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Supplementary information to:

Familial pericentric inversion of chromosome 18: behavioral abnormalities in patients heterozygous for either the dup(18p)/del(18q) or dup(18q)/del(18q) recombinant chromosome

Stefan Vermeulen1, Frank Speleman1, Leen Vanransbeeck1, Jasmien Verspeet1, Björn Menten1, Marie-Rose Verschraegen-Spae1, Philippe De Wilde1, Ludwine Messiaen1, Ron C. Michaelis2,Jules G. Leroy1,2

1Center for Medical Genetics, 0K5, Ghent University Hospital, Ghent, Belgium and 2Greenwood Genetic Center, Greenwood, South Carolina, USA

Running Title: Familial pericentric inversion chromosome 18

Keywords: recombinant chromosome 18, learning deficit, social interaction deficit, mild phenotype

Correspondence: S. Vermeulen

Center for Medical Genetics

0K5, Ghent University Hospital

De Pintelaan 185

B-9000 Ghent, Belgium

E-mail:

Materials and methods

Psychometric and personality Testing

The Dutch version of the Wechsler Adult Intelligence Scale, edition III (WAIS-III) was used for intelligence testing1. The WAIS-III measures full scale intelligence quotient (FSIQ) as well as verbal (VIQ) and performance IQ's (PIQ). IQ scores of 70 or above indicate normal intelligence, while scores of 50-69 indicate mild MR, scores of 35-49 indicate moderate MR, scores of 20-34 indicate severe MR and scores below 20 indicate profound MR. Other measures derived from the WAIS-III results included the verbal comprehension index (VCI), the perceptional organization index (POI), the working memory index (WMI) and the processing speed index (PSI).

The Symptom Checklist 90 (SCL-90), a multidimensional self-assessment list of questions for ambulant psychiatric patients, was used to measure agoraphobia, depression, anxiety, somatic complaints, interpersonal mistrust, mental disorganization, sleep disturbances and hostility/anger2.

The Nijmegen Personality Questionnaire (NPQ), an abbreviated version of the California Psychological Inventory, was used for testing the following dimensions of personality: mental instability, personal and social inadequacy, rigidity/insistence on routine, level of mistrust in and hostility toward others, social dominance, self-contentment and self-esteem. Results obtained from the SCL-90 and the NPQ are reported as one of seven points along a continuum from very low to very high.

The State Trait Anxiety Inventory (STAI), the Dutch version of the Spielberger STAI was also applied. It is comprised of two independent questionnaires. One measures trait anxiety, the degree to which a person’s personality is marked by anxiety, while the second measures state anxiety, the amount of stress elicited by the environment, people or sudden, unexpected occurrences. Results are reported as the decile within the population distribution into which the patient’s score falls3,4.

The Social Anxiety Inventory (SAI) assesses social anxiety and social interaction skills in adults along several dimensions, including giving critical remarks to others, requiring attention for one's own opinion, expressing appreciation for others, self-appreciation and initiative in starting conversations. The two subscales measure the amount of stress associated with these actions and the frequency with which the subject engages in them. Results are reported on a five-point scale ranging from no stress (1) to extreme anxiety (5).

The Utrecht Coping Questionnaire (UCQ) attempts to link the individual's style of coping with life events to his or her psychosomatic complaints. Coping strategies explored include an active coping attitude, palliative or distractive reaction, avoiding, searching for social support, depressive reaction, expression of emotion/anger, and seeking soothing thoughts. Results are reported on a five-point scale along a continuum from very low to very high5.

The revised Beck Depression Inventory (BDI) is a 21 item instrument designed to assess severity of depression in adolescents and adults. Scores of 0-9 indicate minimal, 10-16 mild, 17-29 moderate and 30-63 severe depression6.

Cytogenetic and Molecular Investigations

Analysis of G-banded prometaphase chromosomes was performed on short-term lymphocyte cultures using standard procedures7. Fluorescence in situ hybridization (FISH) was done as described previously8. Probes were labeled with biotin-16-dUTP (Rocche Diagnostics, Mannheim, Germany) or digoxigenin-11-dUTP by standard nick translation and visualized using neutralite-avidine-FITC (Molecular Probes, Eugene, Oregon, USA) and sheep-anti-mouse dig-TRITC (Biognost, Heule, Belgium), respectively. Chromosomes were counterstained with DAPI. Images were collected using a Zeiss Axioplan epifluorescence microscope with black and white high-resolution camera controlled by ISIS software (Metasystems, Altlussheim, Germany; Characterization of the inversion and recombinant inversion chromosome was performed using the following probes: the YAC probe HTY3045 specific for the subtelomeric region of 18q in combination with a chromosome 18 specific paint. In other experiments the subtelomeric PAC probe GS-964-M9(18qter) was used in combination with PAC probe GS-52-M11 (18pter)9. Breakpoint analysis in the 18p and 18q regions was performed using a 1 Mega base (Mb) set of 12 BAC clones for 18p including RP11-324G2; RP11-769O8; RP11-267C19; RP11-291G24; RP11-419P8; RP11-106J7; RP11-502P1; RP11-92G19; RP11-344B7; RP11-466I16; RP11-146G7; breakpoint; RP11-51B9 (telomere to centromere order) and a set of 10 BAC clones for 18q, RP11-430H7; RP11-45A1; RP11-169F17; RP11-25L3; RP11-238P13; RP11-396D4; RP11-234N1; breakpoint; RP11-118I2; RP11-563B11; RP11-154H12 (centromere to telomere order).The abovementioned probes were retrieved from a set of clones selected from the publicly available Ensembl database encompassing the human genome and spaced at approximately 1-Mb intervals10. Gene locations were derived from the Golden Path based on the July 2003 freeze. The resolution of the breakpoint analysis was limited to a 1 Mb resolution11. Essentially all probes were labeled as described above. To define the breakpoint flanking probes several rounds of FISH were performed with probes 1 Mb apart from each other. A breakpoint was defined between a distal duplicated or a deleted probe and a not altered proximal probe.

