemizygous deletions of NRXN1 and CNTNAP2 as well as SNPs in both genes are associated Phenotypic assessment with an increased risk of schizophrenia, epilepsy, and autism spectrum disorder (Kirov et al. 2009; Friedman For all families enrolling at the Chromosome 18 Clinical et al. 2008) and SNPs of TCF4 have been associated with a Research Center, phenotypic data are compiled from three slightly increased risk of schizophrenia (Stefansson et al. sources. First, upon enrollment, families provide the 2009). In addition, individuals with 18q deletions including Research Center with extensive medical records to doc- TCF4, NETO1 and FBXO15 are more likely to exhibit ument the participant’s medical and developmental his- autistic-like behavior (O’Donnell et al. 2010). These data tories. These data are entered into a relational database point to a potential functional relationship between the which is updated annually with the most recent medical products of these three genes as well as a causal relation- and developmental information obtained from the fami- ship between autism and schizophrenia. lies, providing longitudinal data on each of the study The TCF4 gene produces a basic helix-loop-helix (bHLH) participants. Second, all families are solicited annually by which acts through binding to E-box mail to complete psychological surveys. Parents are asked consensus sequences in the promoter regions of the target to complete the following questionnaires and return them genes. It belongs to a family of proneural bHLH transcription by mail: Behavior Assessment System for Children-Sec- factors controlling differentiation of neuronal subtypes. ond Edition (BASC-2; Reynolds and Kamphaus 2004) The temporal and spatial differentiation of the numerous which provides information regarding the presence of neural cell types is controlled by a relatively small number behavior problems and emotional disturbance; the Vine- of transcription factors acting as homo- and hetero-dimers. land Adaptive Behavior Scales-Second Edition (Sparrow The hetero-dimerization of transcription factors produces a et al. 2005) which asks parents to rate communication, greater diversity of regulatory combinations, thereby a daily living and socialization skills and the Gilliam Aut- greater diversity and specificity of gene expression (Guille- ism Rating Scale-First Edition (GARS; Gilliam 1995)or mot 2007). It is likely that the TCF4 gene product interacts Second Edition (GARS-2; Gilliam 2006) which provides with numerous other transcription factors. In the mouse, Tcf4 an overall probability of autism rating. dimerizes with Math1, another bHLH transcription factor. The third data source is the comprehensive clinical Interestingly, mice hemizygous for Math1 die shortly after evaluation at the Chromosome 18 Clinical Research Center birth from central apnea (Rose et al. 2009). as previously described (Cody et al. 2009).

