WO2000068433A2

WO2000068433A2 - Autism gene

Info

Publication number: WO2000068433A2
Application number: PCT/US2000/012385
Authority: WO
Inventors: Julius Peters; N. Sitara Waidyaratne
Original assignee: Children's Hospital Of Los Angeles
Priority date: 1999-05-06
Filing date: 2000-05-05
Publication date: 2000-11-16
Also published as: WO2000068433A3; AU4989700A

Abstract

A method of screening for autism in a subject comprises detecting the presence or absence of downregulation of expression of active human aromatic L-amino acid decarboxylase (AADC) in said subject, the downregulation of expression indicating said subject is afflicted with or at risk of developing autism. The method may be carried out by detecting the presence or absence of a mutation in the first neuronal exon of the human aromatic L-amino acid decarboxylase (AADC) gene. Subjects may be heterozygous or homozygous for the mutation. The mutation screened for is preferably a deletion mutation, and is most preferably a delta 26-29 deletion mutation. The presence of the mutation indicates that the subject is afflicted with autism or is at increased risk of developing autism. Isolated DNA encoding mammalian, preferably human, AADC, which isolated DNA contains a mutation as described above, is also disclosed, along with vectors containing such DNA and cells containing and expressing such DNA.

Description

AUTISM GENE

Related Application Information

This application claims the benefit of U.S. Provisional Application No. 60/132,845, filed May 6, 1999, and U.S. Provisional Application No. 60/150,087, filed August 20, 1999, which are incorporated by reference herein in their entireties.

Field of the Invention

The present invention concerns genes containing mutations indicative of autism, and methods of screening for autism in human subjects.

Background of the Invention

Autism is a pervasive developmental disorder involving language delay and dysfunction in reciprocal social interaction. Autism includes a spectrum of disorders which may or may not involve mental deficit. Autism is typically a life-long disorder. Autism is characterized by a behavioral syndrome often recognized between two and three years of age. The core of the syndrome is a deviant and/or retarded development of cognitive capacities and skills necessary for social relations, communication, fantasy, and symbolic thinking. Almost all autistic children do not rέach independence as adults and 75% are deemed mentally retarded. Diagnosis of autism presents difficulties in its own right, and a number of modalities have been proposed primarily based upon psychiatric evaluations.

A number of different therapies have been attempted in an effort to cure autism or at least lessen the clinical symptoms thereof. Such have included drug therapies as well as psychiatric care and attempted counseling. In general, results of such treatments have been disappointing, and autism remains very difficult to effectively treat, particularly in severe cases.

W. Shaw, Diagnosis of Autism and Treatment Therefor, U.S. Patent No. 5,686,311 (assigned to The Children's Mercy Hospital), describes a method for diagnosing the likelihood of autism in patients by obtaining a sample of body fluid and detecting at least one marker compound therein.

D. Comings, Tourette Syndrome, Autism and Associated Behaviors, U.S. Patent No. 5,405,943 (Assigned to City of Hope), concerns the human tryptophan oxygenase gene sequences, and probes for the diagnosis of psychiatric disorders employing the same.

Presently, the genetics of autism is not well understood. Hence, there is a need for new ways to screen for autism.

Summary of the Invention

A first aspect of the invention is a method of screening for (e.g., diagnosing, prognosing) autism in a subject. The methods comprise detecting downregulation of expression of active human aromatic L-amino acid decarboxylase (AADC) in the nervous tissue of a subject. The downregulation of expression is indicative of the likelihood of occurrence of autism in the subject. Any suitable nerve tissue sample may be used, with samples from the central nervous system being more preferred, e.g., cerebellar samples, cerebral samples, and the like.

The detection step need not be carried out by determining enzyme activity directly, but may be carried out by detecting AADC DNA or RNA expression in a sample obtained from the subject.