DNA was extracted from lymphocytes in peripheral blood samples obtained from all consenting and available subjects in the family using a QiaAmp DNA blood kit (Qiagen GmbH, Hilden, Germany). Microsatellite markers, D18S452-D18S1138-D18S59, ordered from 18p11.31 to 18pter and D18S488-D18S1161-D18S554-D18S461-D18S70, ordered from 18q22 to 18qter were studied as described previously12. Briefly, the microsatellites were PCR amplified using fluorescent primers. Allele sizes were determined using an ALF fragment analyzer (Amersham-Biosciences, NJ).

Results

FISH and Marker analysis

The breakpoints were defined using a 1 Mega base (Mb) set for 18p and for 18q. The breakpoints were situated on the 18p arm between RP11-146G7and RP11-51B9 (telomere to centromere order) and on the 18q arm between RP11-234N1 and RP11-118I2 (centromere to telomere order). Markers D18S452, D18S1138 and D18S59 (18p11.31-pter) each showed one maternal and two paternal alleles in the proband, while markers D18S554, D18S461 and D18S70 (18q22-qter) each showed one maternal allele only (eFigure 2). The parallel analysis in the brother with dup(18q)/del(18p) yielded the inverse results, confirming partial monosomy for 18p and partial trisomy for 18q, representing the alternate consequence of meiotic recombination within the paternal inv(18) chromosome (eFigure 2).

Psychometric testing

eTable 1 illustrates the results of psychometric testing of the proband (VI.1) and her paternal uncle (V.3), both with dup(18p), and of the proband’s brother (VI.2) with the alternate dup(18q). Scores on all tests were noticeably lower in the two individuals with the dup(18p) recombinant chromosome than in the one with the dup(18q) recombinant chromosome, and all three individuals had higher VIQs than PIQs. In both the proband and her uncle, FSIQ, VIQ and PIQ were within the normal range. Working memory and processing speed gave scores significantly below average but the PSI score for the proband’s uncle was the only cognitive testing score that fell below the normal range in any of the subjects tested. In addition, scores significantly below average were recorded in subject accumulation and in verbal arithmetic subtests in both the proband and her uncle (results not shown). An attention deficit and often failing audiogenic memory were also recorded for the uncle. In addition, some subtest scores as well as direct observation suggested that the uncle was dyslexic.

The proband’s brother had FSIQ and VIQ scores close to the population average. However, like his sister and uncle, his PIQ was significantly below his VIQ score. In some verbal arithmetic subtests (results not shown) he scored much below his FSIQ profile. Thinking in logical sequence was often problematic, as suggested by his low score in the picture arrangement test (result not shown), and logical thinking using numbers often represented an insurmountable challenge. Information obtained by family history taking provided indications of similar learning difficulty in grade school and high school in the proband’s second cousin (Figure 1 in published section, VI.3), who also has the dup(18p) recombinant chromosome but who was not available for psychometric testing.

Behavioral and Personality Testing

Scores on all tests were noticeably lower in the two individuals with the dup(18p)/del(18q) recombinant chromosome than in the subject with the dup(18q)/del(18p) recombinant (eTable 1). The parents both scored within normal limits in the psychometric and personality tests applied to the affected.

The proband’s behavioral phenotype is marked by significant anxiety and intense stress associated with social interactions. She scored above mid-score on the anxiety scale of the SCL-90, and high or very high on the agoraphobia, hostility and interpersonal mistrust scales of the SCL-90. On the STAI, she scored in the seventh decile on the trait anxiety scale and the ninth decile on the state anxiety scale. These scores indicated that the proband showed not only significant baseline trait anxiety but also a further increase in her anxiety level in the face of social interactions and other external events. Consistent with this, she scored high on the social incompetence scale and very high on the hostility/mistrust scale of the NPQ. On the IOA, she scored high to very high on the social anxiety subscales related to initiating conversation, giving compliments and asking attention for one’s own opinion, and scored in the midrange on the subscale pertinent to giving critical remarks to others.

