CATECHOLAM I N ERG I C M ETABOL I S M AND AUTISM

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Joelle MartinOair Josiane Herairlt Elisabetlt Petit Pascaline Guerin Laurence Harneury

Anne Perrot Jucyires Mallet Dominique Sair vage Gilbert Lelord Jean- Pierre Muh

In his original description of autism, Kanner (1943) first considered that this syndrome was linked with a ‘biological inability for social relatedness’. With regard to the aetiology of autism, prevailing opinion today is that it has a biological basis (Rutter and Schopler 1987). Autism has been recently redefined in the DsAI-HI-R (American Psychiatric Association 1987) as a pervasive developmental disorder affecting social, communicative and imaginative develop- ment.

I t has been suggested that catechol- amines-dopamine (m) and norepine- phrine plus epinephrine (NE + E)-are involved in the mechanisms of autistic symptoms, because these neurotrans- mitters appear to be involved in be- havioural disturbances (Salamone 1992). The main arguments have been recently reviewed in Martineau ef al. (1992).

Concerning enzymatic mechanisms, no study has been performed on the activity of tyrosine hydroxylase (TH) in autistic children. TH is the enzyme that converts tyrosine to dopa, the precursor of dopamine, and which is the rate-limiting enzyme for the synthesis of catecholamines (Nagatsu ef al. 1964). Conflicting results have been reported on the activity of dopamine beta hydroxylase (DOH) in children with autism; this enzyme converts dopamine to

norepinephrine (Kaufman and Friedman 1965). Goldstein el al. (1976) and Lake et al. (1977) found lower DB11 activity in subjects with autism than in controls, whereas Young ef al. (1980) found no difference. More recently, Garnier er al. (1986) also reported no difference in DOH activity between controls and children with autism as a group. However, the authors noted significantly more U ~ H activity in non-retarded autistic children than in children with mental retardation and autism.

Genetic factors are among the possible causes of the ‘biological inability’ suggested by Kanner. Although autism is a rare disorder, i t is much more prevalent among males than females (Ritvo ef al. 1989~. Fombonne and Mazaubrun 1992). By studying home movies, Sauvage et al. (1988) demonstrated the existence of early symptoms in infants. Over the last five years, the frequency of autism among siblings of autistic probands has been evaluated in several family studies (Ritvo ef al. 1989a, b; Fombonne and Mazaubrun 1992). The risk of siblings of children with autism having the same disorder is between 2 and 5 per cent, i.e. 60 and 100 times more frequent than in the average population. However, we know that there are substantial variations in procedures across studies that could distort the pooled estimates (Smalley et a/.

1988). Twin studies look particularly convincing (Folstein and Rutter 1977, Kitvo el ul. 1985, Stcffenburg et al. 1989). Despite some variability i n results, twin concordance for autism is consistently greater for monozygosity than dizygosity in all studies, but never reaches 100 per cent. This finding could be explained by the postconception environmental differ- ences that occur in monozygotic twins. Other studies have researched the co- existence of autism with autosomal or sex chromosomal problems and even simple gene disorders. Fragile sites on the x chromotomc have been found in 10 per cent of boys with autism in a pooled estimate from several studies (Wahlstrom et al. 1986, Cohen et al. 1991, Tranebjacrg and Kurc 1991). Some cases of autism are linked with chromosomal determinism diseases (Friedman 1969, Valente 1971, Gillberg and Forscll 1984, Rciss et a/ . 1986, Smalley el al. 1992).

We performed a study using whole blood and urine assays and molecular biology techniques. The first aim was to confirm previous results obtained on whole blood and urinary assays of DA and metabolites (DOPAC, HVA, 3 a l l ) and KE t E i,i children with autism (Martineau et al. 1992, Herault e f a/. 1993~) . The second aim was to test for an association between markers coding for the TH. DOH and dopamine receptor D3 (DRD3) genes and autism. An association means that there is an increased frequency of a specific genotype among individuals with a disorder in the population. Here, we used restriction fragment length poly- morphisms (RFLPS) as markers to identify haplotypes composed of gene poly- morphisms. We studied the Ti1 gene located on chromosome 11 (Powell el a/. 1984), the DBH gene located on chromosome 9 (Craig el a/. 1988) and the D R D ~ gene located on chromosome 3 (Sokoloff et a/. 1990).

Method Subjects Our study involved 50 children with autism (20 girls and 30 boys) aged between 23 months and 16 years, with a mean age of 6 years 8 months and a mean IQ of 54, and attending the Child Psychiatry Day Care Unit of the Centre

Hospitalier Regional Universitairc, Tours, France. The controls comprised 50 children (19 girls and 31 boys, with a mean age of 8 years 2 months) with no known neurological dysfunction or treatment, selected from a local and normal school population, according to place of birth. The controls were used for the molecular biology studies. All control children wcre of normal intelligence and doing school work appropriate for their age. All patients and controls were from families who had lived in central or western France for at least two generations.

Clinical evaluation Each child with autism received an extensive evaluation, including a detailed developmental history using a ques- tionnaire, a videotaped psychiatric assessment, psychological and linguistic testing, paediatric and neurological examination and audiological assessment. Examinations were carried out by a professional team of child psychiatrists and child psychologists, language pathologists, a neurologist, a social worker and a paediatrician, all expert in dealing with autistic children. Careful developmental, past and family histories were taken. A diagnosis of infantile '

autism was reached only i f all, or all but one, members of the team agreed that the child's condition met all the criteria for early infantile autism listed in the DSM-111-R. These criteria include a quali- tative impairment of reciprocal and social interaction, a qualitative impairment of communication and imaginative activity, and a markedly restricted repertoire of activities and interests. All children were in excellent physical health and none had a history of endocrine or systemic disease or fragile x syndrome.