As a further aspect, the present invention provides a method of screening for autism in a subject, comprising detecting the presence or absence of a mutation in the AADC gene, where the presence of such mutation indicates that the subject is afflicted with, or is at increased risk of developing, autism. In particular embodiments, the foregoing method may be carried out by detecting the presence or absence of a mutation in the first neuronal exon of the AADC gene. Subjects may be heterozygous or homozygous for the mutation. The presence or absence of the mutation may be detected in any suitable cell or tissue sample from the subject, e.g., peripheral cells such as B-lymphocytes, skin samples, tissue biopsies, and the like. The mutation may be a missense mutation, nonsense mutation, insertion mutation, or deletion mutation and may occur in exon or intron sequences, or in upstream or down-stream regulatory regions of the ADDC gene. Preferably, the mutation results in a down-regulation in expression of the AADC gene. The mutation screened for is preferably a deletion mutation, and is most preferably a delta 26-29 deletion mutation (i.e., deletion of nucleotides 26-29) in the first neuronal exon of the human AADC gene.

The foregoing method may also be carried out by detecting elevated levels of expression of the inactive splice variant of AADC mRNA in brain tissue of a subject, which inactive splice variant is described in Y.T. Chang et al., Neuroscience Letters

202, 157 (1996).

A still further aspect of the present invention is an isolated DNA encoding mammalian, preferably human, AADC, which isolated DNA contains a mutation as described above. Vectors containing such DNA and cells containing such DNA are also provided, which cells are useful for screening oligonucleotide probes to be used in detecting a mutation for diagnostic and/or prognostic screening techniques as described above, and which cells are also useful for screening compounds for use in treating autism in subjects afflicted therewith. These and other aspects of the invention are set forth in more detail in the description of the invention below.

Brief Description of the Drawings

This application includes Figures 1-10, which are described in the specification below.

Detailed Description of Preferred Embodiments

All nucleotide sequences shown herein are presented from the 5' to the 3' direction. Standard nucleotide abbreviations are used. Except as otherwise indicated, standard techniques may be used for production and manipulation of cloned genes, vectors, and transformed cells (and cell culture) according to the present invention. Such techniques are known to those skilled in the art (see e.g., SAMBROOK et al, EDS., MOLECULAR CLONING: A LABORATORY MANUAL 2d ed. (Cold Spring Harbor, NY 1989); F.M. AUSUBEL et al, EDS., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York); U.S. Patents Nos. 4,761,371 to Bell et al; 4,877,729 to Clark et al.; 4,912,038 to Schilling; and 4,879,224 to Wallher. The term "autism" as used herein has its conventional meaning in the art (see, e.g., U.S. Patents Nos. 5,686,311 and 5,405,943)(applicants specifically intend that all U.S. Patent references cited herein be incorporated herein by reference). In general, autism is a pervasive developmental disorder involving language delay and dysfunction in reciprocal social interaction, and includes a spectrum of disorders which may or may not involve mental deficit or retardation. Thus, high functioning individuals (i.e., individuals with normal intelligence) may be afflicted with autism. Autism is typically considered a life-long disorder, and thus may be present in infant, juvenile, adolescent and adult subjects. Mammalian subjects are preferred, with human subjects being more preferred. The subjects may be male or female, but are preferably male subjects, more preferably, human male subjects.

The aromatic L-amino acid decarboxylase (AADC) gene is known, and the first neuronal exon comprising the 5-end thereof is known. See A. Le Van Thai et al., "Identification of a neuron-specific promoter of human aromatic L-amino acid decarboxylase gene", Brain Res. Mol. Brain Res. 17, 227-238 (1993); GENBANK Accession Number L05075; see also Y.T Chang et al., "Alternative splicing in the coding region of human aromatic L-amino acid decarboxylase mRNA", Neuroscience Letters 202, 157 (1996). A mutation therein that is indicative of autism may be a deletion mutation, and is most typically a delta 26-29 deletion mutation of the sequence GAGA, taking the first nucleotide of neuronal exon 1 of the DNA of Le Van Thai above as nucleotide 1. Thus, the first 40 nucleotides of the AADC gene neuron exon 1 are:

CTTAACTGTC ACTGTGGAGA GGAGAGAGAG AGGACAGAGA (SEQ ID NO: 1)

and the same sequence containing a delta 26-29 deletion mutation has the sequence:

CTTAACTGTC ACTGTGGAGA GGAGAGAGGA CAGAGA (SEQ ID NO: 2)

Thus, a preferred DNA of the present invention is a DNA as described in A. Le Van Thai et al., above, and with a mutation in the first neuronal exon, preferably a deletion mutation and most preferably a delta 26-29 deletion mutation. Oligonucleotide probes that specifically bind to an AADC DNA or RNA that contains a mutation as described above, but do not bind to an AADC DNA or RNA that does not contain a mutation as described above, may be produced in accordance with known techniques. Such probes are typically from 5, 8 or 10 nucleotides in length to 20, 30 or 50 nucleotides in length or more. Such probes may be natural or synthetic.

As noted above, the present invention provides a method of screening (e.g., diagnosing or prognosing) for autism in a subject (typically, a human subject). The method comprises detecting the presence or absence of a mutation as described above in the subject. The presence of such a mutation indicates that the subject is afflicted with autism or is at risk of developing autism. Suitable subjects include those which have not previously been diagnosed as afflicted with autism, those which have previously been determined to be at risk of developing autism, and those who have been initially diagnosed as being afflicted with autism where confirming information is desired. Thus, subjects may be of any age, including adult, adolescent, juvenile, infant, and even prenatal or in utero subjects. Preferably, the subjects are male subjects.

Affliction with autism is more likely if a mutation described above is present. A subject with the mutation at increased risk of developing autism over subjects in which the mutation is absent. A subject who is "at increased risk of developing autism" is one who is predisposed to the disease, has genetic susceptibility for the disease or is more likely to develop the disease than subjects in which the mutation is absent.

Further, the methods of the present invention can be used to aid in determining the prognosis of a subject afflicted with or at risk for autism based on the observation of how many alleles containing the mutation are detected in the subject. The subject's prognosis is more negative if the presence of the mutation is detected than if it is absent. In particular embodiments, the subject's prognosis is most negative if the presence of more than one allele containing the mutation is detected (i.e., if the subject is homozygous as opposed to heterozygous). In other embodiments, homozygous subjects do not appear to be at a substantially higher risk than heterozygous subjects. It is contemplated that the methods described herein be used in conjunction with other clinical diagnostic information known or described in the art which are used in the evaluation of subjects with autism or suspected to be at risk for developing such disease. The step of detecting the mutation described above may be carried out either directly or indirectly by any suitable means. A variety of techniques are known to those skilled in the art. All generally involve the step of collecting a sample of biological material containing DNA, and then detecting whether or not the subject possesses DNA containing such a mutation from that sample. Any biological sample which contains the nucleic acid (eg., DNA, RNA) of that subject may be employed, including tissue samples and blood samples, with blood cells being a particularly convenient source.

Determining the presence or absence of nucleic acid containing the mutation described above may be carried out with an oligonucleotide probe labelled with a suitable detectable group, or by means of an amplification reaction such as a polymerase chain reaction or ligase chain reaction (the product of which amplification reaction may then be detected with a labelled oligonucleotide probe or a number of other techniques). Further, the detecting step may include the step of detecting whether the subject is heterozygous or homozygous for the mutation described above. Numerous different oligonucleotide probe assay formats are known which may be employed to carry out the present invention. See, e.g., U.S. Pat. No. 4,302,204 to Wahl et al.; U.S. Pat. No. 4,358,535 to Falkow et al.; U.S. Pat. No. 4,563,419 to Ranki et al.; and U.S. Pat. No. 4,994,373 to Stavrianopoulos et al. (applicants specifically intend that the disclosures of all U.S. Patent references cited herein be incorporated herein by reference).