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The evaluation completed at the Research Center large interstitial deletions (between 24.02 and 27.58 Mb in includes a comprehensive neuropsychological evaluation size) (Fig. 1, Panel c). comprised multiple measures. To obtain a measurement of Within our cohort, there are 132 individuals with ter- estimated cognitive functioning, an individually adminis- minal deletions of 18q with breakpoints distal to the TCF4 tered measure of ability is given. If the participant is able to gene (TCF4?/?). The comparison group was selected from understand the task demands to the extent that it is possible these individuals. We selected the TCF4?/? individuals to obtain a reliable and valid estimate of ability, then with the specific aim of creating a comparison group of measures based on the chronological age are given: Bayley similar size to the TCF4?/- group. Because we had cog- Scales of Infant and Toddler Development-Third Edition nitive data on 8 of the TCF4?/- individuals and adaptive (Bayley 2006); Differential Abilities Scales-Second behavior data on 11, we selected nine TCF4?/? individuals Edition (Elliot 2007) or the Wechsler Adult Intelligence with breakpoints as close as possible to TCF4 to serve as a Scales-Third Edition (Wechsler 1997). If it is not possible control group. Parenthetically, the next smallest deletion to evaluate the intellectual functioning using tasks and contained several more genes than other members of the activities based on their chronological age, it is necessary control group. The photographs of six of these individuals to employ standardized measures which are routinely used are shown in Fig. 2n–s. to assess the cognition, language, and motor abilities of In this study, the TCF4?/? and TCF4?/- groups were infants and toddlers. Because some participants are sig- compared with regard to both physical and behavioral nificantly older than the normative comparison group for phenotypes. The TCF4?/- group included 27 individuals: these evaluations no standard comparison was possible. 12 males and 15 females. Since this is a longitudinal study, Therefore, estimates of cognitive functioning are generated the information was gathered over a period of time. from age equivalent scores. However, the age range at the most recent assessment was To gain additional information about the group of par- 10 months to 24 years 7 months, with an average age of ticipants described in this project, it was necessary to 10 years 10 months. The TCF4?/? group of nine individ- re-contact participating families to fill in gaps in the data uals included four males and five females with an average and to address new questions. All families participating in age range from 10 months to 28 years 7 months with an this study were contacted by telephone to obtain additional average age of 13 years. information about the participant’s respiratory history, We aimed to (1) identify which features are found only including abnormal breathing patterns. in the TCF4?/- group, and (2) determine the incidence of features associated with the Pitt–Hopkins phenotype in Genotypic assessment both the TCF4?/- group and the TCF4?/? group. The physical features that distinguish the TCF4?/- group from The DNA of all participants was evaluated using custom the TCF4?/? group are listed in Table 1 (Zweier et al. designed oligonucleotide microarray comparative genomic 2007, 2008; Pitt and Hopkins 1978; Rosenfeld et al. 2009; hybridization as previously described (Heard et al. 2009). Amiel et al. 2007; Andrieux et al. 2008; Brockschmidt All participants in the study cohort assessed here had a et al. 2007; de Pontual et al. 2009; Giurgea et al. 2008; hemizygous region of 18q without other major chromo- Kalscheuer et al. 2008; Peippo et al. 2006; Singh 1993; somal copy number changes. Van Balkom et al. 1998). Only abnormalities of the corpus callosum (64%) were found in more than half of the TCF4?/- group. Other features were found in fewer than Results 35% of participants with TCF4 hemizygosity, yet were unique to this group. To determine the contribution of TCF4 hemizygosity to the We then compared the non-unique phenotypic features overall phenotype of 18q-, we compared the phenotypes between the two groups. The features previously described of those with and without hemizygosity of TCF4. We have as associated with Pitt–Hopkins syndrome are indicated in a cohort of over 200 individuals with hemizygosity for a italics (Rosenfeld et al. 2009). These findings are shown in portion of chromosome 18q, each with a unique hemizy- Table 2 and reveal that the majority are non-specific find- gous region (Heard et al. 2009). Within this cohort, there ings often associated with many different conditions are 27 individuals with a deletion that includes TCF4 (Zweier et al. 2007, 2008; Pitt and Hopkins 1978; Rosen- (TCF4?/-). As shown in Fig. 1 and pictures in Fig. 2 (a– feld et al; Amiel et al. 2007; Andrieux et al. 2008; m), this is a very heterogeneous group including 13 people Brockschmidt et al. 2007; de Pontual et al. 2009; Giurgea with terminal deletions between 24.67 and 30.71 Mb in et al. 2008; Kalscheuer et al. 2008; Peippo et al. 2006; size (Fig. 1, Panel b), 8 people with small interstitial Singh 1993; Van Balkom et al. 1998; Taddeucci et al. deletions (between 5.6 and 16.88 Mb in size) and 6 with 2010). 123 Author's personal copy

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Fig. 1 Panel a Chromosome 18 ideogram. The box indicates the b Individuals with terminal deletions of 18q, who have one copy of region of the chromosome shown in panels b–d. Panels b–d Display TCF4. Panel c Individuals with interstitial deletions of 18q who have the aCGH data from study participants with distal 18q deletions. The one copy of TCF4. Panel d The nine individuals in our study cohort light bars indicated the presence of the intact chromosome. The dark with terminal deletions and breakpoints closest to but not including band at the end of the light bar indicated the breakpoint region. The TCF4. These individuals phenotypic data were used as the compar- participant’s study number is to the left of their data. Panel ison group

To isolate the effect of TCF4 hemizygosity on cognitive each intellectual assessment instrument designed to assess functioning, we wanted to compare the TCF4?/- group typical children under 1 year of age. Therefore, the most with terminal deletions (N = 13) to the control group of informative indication of their cognitive function was to nine TCF4?/? individuals who also had terminal deletions. report their age equivalent; the age at which the skills are However, we had cognitive data from in-person assess- acquired in a typically developing child. These pronounced ments on only 8 of the 13 individuals in the TCF4?/- deficits were present in children under 5 years of age and group. Table 3 presents the estimated intellectual abilities continue to be present across the lifespan into child and of these two groups by age. As Table 3 indicates, the young adulthood. In general, the cognitive and motor estimated intellectual abilities for the TCF4?/? group range growth pattern is essentially flat across the lifespan from mild intellectual disability to low average cognitive meaning that they did not acquire additional skills beyond functioning. This is quite mild in comparison to the eight that of a 12 months old. persons in the TCF4?/-group. In this group, cognitive and We also compared the language development of these motor development is very significantly delayed with same two groups of individuals. All persons in the standard scores within the profound to severe range of TCF4?/- group are nonverbal with receptive language intellectual disability. In fact, the individuals in the limited to reactions to sounds in the environment, calming TCF4?/- group were unable to do even the easiest items of when spoken to and recognition of caregiver’s voice with