Evaluations were made of autistic behaviour (Barthelemy and Lelord 1991), cognitive disorders (Adrien 1986), neuro- logical syndromes (Garreau el af. 1987), language disorders (Dansan et al. 1988) and environmental factors (Hameury et af. 1989). Signs of autism were evaluated by means of a thorough clinical examination and by a synthesis largely based on the terminology of the DSM-Ill-R classification. A complete psychological examination was performed to evaluate the

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cognitive disorders, using the Brunet- Lezine (1976) psychomotor development test (French version adapted from Gesell and Amatruda's 1944 scale). The degree of retardation was scored using the DSXI-111-R criteria (IQ>70, very slight or no retardation; 10 50 to 69, slight retardation; IQ 35 to 49, moderate retardation; IQ 20 to 34, severe retar- dation; IQ< 20, profound retardation). Neurological syndromes were investigated by means of a complete neurologicat examination, t t c ; , brain mapping, scanner and brain imaging. Scoring took into account the severity of neurological syndromcs and any associated epileptic disorders (Garreau ef al. 1984). Language disorders were evaluated with a complete speech test and a scale of verbal and preverbal communication (Dansart et al. 1990). Environmental factors were evaluated on the basis of interviews with the parents, relationships with other medical and socio-educative services, and a scale of psychosocial stresses (Hameury ef al. 1989) which evaluated living con- ditions, family environment and support capacities for the child. Each of the five aspects (autistic behaviour, cognitive disorders, neurological syndromes, language disorders and environmental factors) was graded from 1 (not present) to 5 (profound).

The clinical evaluation was completed using the Behaviour Summarized Evalu- ation (ssrr) scale (Lelord ef al. 1981, Barthelemy et al. 1990) which consisted of 29 behavioural items rated on a five-point scale ranging from 0 (never) to 4 (always). Each child with autism was assessed once a week by two independent raters who conferred about the weekly ratings to produce the final score. These two observers were nurses who had known the children for several months and who had daily contact with them.

Biochemical evaluation Blood samples were collected between 10.00 and 12.00 hours in Vacutainer tubes and containing EDTA mixed by gentle inversion; two aliquots of 1 -5ml. each were kept at +4Oc until assayed. Whole blood DA and N E + E were measured in nmol/L.

DA and its derivatives homovanillic

acid (HVA; total, free and conjugated forms), 3-4 dihydroxyphenylacetic acid (DOPA('; total, free and conjugated forms), 3 rnethoxytyramine ( 3 hlT) and NI. t E were assayed in a urine sample taken in thc department ovcr a period of seven hours (from 09.00 to 16.00) after elimination of night-time urine and usual dietary restrictions (tea, coffee, vanilla, chocolate, banana, walnut, grapefruit, Camembert, Coca-Cola, aspirin). IM, 3 bIT and N t c E levels are expressed in pmol/mmol creatininc. Blood and urinary samples were collected on the same day.

Whole blood DA, i\ik t t, and urinary

determined by high-performance liquid chromatography with electrochemical detection (Kiggin and Kissinger 1977, Jouve et (11. 1986). Urinary levels of N E + E cannot be separated in urine with this method.

DA, HVA, DOPAC, 3 hlT and N E t I: were

Molecular biology procedure Cienomic DNA, extracted from leucocytes by using standard techniques (Sambrook ef a/ . 1989), was digested with Bgl I 1 (TH), Taq I ( D I ~ H ) and Bal I ( D R D ~ ) restriction enzymes. For TH and D ~ H , samples were analysed by Southern technique. Samples were placed in 0 .8 per cent agarose gel (Sigma) in I x TBE (0.089 moVL TRIs, 0.089 mol/L boric acid and 0.002 mol/L EDTA disodium) and electrophoresed at 45V for 14 hours. The gels were denaturated in 0.5s sodium hydroxide, 0.15mol/~ sodium chloride and transferred to nylon filters by Southern blotting (Southern 1975). Probes were labelled with 3 2 ~ (Amersham, Arlington Heights, 111, 3000 Ci/mmol) and hybridized to the filters overnight at 65"c. The filters were then washed twice at room temperature in 2 x ssc ( 1 x ssc = 0.1 5 m o l / ~ sodium chloride, 0 * 1 5 m o l / ~ sodium citrate)/0.5 per cent sodium dodecyl sulfate for 10 minutes and twice at 65"c in 0.5 x scc/0.5 per cent sodium dodecyl sulfate for 15 minutes and autoradio- graphed with XAR film (Eastern Kodak 'Co., Rochester, NY) for at least two days. Insert of clones (TH, pPH) used for hybridization were provided by J . Mallet. For DRD3, samples were analysed by the polymerase chain reaction technique.

lpg of the I>NA was amplified with Taq I polymerase on an automated thermal cycler (I'erkin Elmer Cetus). To amplify a 462bp fragment, we used 0 . 5 ~ 8 of the two primes (Schwartz et al. 1992). The samples were denatured at 9 5 " ~ for six minutes. A total of 35 cycles were performed with the following steps: 92"c for one minute, 56"c for one minute and 72"c for one minute. The samples were incubated at 72"c for eight minutes for the final extension. 40~1. of the amplification was digested by Bal I and analyscd by electrophoresis through 13 per cent polyacrylamide gel. Depending on the absence or presence of the Bal I restriction site, a fragment of 304bp or two fragments of 206bp and 98bp were produced. This segment also contained two constant sites at 1 1 1 and 47bp (Lannfelt et al. 1992).