Amplification of a selected, or target, nucleic acid sequence may be carried out by any suitable means. See generally D. Kwoh and T. Kwoh, Am. Biotechnol. Lab. 8, 14-25 (1990). Examples of suitable amplification techniques include, but are not limited to, polymerase chain reaction, ligase chain reaction, strand displacement amplification (see generally G. Walker et al., Proc. Na . Acad. Sci. U.S.A. 89, 392- 396 (1992); G. Walker et al., Nucleic Acids Res. 20, 1691-1696 (1992)), transcription- based amplification (see D. Kwoh et al., Proc. Natl. Acad Sci. U.S.A. 86, 1173-1177 (1989)), self-sustained sequence replication (or "3SR") (see J. Guatelli et al., Proc. Natl. Acad. Sci. U.S.A. 87, 1874-1878 (1990)), the Q beta replicase system (see P. Lizardi et al., BioTechnology 6, 1197-1202 (1988)), nucleic acid sequence-based amplification (or "NASBA") (see R. Lewis, Genetic Engineering News 12 (9), 1 (1992)), the repair chain reaction (or "RCR") (see R. Lewis, supra), and boomerang DNA amplification (or "BDA") (see R. Lewis, supra). Polymerase chain reaction is currently preferred.

Polymerase chain reaction (PCR) may be carried out in accordance with known techniques. See, e.g., U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188. In general, PCR involves, first, treating a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) with one oligonucleotide primer for each strand of the specific sequence to be detected under hybridizing conditions so that an extension product of each primer is synthesized which is complementary to each nucleic acid strand, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith so that the extension product synthesized from each primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer, and then treating the sample under denaturing conditions to separate the primer extension products from their templates if the sequence or sequences to be detected are present.

Ligase chain reaction (LCR) is also carried out in accordance with known techniques. See, e.g., R. Weiss, Science 254, 1292 (1991). In general, the reaction is carried out with two pairs of oligonucleotide probes: one pair binds to one strand of the sequence to be detected; the other pair binds to the other strand of the sequence to be detected. Each pair together completely encompasses the strand to which it corresponds. The reaction is carried out by first, denaturing (e.g., separating) the strands of the sequence to be detected, then reacting the strands with the two pairs of oligonucleotide probes in the presence of a heat stable ligase so that each pair of oligonucleotide probes is ligated together, then separating the reaction product, and then cyclically repeating the process until the sequence has been amplified to the desired degree. Detection may then be carried out in like manner as described above with respect to PCR.

Kits for determining if a subject is or was afflicted with or is or was at increased risk of developing autism will include at least one reagent specific for detecting for the presence or absence of the mutation described above, and instructions for observing that the subject is or was afflicted with or is or was at increased risk of developing autism if the presence of the mutation is detected. The kit may optionally include a nucleic acid or oligonucleotide probe for detection of the mutation in a manner such as described above. The test kit may be packaged in any suitable manner, typically with all elements in a single container or package along with a sheet of printed instructions for carrying out the test.

An isolated DNA as described above may be provided in a suitable vector, including but not limited to plasmids, viral vectors, yeast artificial chromosomes, bacterial artificial chromosomes, naked DNA vectors, and the like. Preferably, the vector is a plasmid. The present invention also provides cells that have been transformed with the vector, and preferably express the DNA therein. Cells according to the present invention may be any suitable cell for replicating and expressing the DNA, including but not limited to bacterial cells, yeast cells, plants cells, and animal cells (e.g., avian, insect and mammalian cells). Mammalian (e.g., human, mouse, rat, canine, simian), insect and bacterial cells are preferred. Such cells may be grown in cell culture using standard techniques. The cells of the invention may be used to screen new oligonucleotide probes for use in the diagnostic and prognostic techniques described above. In addition, such cells may be used to screen for compounds that affect the mutation described herein, which compounds are then candidate compounds for treating autism.

The present invention is explained in greater detail in the following non- limiting examples.

EXAMPLE 1 Screening of Multiplex Families

The DNA from children in eight "multiplex" families was studied, with each family being comprised of two autistic children and one unaffected child. DNA was provided by the HBDI Repository (Rutgers, The State University of New Jersey, Cell & DNA Repository, Department of Genetics, FAS-Division of Life Sciences, Nelson Biological Laboratory, 604 Allison Rd., Piscataway, NJ 08854-8082). The four base delta 26-29 deletion mutation described above was present in all 16 of the autistic children but absent in the unaffected sibs. EXAMPLE 2 Rapid Assay Procedure