In addition the proband also scored high on the SCL-90 depression scale and indicated a high incidence of depressive reactions when coping with stressful events by the UCQ. It should be noted that these scores contradict her mild score on the BDI. However, in all three individuals the BDI scores were consistently lower than the indices of depression from the other testing instruments used. However, the relative severity of depression for the three patients was similar on all measures of depression. The proband’s personality profile was also marked by significant mental disorganization. She scored high on scales of psychoneurosis and mental disorganization in the SCL-90, and very high on the mental instability scale of the NPQ.

The proband’s uncle’s personality was marked most prominently by depression and to a lesser degree by moderate anxiety. He scored very high on the depression scale of the SCL-90 and reported a very high incidence of depressive reactions in the face of stressful events. As was true for the others, his score on the BDI indicated less depression than his score on the other instruments. However, his BDI score (moderate depression) was significantly higher than those of his niece and nephew.

The uncle’s personality testing scores indicated moderate to high levels of anxiety, but little if any increased stress in the face of social interactions. He scored above mid-score on the anxiety scale of the SCL-90, and in the tenth decile on the trait anxiety scale of the STAI. In spite of high and very high scores on the hostility and interpersonal mistrust scales in the SCL-90 and the NPQ, respectively, as well as a high score on the personal inadequacy scale in the NPQ, this person had almost no subjective difficulty upon engaging in social interactions. He had a midrange score on the agoraphobia scale of the SCL-90 as well as the social incompetence scale of the NPQ. In addition, despite scoring in the tenth decile on the trait anxiety scale of the STAI, he scored in the seventh decile on the state anxiety scale. He had a low overall score on the SAI, indicating that he did not find social interactions such as criticizing or praising others or asking attention for his own opinion unusually stressful. In spite of the relative ease with which this subject engaged in social interaction, he had a history also of dysfunctional relationships with coworkers, acquaintances and relatives. His lack of success in social interactions may have been due to his high level of psychoneurosis and mental disorganization. The SCL-90 indicated very high levels of both these traits, and he scored high on the mental instability scale of the NPQ.

The proband’s brother was clearly better mentally organized and more at peace with himself than his sister or uncle. Unlike his sister and uncle, who demonstrated strong feelings of personal inadequacy and low levels of self-contentment, the proband’s brother’s score on the personal inadequacy scale of the NPQ was average and his level of self-contentment, according to the NPQ, was high. However, the results of the personality tests were somewhat inconsistent in him. Although he scored high on the depression and anxiety scales of the SCL-90, he showed minimal depression according to the BDI, and obtained a midrange score regarding the incidence of depressive coping reactions in the face of stressful events according to the UCQ. His score on the anxiety scale of the SCL-90 was higher than the other two individuals tested, but his scores on both the trait and state anxiety scales of the STAI were significantly lower than those of the two other subjects.

In spite of this relative self-contentment, the brother’s feelings of social incompetence were strong. In addition, he was highly agoraphobic and had very high scores on the hostility and mistrust scales of the SCL-90 and the NPQ. Like his depression and anxiety scores, the scores indicating his level of stress in the face of social interactions were inconsistent. According to the SAI, initiating conversation, requesting attention for his own opinion and criticizing others were only mildly stressful. In contrast, expressing appreciative thoughts of himself or of others was very stressful. His coping with stressful events was often highly emotional (UCQ). In spite of his apparently adequate level of mental organization, high overall levels of psychoneurosis were detected by the SCL-90.

Electronic databases:

Accession numbers, BAC library addresses and URLs for the data presented are from the following databases: Ensembl Genome Browser: sapiens/; The Golden Path (July 2003 Freeze): and Mapview:

Legends to eFigures

eFigure 1

Clinical picture A of proband (subject VI.1) and B of proband’s brother (subject VI.2).

eFigure 2

CA repeat fragment separation by PAGE after PCR with fluorescent primers for microsatellite marker D18S452 representative of 18pter and marker D18S70 representing 18qter, in V.2 (mother) with normal karyotype (top row), V.1 (father) with inv(18) (second row), VI.1 (proband) dup(18p)/del(18q) (third row) and VI.2 (brother) dup(18q)/del(18p) (bottom row). Fragment size in bps given on X-axis. Polymorphic pattern of alleles (defined by square dot at highest fluorescence intensity) in parents used as reference to findings in offspring. In VI.1, D18S452 is represented by two paternal alleles and one maternal allele (trisomic state). Only one maternal D18S70 (18qter) allele is represented in her genome (monosomic state). Exactly the reverse is observed in VI.2 with two paternal D18S70 alleles and one maternal allele (trisomic state) and one maternal D18S452 (18pter) allele only (monosomic state).

eFigure 3

Overview of the genes duplicated or deleted in the recombinants derived from the pericentric inversion inv(18)(p11.22q23). The aneuploid regions and their underlying genes are indicated in red. Basepair number is indicated above chromosome bands. The relative size and position of the genes are indicated graphically is as in the Golden path, ( The rectangles underneath the chromosome bands depict the relative position and size of the BACs used.

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