Slatistical tests Kruskall-Wallis, Mann-Whitney tests and correlation coefficients were used as statistical tests.

'The term 'frequency of the B allele' (for example for the TH gene located on chromosome 11) referred 10 the gene frequency in which all the 13 alleles were added up and divided by the total number of no. I I chromosomes (Comings et a/. 1991). For example, in the biallelic distribution (R, b), I * ~ I children were BB and NZ children were Bb, so the frequency of B allele was (2N1 t ~ 2 ) / 2 ( ~ 1 +w).

x 2 was used for statistical analyses. Yates correction for continuity was applied.

Results Clinical results 'The clinical results are summarized in Tables I and 11.

Table 1 presents the distribution of scores of the five evaluations in the autistic group. All except nine of these children with autism were rated 3 or above for autistic behaviour. All except one presented with considerable language disorders, Some were severely retarded, 22 of them (15 + 7) having an IQ of <35. This population was not neurologically impaired: nine children scored more than 2 on the neurological scale. Only four scored more than 3 on the environmental

TABLE I Distribution of scores obtained with clinical evaluation in the autistic group, from I (none) to 5 (profound)

Scores

1 2 3 3 5

Autistic behaviour 0 9 16 18 7

Cognitive disorders 6 8 14 I5 7

Ncurologicsl syndromes 13 28 7 2 0 I.anguage disorders 0 I 21 18 10 Environmental factors 34 10 2 2 2

TABLE 11 Clinical profile performed using the BSE scale for the autistic group

lrems Mean SEIM ~~ ~~

1. Eagerness to be alone 2. Lack of response to people 3. Poor social interaction 4. Abnormal eye-conracr 5 . Lack of effort 10

communicate using voice 6. Lack of appropriate facial

expressions and gestures 7. Stereorgped vocal and voice

utterances. echolalia 8. Lack of initiative, poor

activity 9. Relates inappropriately to

inanimate objects or to doll

10. Compulsive and:or ritualistic use of objects

1 1 . Resistance to change and to frustration

12. Stereotyped sensorimotor activity

13. Agitation, restlessness 14. Bizarre posture and gait 15 . Auto-aggressiveness 16. Hetero-aggressiveness 17. Soft anxiety signs 18. Mood difficulties 19. Disturbances of feeding

behaviour 20. No attempt to control urine

or faeces 21. Masturbation 22. Sleep disturbances 23. Short attention span and

distractability 24. Bizarre responses to auditory

stimuli 25. Behavioural variability 26, No imitation of gestures, or

27. Hypotonia 28. Lack of sharing emotion 29. Odd responses to body

voice

contact

~~

1.98 0.17 1.84 0.17 2.62 0.16 1.60 0.16

2.76 0.17

2.32 0.18

1.30 0.19

2.86 0.15

2.40 0.20

2.00 0.23

1.82 0-18

1.96 0.21 1.38 0 - 2 0 2.44 0.19 0.66 0.13 0.90 0.15 1.78 0.14 1.16 0.17

1.68 0.18

1.20 0.21 0 - 7 0 0.16 0.56 0.14

2.64 0.17

1.26 0.20 1.06 0.17

2.26 0.20 0.86 0.16 2.44 0.16

1.38 0.17

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Tyrosine a7C,onj. DOPAC

DOPA

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NOREPINEPHRINE EPINEPHRINE ,- 1 3 1 3 '

Fig. 2. Group means and SEMs /or whole blood levels of dopumine. norepiirephrine and epinephrine (expressed in ntnol/L).

scale, because of poor living conditions. Table 11 shows the mean and standard

error of the mean (SEM) of BSE scale ratings for the autistic group, confirming that i t is a good clinical tool for the observation of autistic behaviour. Elev- ated scores were found for items which

Free HVA A

were related specifically to autistic symptoms-poor social interaction (2-62), lack of effort to communicate using voice (2.76), lack of initiative, poor activity (2.86), bizarre posture and gait (2-44), inappropriate relation to inanimate objects or to doll (2*40)-and also short attention span and distractability (2.64) and lack of sharing emotion (2-44). This group was relatively homogeneous, with intensive' autistic behaviour. .

Biochetnical resiilts Figure 1 shows the group means determined for urinary levels of D.4, H V A (total, free and conjugated forms), DOPAC (total, free and conjugated forms), 3 MT and NE t E for the children with autism. These means were compared with our laboratory values for unaffected children of the same age, and confirmed our previous results (Martineau et a/. 1992) for children with autism: slightly lower levels of DA, higher levels of H V A (for the three forms), no change of DOPAC, higher levels of 3 MT and higher levels of NE + E.