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Fig. 2 Individuals with terminal deletions of 18q, who have one copy Individuals with terminal deletions and breakpoints closest to but of TCF4. a 3 years 9 months, b 6 years, c 6 years 8 months, not including TCF4. n 25 years 9 months, o 14 years, p 7 years d 8 years 4 months, e 4 months 29 days, f 9 months, g 14 months 5 months, q 8 years 9 months, r 6 years 6 months, s 1 year 2 months. 22 days. Individuals with interstitial deletions of 18q who have one One individual with a small interstitial deletion that includes only the copy of TCF4. h 19 years 10 months, i 4 years, j 1 year 5 months, TCF4 gene. t 12 years 8 months k 4 years 2 months, l 2 years 8 months, m 1 year 10 months. increased motor movement (wiggling). Expressive lan- of the TCF4?/? group was rated as falling within the mild guage consisted of undifferentiated throaty or nasal sounds. range of impairment. In contrast, in the TCF4?/? group, six of the nine persons Parents also completed the BASC-2 which provides an communicate verbally; one child uses sign language to evaluation of problem behaviors which fall into three communicate; and two individuals were under the age of general areas: externalizing problems (hyperactivity, two at the time of testing and were not yet speaking. These aggressiveness, conduct problems); internalizing problems two toddlers do however communicate through the use of (anxiety, depression, somatization) and those in the engaged eye contact, facial expressions, gestures and sub- behavior index [atypicality (disconnection from reality), vocalizations. withdrawal and attention problems]. Only problems with Parents in both terminal deletion groups completed an attention were rated by parents in both groups as cause for adaptive behavior questionnaire which compared the concern (average T-Score = 63.18 for the TCF4?/- group communication, daily living, and socialization skill and average T-Score = 60.55 for the TCF4?/? group). development of their children with a normative group of Average parental ratings across all of the other domains same-age peers. We had data on 11 of the 13 individuals in were within normal or typical limits compared with same the terminal deletion TCF4?/- group using this instrument age peers. (Table 4). Parental ratings of both groups across all To determine the presence of behaviors consistent with domains were congruent with the intellectual assessment autism, parents evaluated their children’s communication results obtained through one-on-one assessment. The and social skill functioning along with the presence of adaptive behavior functioning of the TCF4?/- group was stereotyped or perseverative behaviors using one of the rated as severely impaired while the behavioral functioning Gilliam Autism Rating Scales (GARS or GARS-2). The

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Table 1 Unique features of TCF4 phenotypic componentsa Hemizygous for TCF4 those with TCF4 hemizygosity Number %

Abnormal corpus callosum 16/25 64 Atrial septal defect 7/20 35 Sacral dimple 7/23 30 Clubbed or broad fingers 3/11 27 Short neck 5/19 26 Hypertonia 7/27 26 Genital abnormalities-small penis 3/12 25 Camptodactyly of the fingers 5/22 23 Wide spaced nipples 5/22 23 Toe-2nd and 4th overlapping 3rd toe bilaterally 4/21 19 Features in bold italics are those Premature death (\22 y/o) 5/27 18 in the literature as TCF4 Overfolding of the ears 4/22 18 phenotypic components Short philtrum 4/23 17 a Zweier et al. (2007, 2008); Malrotation of intestine 4/25 16 Pitt and Hopkins (1978); Rosenfeld et al. (2009); Amiel Cortical visual impairment 4/26 15 et al. (2007); Andrieux et al. Absence or flattening of the superior fork of the antihelix 3/22 13 (2008); Brockschmidt et al. Optic atrophy 3/26 12 (2007); de Pontual et al. (2009); Accessory nipple 2/22 9 Giurgea et al. (2008); Kalscheuer et al. (2008); Peippo Wolff–Parkinson–White syndrome 2/25 8 et al. (2006); Singh (1993); Van Kyphosis 1/22 4 Balkom et al. (1998)

