TETRASOM Y 15Q11-Q13 IDENTIFIED BY
FLUORESCENCE
IN SITU
HYBRIDIZATION
IN A PATIENT WITH AUTISTIC DISORDER
Ana Elizabete Silva
1, Sheila Adami Vayego-Lourenço
2, Agnes Cristina Fett-Conte
3,
Eny Maria Goloni-Bertollo
3, Marileila Varella-Garcia
4ABSTRACT - We report a female child w it h t et rasomy of t he 15q11-q13 chromosomal region, and aut ist ic disorder associat ed w it h ment al ret ardat ion, development al problems and behavioral disorders. Combining classical and molecular cytogenetic approaches by fluorescence in situ hybridization technique, the karyotype w as dem onst rat ed as 47,XX,+ m ar.ish der(15)(D15Z1+ + ,D15S11+ + ,GABRB3+ + ,PM L-). Duplicat ion of t he 15q proximal segment represents the most consistent chromosomal abnormality reported in association w ith autism. The contribution of the GABA receptor subunit genes, and other genes mapped to this region, to the clinical symptoms of the disease is discussed.
KEY WORDS: tetrasomy 15, 15q11-q13, autism, fluorescence in situ hybridizat ion, GABA recept ors.
Identificação de tetrassomia 15q11-q13 por hibridação in situ fluorescente em uma paciente com distúrbio autístico
RESUM O - Relat am os um a criança do sexo f em inino com t et rassom ia da região crom ossôm ica 15q11-q 13 e d ist úrb io aut íst ico associad o com ret ard o m ent al, p rob lem as d e d esenvolvim ent o e d ist úrb ios com port am ent ais. A co m b in ação d e m et o d o lo g ias d a cit o g en ét ica clássica e m o lecu lar p ela t écn ica d e h i b r i d ação in situ f l u o r escen t e, d em o n st r o u o car i ó t i p o co m o 4 7 , XX, + m ar . i sh d er (1 5 ) (D15Z1+ + ,D15S11+ + ,GABRB3+ + ,PM L). Du p licação d o seg m en t o 15q p ro xim al rep resen t a a m ais consist ent e anom alia crom ossôm ica relat ad a em associação com aut ism o. A cont rib uição d os g enes das subunidades do recepet or GABA, assim com o out ros genes m apeados nessa região, para os sint om as clínicos da doença é discut ida.
PALAVRAS-CHAVE: t et rassomia 15, 15q11-q13, aut ismo, hibridação in situ fluorescent e, recept ores GABA.
1Professor Assist ent e Dout or - Depart ament o de Biologia, IBILCE, St at e Universit y of São Paulo (UNESP) Campus de São José do Rio Pret o, SP, Brasil; 2Prof essor Assist ent e Dout or - Cent ro Universit ário de Vot uporanga, CEUV, Vot uporanga, SP, Brasil; 3Prof essor Adjunt o -Depart ament o de Biologia M olecular, FAM ERP, São José do Rio Pret o, SP, Brasil; 4 Associat e Professor - Depart ment of M edicine, Univer-sit y of Colorado Healt h Sciences Cent er, Denver, CO, USA. Financial support : Part ially support ed by grant s from FUNDUNESP (Brazil) t o AES, and t he Nat ional Cancer Inst it ut e [CA46934] t o M VG.
Recevied 8 Agust 2001, received in final form 26 Oct ober 2001. Accept ed 5 November 2001.
Dra. Ana Elizabete Silva - Departamento de Biologia - IBILCE/UNESP - Rua Cristovão Colombo 2265 - 15054-000 São José do Rio Preto SP - Brasil. E-mail: anabete@bio.ibilce.unesp.br
Aut ism is a severe disorder t hat predominant ly affects males (4 males:1 female)1. The reported
popu-lat ion prevalence ranges from 1 in 1,000 2 t o 1 in
10,000 live birt hs1, t his large range probably being
due t o inaccurat e dat a collect ion based on Kanner’s descript ion of t his syndrom e. The diagnosis of au-t ism is based upon a number of behavioral criau-t eria including abnorm al social behavior, language and cognit ive developm ent3. Similar t o most behavioral
syndromes, autism is etiologically heterogeneous and has been associat ed w it h a diverse set of inf ect ious diseases such as rubella embryopat hy and herpes encephalit is, or genet ic condit ions such as
phenylke-t onuria, phenylke-t uberous sclerosis, and fragile X syndrome1.