Group means determined for whole

TAR1.E I l l Corrclation coefficients betHern urinary H V A , DOPAC, 3 MT. D A and NE + E levels in children Hith autism

f I I V A c HVA I I>OPAC f I>OPA<’ c DOPAC 3 3IT I > A Nt 4 t

t 111’11

0.8754 0.4401 0.4898 0.3985 0.4152 0,3468 0.4314 0.3743

._ -

0.5019 0.4442 0.8319 0 . 3 ~ 9 3 0.8294 0.3798 0.3299

0 . 3 7 5 1 0.7340 0.51 I6

I = total. f free, c - conjugated forms

blood levels of DA and NF. t E for the TABLE IV children with autism arc shown in Figure Frequencies and x’ values for alleles of TH. DllH and

DRD3 genes. Restriction polymorphism of TH gene with BgI I I enzyme, of DOH gene with Tap I enzjme and of D R D 3 gene with Bal I enzyme

* * These wi th Our laboratory values for normal children of the same ages. They also confirmed our previous results (Hcrault et a/ . 1993a) Allelic frequencies

observed in the blood of children with autism: higher levels of DA and E and no change for NE.

There was no significant correlation between whole blood and urine mono- amine levels in children with autism. As previously observed (Herault et al. 1993a), there was a significant relationship between the whole blood NE L E levels (r=0.7114, df 49, p < O . O O I ) , but also between the whole blood DA and NE levels (r=0-5615, df 49, p<O.OOI), and between the whole blood I)A and E levels (r=0.6374, df 49, p c 0.001).

Table 111 presents the coefficient correlations (with statistical significance) obtained between urinary parameters in the children with autism. I t may be noted that total HVA is a good marker of the dopaminergic pathway because of the numerous correlations obtained between urinary total HVA levels and the other parameters.

Molecular biology results The genetic results given in Table 1V indicate the allelic frequency poly- morphisms of T H , IIfiH and DRD3 markers. No significant difference by x2 analysis was found in allelic frequencies of these markers between children with and without autism. Nor was any relationship found between the allelic

- TH B

b

DRH A l A2 BI €32

DKD3 I 2

Unaffected children

0.26 0.74

0.64 0.36 0.36 0.64

0.72 0.28

- -- Children 1’

with autism

0.28 0.72 0.02

0.74 0.26 1.89 0.33 0.67 0.09

0.66 0.34 0.58

frequency polymorphisms of these markers and clinical assessment or biochemical measurements.

Discussion These results obtained in a homogeneous group of autistic children show dif- ferences in the whole blood and urinary excretion of catecholamines, but no association between aut.istic disorders and loci for genes coding for enzymes involved in the biosynthesis of monoamines.

The biochemical results confirm, in a larger sample, the slightly lower levels of urinary DA, and the higher levels of urinary HVA, urinary 3 Mf and urinary NE + E previously observed (Martineau et al. 1992), but also higher levels of DA and

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t in the whole blood (Herault ef a/. 1993~) of autistic children compared with normal childrcn of the same age. Evcn if these measurements cannot be used as a diagnostic tool because of their lack of specificity (Martineau et a/. 1992), they may be useful to appreciate relations between biological and clinical parameters, and may contribute to the assessment of drug efficacy (Martineau et a/ . 1985, Barthelemy ef a/ . 1989).

The molecular biology findings have both clinical and biological implications. Over the last 10 years, research on autism has clearly implicated a clinical genetic component in the actiology of this disorder. Most research has centred on the family, dealing with early detection, twin studies and cases of autism associated with chromo5ome abnormalities or other genetic disorders. I n the course of these studies, other kinds of. developmental disorders (e.g. cognitive and language impairment) have been observed particularly often among siblings of patients with infantile autism (Baird and August 1985, .MacDonald ef a/. 1989). However, the sibling, twin and population data are not consistent with a simple monogenic mode of transmission for all cases of autism: none of the four ordinary modes of genetic transmission alone can explain the general results of these studies (Ritvo et al. 1985). This has encouraged us to refine clinical procedures such as multi-axial evaluation and the RSF scale to identify homogeneous groups of children. In the same way, our group is elaborating a ‘genetic’ scale scored from 1 to 5 (Lenoir 1989).

Identification of specific DNA or gene markers in autism is a promising area, given the increased sophistication of molecular biology techniques. The literature currently contains one linkage investigation of autism using blood polymorphisms in a subset of multiplex families (Spcnce ef a/. 1985). They found no evidence of linkage for autism with 30 blood polymorphisms and could rule out linkage with 19 of these markers.

The association of autism with specific gene markers has also been examined. Spence ef al. (1985) tested for an association of HLA with autism by comparing the number of shared HLA haplotypes in autistic siblings. If an

association is present, the siblings with autism should share H I . A haplotypes more often than expected o n the basis of mendelian segregation. They found no evidence to support an association of HLA and autism. Recently a positive association has been observed between autism and a marker located on the tip of the short arm of chromosome 1 1 (c-Harvey ras oncogene: HKAS gene) (Herault et a/. 19936).

Although the monoamines appear to be involved in the behavioural disturbances of autism, our study did not demonstrate a positive association between autistic disorders and loci containing genes for enzymes of the biosynthesis of mono- amines, nor the dopamine D3 receptor gene. As the HRAS gene is closely linked to the gene encoding TH, this association suggests that the D N A region of chromosome 1 1 may confer susceptibility for autism. The gene of the D4 dopamincrgic receptor, located by van To1 et al. (1991) on the short arm of chromosome 1 1 linked to the HRAS gene, could also be considered a good candidate. Recently Comings el a/. (1991), studying the DZ doparninergic receptor located on the long arm of this chromosome 11, found an association between a marker of the dopamine D2 receptor gene and autism, and many other neuropsychiatric disorders as well (i.e. Tourette syndrome, attention deficit hyperactivity disorder, alcoholism and post-traumatic stress disorder).