The sequence of the two primers surrounding the deletion which have been used for sequencing and also for rapid screening of the homozygous deletion, heterozygous deletion, and wild type sequences are:

Sense primer: Pr3/s (located in the neuronal promoter):

GCC CTG ATG CTC CTC TCC A (SEQ ID NO: 3) Antisense primer: Intl/as (located in first intron):

GCA CAG CTA GAA TCA ACC G (SEQ ID NO: 4) For a rapid screening method the sense primer is labelled at the 5' end with a fluorescent tag (hexachlorinated 6-carboxyfluorescein) for tracking by fluorescence in the ABI 373A DNA sequencer in accordance with known techniques. PCR was carried out under standard conditions using the fluorescently tagged sense primer and the unmodified antisense primer resulting in the quantitative 5'end labeling of the sense strand of the product. The denatured PCR product was run on a sequencing gel, and the size of the product was determined relative to in-lane rhodamine labeled molecular weight standards. The homozygous wild type sample yields a single- stranded sense sequence of 349 bases.

EXAMPLE 3

Additional Primers for the First Neuronal Exon (Exon I) Deletion Mutation

The deletion mutation described in Example 1 above is screened by standard amplification procedures with a pair of primers. The sequence of the sense primer located in neuronal Exon I is:

TAACTGTCACTGTGGAGGAG (SEQ ID NO: 5) The sequence of the antisense primer located in Exon II is:

GATGTCCTCAAACGTGTCTGGC (SEQ ID NO: 6). These primers produce a 255 bp product for the wild type sequence and a 251 bp product if the deletion is present. EXAMPLE 4 Elevated Expression of Inactive Splice Variants in Autistic Subjects

A set of experiments was completed with autistic and control cerebellar RNA samples provided by Jonathan Pevsner (Department of Neurology, Johns Hopkins School of Medicine, Baltimore, Maryland, USA). Significant differences in the relative level of AADC-specific m-RNA was found between samples and also in the percentage of the m-RNA with exon III spliced-in or out. Based on these two parameters we have classified 11 of the samples as originating from autistic individuals and 11 as control samples. Two PCR primers flanking exon III were used. The sense primer is in exon II and has a fluorescent label at the 5 '-end quantitatively labeling the sense strand of the PCR product. The antisense primer is in exon IV and is unmodified. The expected size of the PCR product is 300 bp for the spliced-out variant and 414 bp with exon III spliced-in. These primers are known and described in Y. Chang et al., Neurosci. Lett. 202, 157-160 (1996). The sequence of the sense primer is:

CGCAAGTGAATTCCGAAGGAG (SEQ ID NO: 1). The sequence of the antisense primer is:

CCTTCTCAGCTTTCTCATTCA (SEQ ID NO:2).

Figure 1 and Table 1 show the output of the ABI 373 A DNA sequencer using Genescan software to size and quantitate the fluorescent PCR products which have been resolved under denaturing conditions. Peak sizes are about 302 and 416 nt for the two exon III splice variants close to the predicted values. Peak areas were used to quantitate the relative amounts of splice variants and product accumulation as a function of PCR cycle number.

Table 1

Prior to receiving the cerebellar specimens, we have been studying the peripheral expression of AADC using EBV-transformed B cell lines from autistic and control children as the source of the RNA. Figure 2 shows the percent spliced-out variant determined at cycles 30, 35, and 40. Bl is the control B cell line with about 42% exon III spliced-out variant. B2, B3, and B4 are cell lines from three unrelated autistic children with the spliced-out variant clustered around 65%. Thus expression is skewed toward the spliced-out variant in B cells from autistic samples. Homogenates from transfectants with expression vectors containing cDNA for the spliced-in transcript have been found to be enzymatically active in decarboxylating both L-DOPA and 5-hydroxytryptophan while transfectants containing the spliced-out from ere enzymatically inactive toward both substrates (K. O'Malley et al, J Neurochem. 65, 2409 (1995)). The function of the "inactive" spliced-out form is unknown. It may have different substrate requirements or it may form a heterodimer with the spliced-in isoform modifying the regulation or activity of the complex.