How ever, t risomy or t et rasomy of t he 15q11-q13 region has also been report ed in some aut ist ic pa-tients w ith varying degrees of mental retardation4-12.
Abnormalities involving this chromosomal region are commonly ident ified in associat ion t o Prader-Willi syndrome (PWS) and Angelman syndrome (AS), usu-ally as chrom osom al delet ion. Addit ional copies of t his chrom osom al segm ent are less f requent ly de-pict ed and have been found in dist inct phenot ypes, including aut ist ic disorder, a few PWS and AS ca-ses5,13, and even apparent ly normal individuals5,6,14.
is-tic disorder w hose karyotype displayed an extra chro-m osochro-m e, ident if ied by t he f luorescence in sit u hy-bridizat ion t echnique as a der(15) w it h a duplica-t ion of 15q11-q13. This case reporduplica-t reinforces duplica-t he hypot hesis t hat addit ional copies of t his chrom o-somal segment may be causally relat ed t o aut ism.
M ETHOD
Case
The pat ient w as a female, born at full t erm in 1988 by normal delivery, w it h a birt h w eight of 2,650 g and a body lengt h of 46 cm. The 29-year-old mot her and t he 30-year-old fat her w ere non-consanguineous and report ed a pre-vious first t rimest er spont aneous abort ion. At 4 years of age, t he pat ient present ed w it h developm ent al delay, ment al ret ardat ion, hyperact ivit y, poor mot or coordina-t ion, scoordina-t ereocoordina-t yped movemencoordina-t s, ancoordina-t i-social behavior, abnor-mal language skills, and inabilit y t o maint ain social ap-proaches. She w as diagnosed as having infant ile aut ism (DSM -IIIR)15, and a mult iprofessional t herapy w as init iat ed
w hich result ed in pronounced improvement in t he clinical pict ure. Physical examinat ion at 8 years of age revealed hyperlaxit y of joint s, hypot onia, dolichocephaly,
promi-nent occiput, malformed auricles, bilateral epicanthal folds, dow nslant ing palpebral fissures, st rabismus, high-arched palat e, m icrognat hia, kyphoscoliosis, pect us excavat um , and bilat eral fift h finger clinodact yly. Brain t omography, X-ray, and nuclear magnet ic resonance evaluat ions w ere all normal. At t hat t ime, t he diagnosis of aut ist ic disorder (DSM -IV)3 w as est ablished.
Cytogenetic studies
Peripheral blood lymphocyt es from t he pat ient and her parent s w ere cult ured using PHA st imulat ed procedure, harvest ed and GTG banded follow ing st andard prot ocols. To confirm t he origin of t he marker chromosome t hat w as found, dual t arget , single color fluorescence in situ hy-bridizat ion (FISH) assays w ere perf orm ed. Slides w ere w ashed 1 min in 70% acet ic acid, rinsed in PBS, digest ed at 37oC in RNase A (100 µg/ml in 2xSSC) for one h and in
pepsin (0.01% in 0.01M HCl) for 5 min. Slides w ere t hen f ixed in f orm aldehyde (1% in PBS) at 37oC f or 5 m in,
w ashed in PBS, dehydrat ed in an et hanol series, and de-nat ured in 70% formamide/2xSSC for 3 min at 72oC.
Three set s of DNA probes m anuf act ured by Oncor (Gait hersburg, M D) w ere used: (a) D15Z1; (b) D15S11 and PM L; (c) GABRB3 and PM L. D15Z1 is cent romeric, D15S11
Fig 2. Metaphases hybridized with D15Z1 (A) and partial metaphases hybridized with D15S11/PML (B) and GABRB3 (C). Two normal copies of chromosome 15 displayed the expected centromeric signals and signals mapped at q11-13 and q21-22. The der(15) has two copies of the centromeric signal and a duplicated signal for D15S11 and GABRB3, suggesting tetrasomy 15q11-q13.