We consider that the negative results concerning genes of TH, DOH and D R D ~ are provisional. This preliminary study should be continued using a larger sample of children in order to determine subgroups separated by the intensity of autistic symptoms, cognitive and neurological disorders, and language disturbances (Hameury et al. 1989). A very careful study of other genes located on chromosome 1 1 should also be performed.

Accepred f o r publicorion 20th January 1994.

Acknowledgements This study was performed by INSERM U316. INSERM Nefwork No. 493001, CRAMST No. 99831 3, Convention INSERM-FRANCE TELECOM and Fondation Langlois. We are very grateful to Professor J . C. Schwartz for his critical reading of the manuscript. The study received the approval of the Ethics Committee of the Centre Hospitalier Regional, Tours.

(1 urhorr ’ Apporrrrrnrnrs Gilbert Lelord, M . D . , Ph.t>. . Professor of

*JoelIc Alarrineau. I’h.I).. Chargee de recherche< Jean-Pierre Muh. M . D . . P1i.D.. Professor of INStIK\l; Biochemistry; Jo5iane Cierault, Ph.D.; Elisabeth f’ctit. Ph.D. Studcnt; Pa5calinc Ciuirin, M.D.; 1-aurcnce tlarneury. M.D. ; ‘Correspondence lo firs, aurlror at INSEKM U3 16, Anne Perroi. X1.D.; Department de Neurophysiologie et de Psycho- Jacques Slallet. Ph.11.. Research Director; pathologie du diveloppement, CHU Rretonneau, Dornrniquc Sauuge, k1.D.. Professor of Psychiatry; 37044 Tours Cedex, France.

Physiology;

INSERhl U316, Tours.

su Jl x1 A K 1’ The author\ determined Icvels of dopamint: (DA) and its derivatives homovanillic acid (HVA), 3-4 dihydrouyphenylacetic. acid (DOPAC), 3 rnethosytyramine and norepinephrine + epinephrine (KE + E) in the urinc. and [)A, E and NE in the whole blood of 50 autistic children aged between I year I I months and 16 ycars. An association was tested for between markers coding for the enzymes and D3 dopamincrgic receptor genes implicated in the monoaminergic pathway and autism. using restriction fragment-length polymorphism. There were significant modifications of catecholamine metabolites, but n o difference for allele frequencies of the genes coding for tyrosine hydroxylase, dopamine beta hydroxylate and DRD3 in this population compared with a healthy school population matched for chronological age. However, somc of the data encourage a more complete study of chromosome I I .

Krisualc Merubolisme carecholat,ritier~iqrce er auristire Les autcurs ont mesurc les taux de dopamine (DA) et de ses derives, acide homovanillique (HVA), 3-4 acide dihydroxyphtnylacetique (DOPAC). 3-methoxytyramine, el noradrenaline t adrenaline (SE + E). dans les urines, et ceux de DA, E et NE dans le sang total de 50 enfants autistiques Pgcs de 23 mois B 16 ans. L n e association a ete recherchce avec des marqueurs codant pour les genes des enzyrncs et des recepteurs dopaminergiques D3, en utilisant le polymorphisme de longueur du fragmenr de restriction. I I fut constate des modifications significatives des metabolites catecholaminergiques, mais pas de difference pour la frequence des alleles pour les genes correspondant a la tyrosine-hydroxylase, la dopamine beta-hydroxylase, et la DRD3 de la population etudiee en comparaison avec une population scolaire en bonne sante et appariee pour I’Pge. Cependant. cerraines des donnces obtenues cncouragent h une etude plus complcte du chromosome 1 1 .

ZUSAb13lESFASSUNG Karrclrolanrin-iMetabolistnus und A utismics Die Autoren bestimmtem bei 50 autistischen Kindern im Alter zwischen I Jahre und 1 1 Monaten und 16 Jahren die L‘rinspiegel von Dopamin (DA) und seiner Derivate Homovanillinsaure (HVA), 3-1 Dihydrosyphenylessigsaure (DOPAC), 3-Methoxytyramin und Noradrenalin und Adrenalin (NE und E). sowie die Blutspiegel von DA, E und NE. Anhand des Restriktions-T;ragmentlangen- Polymorphismus wurde eine Ascoziation zwischen Autismus und DNA Markern untersucht, die fur Enzym- und D3 dopaininerge Rezeptorgene codieren und im monoaminergen Stoffwechsel eine zcnrrale Rolle spielen. t3ei diesen Patienten fanden sich im Vergleich LU gesunden altersentsprechenden Schulkindern signifikante Veranderungen der Katecholaminmetaboliten, aber keine Unterschiede der Allelfrequenz der Genloci fur Tyrosinhydroxylase, Dopaminbetahydroxylase und DRD3. Allerdings sprechen einige Daten f u r eine genauere Untersuchung des Chromosoms 1 1 .

RESGMEK Metabolistno corecolaminergico y aurismo Los autores determinaron en 50 nillos autisticos de entre I arlo y 1 1 mews de edad y 16 allos, 10s niveles de dopamina (DA) y sus derivados: acido homovanilico (HVA), 3-4 acido hidroxi-fenilacetico (DOPAC), 3 metoxitiramina y norepinefrina t epinefrina en la orina, asi como DA, E y NE en sangre total. Se investigo una asociacion entre 10s marcadores del codigo para 10s enzimas y 10s genes del receptor D3 dopaminergico implicados en la via monoaminergica, utilizando la restriccion del polimorfismo de la longitud drl fragmento. Habian modificaciones significativas de 10s catabolitos catecolaminicos, pero ninguna diferencia en las frecuencias alelicas de 10s genes loci- contenedores para la hidroxilasa tironina, dopamina-beta-hidroxilasa y DRD3 en esta poblacion, en comparacion con una poblacion escolar sana de la misma edad. Sin embargo, algunos de 10s datos, animan ha acometer un estudio mas completo del cromosoma 11.