Table 2 Features NOT unique TCF4 phenotypic componentsa Hemizygous for Nine largest terminal deletions with two to TCF4 hemizygosity TCF4 copies of TCF4 Number % Number %

Hypotonia 26/26 100 9/9 100 Microcephaly 16/26 62 3/9 33 Features in bold italics are those Postnatal growth retardation 15/27 55 1/9 11 in the literature as TCF4 Single palmar crease 12/22 54 1/9 11 phenotypic components Seizures 14/27 52 4/9 44 a Zweier et al. (2007, 2008); Myopia 14/27 52 3/9 33 Pitt and Hopkins (1978); Rosenfeld et al. (2009); Amiel Intra uterine growth retardation 13/25 52 1/9 11 et al. (2007); Andrieux et al. Constipation 11/25 44 2/9 22 (2008); Brockschmidt et al. Central apnea 9/25 36 2/9 22 (2007); de Pontual et al. (2009); Drooling 7/25 28 3/9 33 Giurgea et al. (2008); Kalscheuer et al. (2008); Peippo Genital abnormalities-cryptorchidism 3/12 25 1/4 25 et al. (2006); Singh (1993); Van Strabismus 6/27 22 2/9 22 Balkom et al. (1998); Taddeucci Scoliosis 2/14 14 1/9 11 et al. (2010)

GARS is a screening tool and should not be used alone to the ratings of parents of children in the TCF4?/? group make a definitive diagnosis of autism. Therefore, scores indicated that it was a possibility. from this instrument are cataloged as to the probability of Within the group of TCF4?/-, children who had been having an autistic diagnosis. Parental ratings of children in evaluated at the Research Center, there is great genotypic the TCF4?/- group indicated a very likely or high proba- variability between the individuals with regard to the bility of autism being part of the diagnostic picture while number of other genes involved in the deletion. Although

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Table 3 Comparison of intellectual abilities 18q-, TCF4?/? Intellectual CA less than CA between CA 13 years Overall IQ and overall abilities 5 years 6–12 years and older range of scores N = 9 N = 3 N = 4 N = 2

Full scale IQ 55a 63a 62a 61a (50–82) Verbal IQ 57a 66a 59a 63a (50–81) Nonverbal IQ 55a 75a 70a 69a (50–90) 18q-, TCF4?/- Intellectual abilities CA less than 5 years CA between 6–12 years CA 13 years and older N = 8 N = 3 N = 4 N = 1

Cognitive abilities AE = 1.3 months of age AE = 7 months of age AE = 6 months of age Motor abilities AE = 2 months of age AE = 6 months of age AE = 6 months of age

CA chronological age, AE age equivalent a Average standard scores with a mean of 100 and a standard deviation of 15

Table 4 Average vineland Type Communication Daily living Socialization Overall adaptive adaptive behavior scales-second skills behavior edition parental ratings TCF4?/? 60.22a 57.44a 61.22a 56.77a N = 9 a Average standard scores with Terminal deletion, TCF4?/- 36.38a 36.84a 40.15a 36.46a a mean of 100 and a standard N = 11 deviation of 15 the behavioral and cognitive data presented in Tables 3 and in our analysis. Again, age equivalent scores were gener- 4 are from only those individuals with terminal deletions, ated for the three groups because the use of standard peer- we wished to evaluate whether other regions of hemizy- based assessment measures was not possible due to the gosity of 18q had an impact on the effect of TCF4 hemi- participants’ very low cognitive and motor functioning. As zygosity. For this analysis, we now included the cognitive Table 5 indicates, the cognitive functioning of the three data from both the interstitial as well as the terminal groups is not significantly different. All three groups of deletions in the TCF4?/- group. Again, these data were not people hemizygous for the TCF4 gene have significantly available on every individual whose genotype data are delayed cognitive and motor functioning. This suggests shown in Fig. 1 and phenotype data shown in Tables 1 and that the size of the hemizygous region has little to no effect 2. Because there are no other genes on 18q specifically on the developmental impact of the TCF4 gene. In essence, identified as haploinsufficient, the comparison between the the effect of TCF4 hemizygosity is so profound that, with TCF4?/- subgroups was made based on grouping them by regard to development, children with large regions of the size of their deletion. We created three sub-groups: hemizygosity including many other genes are not more those with terminal deletions (N = 13, 8 with cognitive developmentally delayed than children with hemizygosity data), those with large interstitial deletions (N = 8, 5 with for the TCF4 gene alone. cognitive data) and those with small interstitial deletions One of the more striking findings was the realization (N = 6, 4 with cognitive data). The behavioral perfor- that, within our large cohort of people with simple terminal mance of these three groups was compared to each other. 18q deletions (N = 132), the majority of individuals who In addition, we have one participant, age 12 years and died before the age of 18 had a deletion that included the 8 months who has a small interstitial deletion of only the TCF4 gene. Figure 3 illustrates this point. The figure shows TCF4 gene whose data have not been included elsewhere either the current age or the age of death for the two