and GABRB3 are m apped at 15q11-q1316, and PM L is
mapped at 15q21-22. All probes w ere labeled w it h digo-xigenin, prepared follow ing the manufacturer’s instruct ions, and applied t o select ed areas of t he slides. These areas w ere covered w it h glass coverslips, sealed w it h rubber ce-ment and placed in a humidit y chamber at 37°C w here hy-bridizat ion w as allow ed t o occur for 20 h, aft er w hich t he slides w ere w ashed in 2xSSC at 72oC for 5 min. Probe D15Z1
w as detected w ith fluorescein isothiocyanate (FITC) and the tw o other probes w ith Rhodamine. Digoxigenin-Rhodamine detection w as performed at 37oC using consecut ive 30 min
incubat ions in Rhodamine-sheep ant i-digoxigenin, rabbit ant i-sheep, and Rhodamine-ant i-rabbit ant ibodies (Oncor’s Digoxigenin-Rhodamine Det ect ion Kit ). Digoxigenin-FITC det ect ion used FITC-avidin and biot inylat ed ant i-avidin antibodies (Oncor Digoxigenin-FITC Detection Kit). Finally, slides w ere w ashed in PBS, dehydrat ed in an et hanol series, air dried, and count erst ained w it h DAPI in Vect ashield M ounting M edium (Vector Laboratories).
The slides w ere examined in an Olympus BX60 micro-scope using single int erference filt er set s of red (Texas red), green (FITC), blue (DAPI), dual (red/green), and t riple (blue/ red/green) band-pass filt ers. For document at ion, gray scale im ages f or single color signals w ere capt ured using a cooled CCD camera (SenSys, Phot omet rics, Tucson, AZ), and w ere pseudocolored and merged using a Quips XL Genet ics Workst at ion (Vysis, Inc.).
RESULTS
GTG-banding analysis of t he pat ient ’s chromsom es revealed one sm all supernum erary chrom o-some in 20 cells scored, and t he pat ient ’s karyot ype w as designat ed 47,XX,+ mar (Figure 1A-B). This ex-t ra chromosome w as acrocenex-t ric and, as w as re-vealed by NOR-banding, show ed sat ellit es only at t he ends of t he long arm (Figure 1C). The CBG-band-ing highlight ed t he primary const rict ion plus an ex-t ra region aex-t ex-t he end of ex-t he long arm, w hich appea-red t o be an inact ive cent romere (Figure1D). GTG-banding karyot ypes of bot h parent s w ere normal.
DAPI band bet w een t he t w o cent romeric signals, indicating that the der(15) chromosome has segment 15q11-q13 duplicat ed and t hat t he karyot ype of t he p at ien t is 47,XX,+ m ar.ish d er(15) (D15Z1+ + , D15S11+ + ,GABRB3+ + , PM L-).
DISCUSSION
Alt hough a variet y of chromosomal anomalies have been described in individuals w it h aut ist ic dis-order9,11,17, t he occurrence of ext ra copies of
15q11-q13 has been t he one most frequent ly report ed. Despit e t he great clinical variat ion amongst aut ist ic pat ient s w it h addit ional copies of t he proximal re-gion of 15q, our pat ient shared feat ures such as ment al ret ardat ion, hyperact ivit y, epicant hal folds, high-arched palat e, kyphosis, and st rabismus w it h anot her cases. Spont aneous abort ion in t he family, as observed in t he present case, w as also previously report ed6,7. The num ber of addit ional copies of t his
region and t he ext ent of t he duplicat ed region are some of the major factors tentatively associated w ith clinical severit y in t hese pat ient s. In m ost cases, in-cluding t he pat ient report ed here, an invert ed du-plicat ion [inv dup(15)(pt er-q13)] in an addit ional chromosome w as responsible for t he t et rasomy of t his region6,7,9,14. In a smaller number of cases t his
region w as found in t risom y4,8,9. Trisomic pat ient s
usually displayed a milder phenot ype w hile t et ra-so m ic p at ien t s h ave m o re severe clin ical f ea-t ures4,5,13,14.
A considerable het erogeneit y in size, shape, and molecular composit ion of t he 15q proximal region w as observed in t he derivat ive chrom osom es. De-spit e t his, it is suggest ed t hat more severe feat ures such as ment al ret ardat ion and aut ist ic behavior are associat ed w it h a longer ext ension of t he duplica-t ion5,6. The der(15) found in t he present pat ient is
similar to the derivatives classified as type 3 in Cheng et al.5 and as type 1 in Crolla et al.6, w hich correspond
t o bi-sat ellit ed, dicent ric marker w it h inv dup(15), including t w o addit ional copies of all loci ext ending from t he cent romere dist al t o D15S12, differing only in t he presence of a single sat ellit e at t he end of t he long arm.