Hejerences Adrien, J . L. (1986) ‘Interit des evaluations Baird, 7’. D., August, G . J . (1985) ‘Familial

psychologiques dans Ies troubles graves du heterogeneity in infantile autism.’ Journal of developpement .’ Neuropsychiarrie de I’Enfance el Autism and Developmental Disorders. 15,

American Psychiatric Association (1987) Diagnosric Barthelemy, C., Lelord, G. (1991) Les echelles and Srurisric-a1 Manual of Menrat Disorders. 3rd d’Evaluarion Clinique en Psychiarrie de I‘EnJant. Edn-Revised. Washington, DC: APA. Paris: Expansion Scientifique Franqaise.

de l’Ado1escent.e. 34, 63-YI . 315-321.

2 E L,

-2 U

695

< b a2 c

U m

696

- Bruneau. N., Jouve, J.. Martineau, J . . Muh. J . -P . . Lelord, G. (1989) ‘Urinary dopamine metabolites as indicators of the responsiveness to fenfluramine treatment in children with autistic behavior.’ Journal of Auiisni and Developmental Disorders. 19, 241-254.

- Adrien. J . L.. Tanguay. P., (iarrrau, H., Fermanian. J.. Roux. S . , Sauvage. D., Lelord, G . (1990) ‘The Behavioral Summarired Evaluation: validity and reliability of a scale for the assessment of autistic behaviors.’ Journal of Auiism and Developrnenlal Disorders. 20. 109-203.

Brunet , D . . Lezine, I . (1976) ‘2chel le d e developpement psychomoteur de la premiere enfancc.’ In Rrunet. 0. . Lezinc. 1 . (Eds.) 1.c De veloppan en i Psjcholog iqiie de la Premiere Enfance. 2nd Edn. Park: Presm Unrversiraires dc France.

Cohen, I . L . , Sudhalter, V., Pfadt. A,. Jenkins, C. , Brown, W . T.. Vietze, P. M. (1991) ‘Why are autism and the fragile-X syndrome associated? Conceptual and methodological issues.’ American Journal of Human Generics, 48, IY5-202.

Comings, D. E.. Comings, B. G. , hluhleman, D., Dietz. G . , Shahbahrami, B., Tast. D., Knell, E . , Kocsis, P.. Haumgarten, R. , Kovacs, B. W. el al. (1991) “fhe dopamine D2 receptor locus as a modifying gene in neuropsychiatric disorders.’ Journul v f ihe American Medical Associalion, 266,

Craig, S. P., Huckel. V . J.. h n i o u r o u x , A. 31. C.. hlallet, J . , Craig, I . W. (1988) ‘1.ocalization of human dopamine beta hydroxylase (Dot{) gene to chromosome 9q34.’ Cyrogenerics atid Cell Genetics.

Dansart, P.. Barthelemy, C.. Adrien, J . L., Sauvage, D., Lelord. G. (1988) ‘Troubles de la com- munication pre-verbale chez I’enfant autistique: mise au point d’une &helle d’evaluation.’ Aciualiies Psychrarriques, 4, 38-43.

Pallado. I . . , Lenoir, P . (1990) ‘Evaluation du langage.’ In Lelord, G., Sauvage, D. (Eds.) L ’Aitirstne de I’Enfanr. Paris: Xlasson. pp.

Fol5tein. S . . Rutter, M. (1977) ‘Infantile autism: a genetic study of 21 twin pairs.’ Journal of Child Psychology and Psychiarry, 18, 297-321.

Fombonne, E., Ma7aubrun, C. (1992) ‘Prevalence of infantile autism in four French regions.’ Social Psjchiairy and Psychiairic Epideniio/ogy. 27,

Friedman, E. (1969) ‘The autistic syndrome and p hen y I ket on u r i a . ’ Schi;op hrenia Rulleiin. I ,

(iarnier. C . . Comoy. E.. Barthelemy. C. , Leddet, I . , Garreau, B.. Muh, J.-P.. Lelord. G. (1986) ‘Dopamine B hydroxylase and homovanillic acid in autistic children.’ Journal of Aurism and Developmenial Disorders, 16, 23-29.

Garreau, H., Rarthelemy. C.. Sauvage. D.. Leddet, I . . Lelord, G. (1984) ‘A comparison of autistic syndromes with and without associated neurological problems.’ Journal of Auiism and Developmenial Disorders, 14, 105-1 1 1 .

Bruneau, S. . Martineau, J . (1987) ‘Aulisrne et psychoses de I’enfant. Signes neurologiques et examcns complementaires.’ Soins Psychiarrie, 82,

Gesell, A., Amatruda, C. S. (1944) Normal and Abnormal Child Developmenr: Clinical Methods and Pediatric Applications. New York: Haeber.

Gillberg, C., Forsell. C . (1984) ‘Childhood psychosis and neurofibromatosis. More than a coincidence?’ Journal of A urism and Developmenral Disorders.

Goldstein, M.. Mahanand. D., Lee, J . , Coleman, M.