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Table 5 Average age equivalents of the three groups missing the TCF4 gene Terminal deletion Large interstitial deletion Small interstitial deletion Deletion of only TCF4 N = 8 N = 5 N = 4 N = 1

Cognitive abilities AE = 3.8 months of age AE = 2.4 months of age AE = 5 months of age AE = 11 months of age Motor abilities AE = 3.4 months of age AE = 2.6 months of age AE = 6.25 months of age AE = 10 months of age subgroups within our entire cohort; those with one copy of TCF4 and those with two copies of TCF4. We investigated the cause of death by reviewing medical records and interviewing the parents. While a variety of reasons were given for the cause of death, almost all of the children who died had a history of multiple pneumonias, primarily thought to be due to chronic aspiration, as is common in such severely delayed children. This may have been complicated by the breathing abnormalities that are com- mon in the affected individuals.

Discussion

The analysis revealed three key observations. First, the features unique to those with TCF4 hemizygosity were abnormal corpus callosum, small penis, accessory nipples, broad or clubbed fingers, sacral dimple, short neck and wide spaced nipples. The potentially most clinically sig- nificant of these physical findings is an abnormally thin or absent corpus callosum. It would be reasonable to expect that such an abnormality would be associated with an inability to walk. However, none of the individuals with Fig. 3 Current age and age at death. The open circles indicate current TCF4 hemizygosity in our cohort were able to walk age and the black diamonds indicate the age at death. TCF4?/?; data regardless of the morphology of their corpus callosum. from 132 individuals with simple distal 18q deletions. Average This should not imply that all individuals with TCF4 current age of those with two copies of TCF4 is 17 years. The two deletions are never able to walk. There is anecdotal evi- individuals in this group who died were a female age 20 years, 6 months and a male 19 years, 9 months. TCF4?/- data from 22 dence that some children with TCF4 are able to walk; subjects average current age is 11.5 years. Average age at death was however, none of the individuals enrolled in this study had 12 years old. The five participants who died included two males and attained that milestone. three females; ages 22 months, 6 years 10 months, 13 years, 20 years Second, it could be anticipated that individuals with 11 months, and 22 years larger deletions that include TCF4 might be more impaired physically and mentally than those with smaller deletions. TCF4 hemizygosity resulted in a developmental ceiling of However, we did not find this to be true. The presence or 12 months irrespective of chronologic age, which ranged absence of TCF4 seems to be more important in predicting from 10 to 238 months. severity than the size of the deletion. We are fortunate to Third, the analysis of the ages and the age at death of have in this sample an individual with TCF4 hemizygosity those with TCF4 hemizygosity and those with deletions of alone. As Table 5 illustrates, this person has significant chromosome 18 not including the TCF4 gene showed that cognitive and motor delay. Given the very small number of TCF4 hemizygosity conferred an increased risk of early persons in each of our three sub-groups missing the TCF4 death. The cause of death was in most cases related to the gene, it is not possible to determine if the differences in age consequences of chronic aspiration. equivalent scores among these three deletion types are The long-term goal of the Research Center is to deter- statistically significant. Functionally, however, the cogni- mine which genes are responsible for which aspects of the tive and motor delays across all groups are significant and phenotype of 18q-. In this study, we analyzed the effect of appear to be lifelong. In fact, in the cohort we evaluated, TCF4 hemizygosity to determine which components of the