Anot her fact or t hat may play a role in t he pheno-t ypic variabilipheno-t y reporpheno-t ed in pheno-t he lipheno-t erapheno-t ure is pheno-t he pa-rent al origin of t he ext ra chromosome. A large num-ber of 15q11-q13 rearrangements of autistic patients w hich w ere evaluat ed for t heir parent al origin w ere ident ified as mat ernally derived6,8,10,12,13. How ever, in
t his w ork is not possible t o est ablish t he parent al
origin of t he ext ra chromosome. In a informat ive family, Cook et al.8 observed t hat pat ernally
inher-it ed duplicat ions of 15q11-q13 led t o a normal phe-not ype, w hereas mat ernally inherit ed duplicat ions led t o t ypical or at ypical aut ism. These findings sug-gest an import ant role for genet ic imprint ing in sus-cept ibilit y t o aut ism , w hich is not unexpect ed since t he 15q11-q13 region is know n t o be crit ically im-print ed in humans. Bot h pat ernal and mat ernal cop-ies of t his region are required for norm al develop-ment , w it h loss of t he pat ernal cont ribut ion leading t o PWS and loss of t he mat ernal cont ribut ion result -ing in t he AS phenot ypes13. Surprisingly, M ohandas
et al.18 report ed a pat ernally derived de novo int
er-stitial duplication of the proximal 15q containing the PWS/AS critical region in a patient w ith developmen-t al delay, w hich raises developmen-t he quesdevelopmen-t ion as developmen-t o w hedevelopmen-t her or not all pat ernal duplicat ions are benign w it h respect t o phenot ype. M ohandas et al.18 hypot hesized t hat
t he breakpoint s generat ing t he 15q duplicat ions, alt hough clust ered in t he proximal 15q, may vary at t he m olecular level result ing in dosage changes f or a different set of genes. It is also possible t hat t he clinical f indings in t his pat ient m ay not be causally relat ed t o t he duplicat ion.
A number of genes pot ent ially relat ed t o aut ism have been mapped at 15q11-q13. These genes in-clude t he pigm ent at ion gene P, w hich is suggest ed t o be relat ed w it h t he hypomelanosis of It o19, t he
ubiquit in-prot ein ligase (UBE3A) t hat is responsible for AS20, and t he t hree GABA
A recept or subunit s,
GABRB321, GABRA522 and GABRG323 . GABA is t he
major inhibit ory neurot ransmit t er in t he mammalian brain w it h synapt ically released GABA act ivat -ing recept ors w hich open chloride-perm eable ion channels and inhibit the electrical activity of the neu-ron, t hus making it less responsive t o an excit at ory input24. Previous st udies indicat ed a role for GABA
in t he suppression of seizure act ivit y and def ect s in GABA recept or funct ion in t he brain might be ex-pect ed t o produce seizures, uncont rolled behavior, and disorders of movement , all effect s relat ed w it h suppression of b3 subunit gene expression21. Wagner
et al.25 report ed t hat GABA can also act as an excit
behavioral problems such as hyperact ivit y and t o epilepsy. Whet her t he GABA recept or gene is caus-ally relat ed t o aut ism is not yet clear, and t he dupli-cat ed chromosomal segment not ably includes sev-eral ot her genes. How ever, Cook et al.26 screened
nine loci on t he 15q11-q13 region f or linkage equilibrium w it h aut ist ic disorder, and linkage dis-eq uilib rium w as f ound only af f ect ing GABRB3 155CA-2 locus, point ing t o t his gene as a causat ive candidat e gene for aut ist ic disorder.
The current ly available dat a underline t he neces-sit y of searching for chromosome 15q11-q13 abnor-malit ies in pat ient s w it h aut ism, and support t he molecular analysis of t his region as an approach t o t he clarif ycat ion of t he et iology of t his disorder.
Acknowledgement - The aut hors acknow ledge t he t echnical support received f rom Josué Rodrigues dos Sant os and Cint ia Fridman.
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