’

1793-1800.

48, 48-50.

-

139-1 57.

203-2 10.

249-261.

-

15-17.

14, 1-8.

(1976) ‘Dopamine-beta-hsdroxyla5e and endo- genous total 5-hydroxyindole levels in autistic patients and controls.’ In Coleman, M . (Ed.) Thc Airrisric Syndrome. New Y o r k : Elsevier.

Hameury, I . . , Adrien. J . t.., Roux, S., Dansart, P., Martineau, J . , Lelord, G. (1989) ‘Standardisation des donnees par classification multi-axiale !ans les troubles du developpement global de I erifant.’ iVeitropsyc/fiarrir, dc I’Enfunce ei de lildolescence. 12, 550-556.

Herault, J . , Martineau, J., Perrot, A, , Jouve, J., Tournade, D.. Barthelemy, C.. Ixlord, G . . Xluh. J.4’. (19330) ‘Investigation of whole blood and urine monoamines in autism.’ European Child und Adolescent Psychiarry. 2, 2 1 1-220.

Perrot, A,, Barthelemy, C.. Buchler, kl. , Cherpy. C. . Leboyer. $1.. Sauvage, D., Lclord, G., Xlallet. J . . kluh, J . - P . (IYY3h) ‘Possible association of c- Harvey-ras-l (HRAS) marker with autism.’ Psychiatry Research, 46, 26 1-267.

Jouve, J., klartineau, J.. Mariotte, N., Barthelemy. C. , Xluh, J . -P. . I.elord. G. (1986) ‘Determination of urinary serotonin using liquid chromatography with electrochemical detection.’ Joiirnul o/ Chrotnarography. 318, 437-443.

Kanner, I.. (1943) ‘Autistic disturbances of affective contact.’ Nervous Child, 2, 217-250.

Kaufman, S., Friedman, S. (1965) ‘Dopamine beta hydroxylase.’ Pharniacological Review. 17,

Lake, C. R., Ziegler, M. G.. Murphy, D. L. (1977) ‘Increased norepinephrine levels and decreased dopamine beta hydroxyla5e activity in primary autism.’ Archives o/ General Psychiatry. 34,

Lannfelt, L., Sokoloff. P., klartres. M. P.. Pilon, C., Giros, B., Jonsson. E.. Sedvall, G . , Schwartz. J . C . (1992) ‘Amino acid substitulion in thedopamine D3 receptor as a useful polymorphism for investigating psychiatric disorders.’ P.Tycl/iairir Generics. 2, 249-256.

Lelord. G. , Xluh, J.-P., RarthdCmy, C . . Martineau. J., Garreau. H. (1981) ‘Effects of pyridoxine and magnesium on autistic symptoms-initial observations.’ Journal of Aitiism and Develop menial Disorders, 1 I , 219-230.

I.enoir, P . ( 1989) Anrecedenrs medicairs fatviliaux dans les rroitbles globaux du devcloppemeni de I’en fan, (326 cas). Eiitde clinique de la place des /acreurs RPnPirques dans I’aulistne. (Thesis.) Medecine d e Tours.

MacDonald, H.. Rutter. kl., Rios, P. , Boliin. P . (1989) ‘Cognitive and social abnormalities in the siblings of autistic and Down’s syndrome probands.’ Paper given or the first World Congress in Psychiatric Generics, Churchill College. Cam bridge.

Martineau. J . , Barthelemy, C.. Garreau, B., 1.elord. G. (1985) ‘Vitamin B6, magnesium and combined B6-Mg: therapeutic effects in childhood autism.’ Biological Psychiarry, 20, 467-478.

- Barthelemy. C. , Jouve. J., Muh, J.-P.. 1.elord. G. (1992) ‘Monoamines ( sero tonin a n d catecholamines] and [heir derivates in infantile autism: age-related changes and drug effects.’ Developmenral Medicine and Child Neurology, 34.

Nagatsu, T.. Levitt. M., Udenfrien, S. (1964) ‘Tyrosine hydroxylase: the initial step in norepinephrine biosynthesis.’ Journal of Bio- logical Chemisiry, 239, 2910-291 7.

Powell, J . F., Boni. C.. Lamouroux, A., Craig. I . W., Mallet, J. (1984) ‘Assignment of the human tyrosine hydroxylase gene to chromosome I I .’ Federarion of European Biochemical Socieiies Lerrers, 175, 37-40.

-

7 1 - 1 0 .

553-556.

593-603.

Rei\\. A. I... I.'einsiein, C.. Rosenbaum. K. N. (1986) 'Auiicm and geneiic dicorders.' Schi:ophrenia f ~ i i l l c * ~ r t i , 12, 724-138.

Kiggiii, K. 51., Kk4ingcr. 1'. T. (1977) '1)ctcrmi- naiioii ol carecholamine\ iii urine by reverse phasc liquid cliromaiography with clectrochcinical dcicciioii.' / l i iuly/icd Clicitiiury, 49, 2109 21 I I .

Kitvo. I!. R.. 1-rccman, 13. .I.. blason-Broihers, A, , hlo. A , , Hiivo. A. X I . (19x5) '('oneordance for ihc \yndronie of auii\nt in 40 pair5 of afflicted twin\.' ,4iti~wcan Jorrriial o f Psychiurry. 142. 74-77

I'ingrcc, C.., Zla\on-Rrother\. A , , Jorde. I.. . Jcn\oii. \ V . R.. Xlcblalion. \V. $ I . , f'cierson. 1'. 13.. 510. A , . Kiivo, A . A l . (19890) 'The IJCI.r\ IJnivcr\iiy of Linli. l..pidcmiologic w r v c y of iiuii\ni: prevalence.' Aiiicr/cun Joicrtiol (11. i'.yw/iicrrr~. 1 46, 194 - 199.