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18q- phenotype can be attributed to the loss of one copy and intervention to prevent aspiration. Our data also of the gene. We compared the TCF4?/- group with the highlight the deleterious and chronic impact that hemizy- TCF4?/? group using data from medical records, parental gosity of the TCF4 gene has on cognitive and behavioral questionnaires and in-person physical and behavioral development. In our study, all individuals hemizygous for evaluations. Comparison to the literature was somewhat the TCF4 gene regardless of their age were similar to hampered by the dearth of detailed phenotypic information typically developing babies less than 12 months old. It is in previously reported cases of Pitt–Hopkins. Much of the critical that parents plan for the long-term 24-h care their syndrome description is limited to dysmorphology, much children will need and refocus their developmental of which is imprecise, (e.g., ‘‘microcephaly’’), and medical expectations. It is important to provide a nurturing envi- record abstraction. In particular, the behavioral and ronment that is rich in sensory stimulation and is one where developmental assessments are very limited in scope. This the child can feel comfortable and secure. made comparisons to the literature difficult. There is an Of note, we are now faced with the challenge of urgent need, now that the molecular genetics are known, to devising a meaningful nomenclature for 18q- that conveys perform a comprehensive multidisciplinary assessment of a both the information about the genotype and as well as its cohort of individuals with hemizygosity of or loss of implications for phenotype. In this study, we used the functions mutations in the TCF4 gene alone. mouse nomenclature as a guide, since our goal is to merely It is interesting to note that, in our population, the only indicate gene copy number. We are indicating the diploid Pitt–Hopkins phenotypic components that were unique to state with regard to the TCF4 gene as TCF4?/? and the those with TCF4 hemizygosity were thin or absent corpus hemizygous state as TCF4?/-. However, as we are able callosum, small penis, accessory nipples, broad or clubbed to classify more genes as being either haplosufficient or fingers, sacral dimple, short neck and wide spaced nip- haploinsufficient, it will become a challenge for the ples. Results of our analysis indicated that many of molecular cytogeneticist to write a clinically meaningful the features of Pitt–Hopkins were present in both the karyotype. We do not envision a diagnostic code for TCF4?/- as well as the TCF4?/? populations. Two of the someone with a segmental deletion to include a long list of reported cardinal features of Pitt–Hopkins syndrome are all the hemizygous genes, but rather an edited list of those irregular breathing and seizures. The breathing abnor- genes that are haploinsufficient, i.e. have clinical signifi- mality phenotype is poorly documented in the literature, cance. Ultimately such a genotype would imply a particular so we were not able to distinguish between documented phenotype and thereby direct a plan of medical surveillance central apnea, episodes of heavy breathing and hyper- and therapy. ventilation. Interestingly, in our population this feature Lastly, these data reinforce the need to eliminate the was not unique to those with TCF4 hemizygosity. Like- word ‘‘syndrome’’ when referring to 18q- for two reasons. wise, seizures were not unique to the TCF4?/- group. First, rather than being defined by a constellation of fea- This is in part due to the fact that many of the features tures, this condition is defined by a genotype, as implied by such as hypotonia, single palmar crease, microcephaly, the fact that it is named after the type of chromosome and seizures are genetically heterogeneous and therefore aberration. Second, this particular chromosome abnormal- non-specific findings. ity is uniquely heterogeneous. No two unrelated individuals Alterations in the TCF4 gene have been implicated in have the same exact region of hemizygosity. Therefore, we schizophrenia, and schizophrenia associated genes have could identify numerous pairs of individuals who both have been linked to myelin-related pathways (Rietkerk et al. 18q- yet have no hemizygous genes in common (Heard 2009). Since a key gene important in the compaction and et al. 2009). The term ‘‘syndrome’’, which implies a col- function of myelin (MBP) is located near the end of 18q, lection of similar phenotypic findings attributable to the and individuals with deletions of a critical region that same genetic cause, is thus not appropriate. Rather, the data includes this gene have dysmyelination of the brain (Cody presented here begin to define the molecular basis of 18q-, et al. 2009), it might be postulated that hemizygosity of highlighting the role of genomic heterogeneity in creating both TCF4 and MBP would exacerbate their individual phenotypic heterogeneity. Thus, the word ‘‘syndrome’’ is effects. However, we did not see significant differences no longer appropriate. between the TCF4 hemizygous participants whose 18q deletion included the myelin basic gene (MBP) Acknowledgments The authors would like to first thank the fami- (terminal deletions) and those that did not (interstitial lies that participated in this study for their willingness to share their knowledge and for answering numerous questionnaires and emails. deletions) as shown in Table 5. This work was funded by the MacDonald family, The Chromosome These data help us to make recommendations for this 18 Registry & Research Society, the Institute for the Integration of unique group of individuals with 18q deletions. The cause Medicine and Science (UL 1RR025767; National Center for Research of death data highlights the need for aggressive detection Resources) and CHRISTUS Santa Rosa Children’s Hospital. 123 Author's personal copy

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