Jordc. 1.. 13.. Zlnson-Broihcr\, A, . f:recman, B. J . , t'ingree. ( ~ . , Jones. 3.1. 13.. Mchlahon. W. N., Pcicrwn, 1'. 13.. Jcnxn. \\I. R.. hlo. A. (19890) 'The I;C".A-Cniversiiy of Utah. fipidcmiologic survey ot autism: recuircncc rish estimates and ge~ir i ic counselling.' Auicmcoii Jotrriiol O J ~

f?\.vc~/iiu/r~~, 146, 1032-1036. Kirllcr. A1.. Schoplcr. E . (1987) 'Auiism and

peria\ive drvclopmenial disordcrs. Concepts and diagnociic iswcs . ' Jotrriial o/ Au/i.\iii und ~ ~ ~ ~ ~ c ~ l i ~ ~ ~ t r i r i i r o / lhorrler.\, 17, 159-186.

Salniiioiic. J. D. (1992) 'C:omplcx motor and cenwrinioior functioiis of ctriatal and nccurnbent dopamine: involvcniunt in inwumcntal behavior proce\w\.' Pr,.chopiiurniuc(r/u~y. 107, 160-1 74.

Sambrooh. J.. Friisch. E. F., Maniaiis, 7'. (1989) 'A~ialysi\ of gcnomic D N A by Southern hybridation.' In Ford, N., Nolan, V. C.. Icrgii\on, 51. (Ed\ . ) .~llo/ecirlur Cloning; A 1.uhoruror.v bfunitol. 2nd Edn. Yo/. 2. New York: ('old Spring tlarhor. pp. 9.31-9.62.

Snuvagc. D., Faure, M., Adrien. J . I... tiamcury, I.., Ih r thd~my, C.. I'crrot. A. (1988) 'Autismc el t i Iin5 tam i l iaii Y . ' An tialcs de P ryclirurrtc. 4, 4 18-424.

Schwnrir. I . C., Giro\. l3 . , Martres, M. P., Soholotl. f'. (1992) 'The dopamine receptor tamily: niolecular biology and pharmacology.' Scviirtiurs i i i /hi. Ncicurosc.rencc~.r. 4, 9Y- 108.

Sniallcy. S. l . . . Asarnow. K . I . , Spcnce, 51. A.

- -

-

(19x8) 'Autisni and genetics. A dccade of research.' Archives oJ Gencrul Psychiarrv. 45,

-- Tanguay. P . I!.. Smith, M., Guttierrcr, G . (1992) 'Auii jm and tuberous derosis.' Joiirtialo/ Aiittsii/ unrl De~elopi~icwrul Disorrlers. 22, 339-335.

Soholoff. P.. Giro<, B.. Martrcs, M. P., Bouthenet. M. l . . . Sch\varix. J . C. (1990) 'hlolecular cloning and characicrization of a norcl dopaminc receptor (D3) a \ a targei for ncurolepiics.' Nulure, 347, 146- IS I .

Southern. F. $1. (1975) 'Detection of cpccific ~equences among DNA fragmenis teparaicd by gel elcctroplioresis.' Joirrnal 0.f Mo/ccitlor

Spcncc. % I . A , , Kitvo. E. R., Maraiota, M. L.. Funderburck. S. J., Sparkes, K. S.. Freeman. B. J. (1985) 'Gene mapping studies with thc syiidrome of auiism.' Behavioral Generics. 15,

Stcffcnburg. S.. Gillberg, C., Hellgren, L . , Anderson. L.. Gillberg. 1 . C., Jakobsron, G.. Rohman, M. (19R9) ' A twin study of autism in Denmark. Finland. Iceland, Norway and Sweden .' Journal o j Child Psychology and P.~ychiatry. 3, 405-4 16.

lranabjaerg, I . . , Kure, P . (1991) 'Prcvalence of Fra(X) and other specific diagnose: in auiistic individuals in a Danish county. Aitierican Joitrtiui o f iMrdical C;eneiics. 38, 2 1 2-2 14.

Valente, M. (1971) 'Autism: symptomatic and idiopathic autism and mental retardation.' Pediatrics. 48, 495-496.

van Tol, H. ti. M.. Runzow, J. R.. Gum, H. C. , Siinahara. K.. Secinan. l'., Nirnik. H . 8. . Civclli, 0. (1991) 'Cloning of the gene for a human dopamine 114 receptor with high affinity for the antipsychotic clozapine.' Narure, 350, 610-614.

Wahlsirom. J . . Gillberg. C.. Gusiavson. K. ff., tlolmgren. G . (1986) 'Infantile autism and the fragile X . A swedish multicenter study.' Anicrican Journal o/ Medical Generics. 23, 403-408.

Young, J . G . . Kyprie, R. M., Ross. N. T., Cohen, D. J. (1980) 'Scrum dopamine beta hydroxylasc ac t iv i t y : clinical application\ in child psychiatry.' Journal o/ A i r i is in and Iler~elopi~ietiral Disorders. 10, IYJ.

953 -96 I .

HioloRy, 98, 503-517.

1-13.

-t 01 2

P

697