At the bottom of Figure 2 are the graphs for two non-autistic control human brain specimens Brl and Br2 (undefined as to the region of the brain) with midpoint values around 15% of the spliced-out variant. These samples provided the baseline values for the analysis of the cerebellar samples with the assumption that autistic cerebellar samples will show lower overall expression of AADC m-RNA and/or higher percentage of the inactive spliced-out variant as was found with B cells.

Figure 3 shows the accumulation of total AADC specific PCR products (both spliced-in and spliced-out variants) for the first set of cerebellar samples. In each case 1.0 ug RNA was used as the template for RT-PCR. Samples were taken at the indicated cycle intervals, and the areas of the fluorescent peaks were used as a measure of relative expression. Sample #3 was classified as autistic. Sample #3 also produced high percentage of the spliced-out variant as shown in figure 4. Sample #1 which could not be differentiated form the other samples on the basis of total expression also produced high level of the spliced-out variant and was therefore classified as autistic. Samples #4, #5, and #6 were classified as controls since they were high expressors of total AADC m-RNA, and all produced 18-20% of the spliced-out variant at cycle 30. The percentage of the spliced-out variant tended to creep up with increasing cycle number indicating that it was amplified somewhat more efficiently than the larger spliced-in variant.

In the second set (7-13) sample #8 is a low expressor of total AADC (Figure 5) and was classified as autistic, and the other samples in the set could not be differentiated on the basis of total expression. Sample #7 was classified as control since it had the lowest level of the spliced-out variant at about 14% (Figure 6).

Sample #13 was classified as a probable control since it had the highest rate of total AADC accumulation in the exponential phase (cycles 28-32) and also had intermediate level (23-24%) of the spliced-out variant. Samples #9, #11, and #12 were clustered around 28% with respect to the spliced-out variant and on this basis alone were classified as probable autistics. In this set of experiments the increase in percentage of the spliced-out variant became more pronounced after cycle 32, so probably the splice variant measurements are more reliable at the earlier cycles when they are relatively cycle-independent. In the third set (14-19) sample #18 is a low expressor (Figure 7) and also produces high level (50%) of the spliced-out variant (Figure 8) and was therefore classified as an autistic sample. Sample #17 produces comparably high level of the spliced-out variant and was classified as an autistic sample, although it is a high expressor of total AADC message. The values for samples #15, #16, and #19 cluster around 20% of the spliced-out variant and these samples were classified as controls. Sample #14 is a high expressor of total AADC message and produces a borderline level of the spliced-out variant at about 24% and was classified as a probable control.

In the fourth set (20-24) sample #24 has the lowest total AADC message (Figure 9) and the highest level of the spliced-out variant at about 31%> (Figure 10) and was classified as an autistic sample. Samples #20 and #23 are intermediate level expressors of total AADC message and also produce intermediate percentage of the spliced-out variant (25%) and were classified as probable autistic samples. Samples #21 and #22 are high expressors of total AADC message and also produce low percentage of the spliced-out variant (19-21%) and were classified as control samples.

This interpretation assumes that the apparent low expression was not due to RNA degradation. The observation that most of the low expressors had higher percentage of the spliced-out variant would argue against this possibility.

Example 5

Multiplex Study to Evaluate Gender-Associated Susceptibility

Based on analysis of 128 multiplex families (as described in Example 1) with autistic and unaffected children, it was found that the deletion mutation described in Example 1 was a susceptibility mutation in boys, but not girls. Risk did not appear to be greater for boys homozygous for the deletion, as opposed to heterozygotes.

The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

We claim:

1. A method of screening for autism in a subject, comprising: detecting the presence or absence of a downregulation of expression of active human aromatic L-amino acid decarboxylase (AADC) in nerve tissue of said subject; the presence of such downregulation indicating said subject is afflicted with, or is at increased risk of developing, autism.

2. A method according to claim 1, wherein said detecting step is carried out by: collecting a biological sample containing nucleic acid from said subject; and determining the presence or absence of said mutation from said nucleic acid.

3. A method according to claim 2, wherein said determining step is carried out by polymerase chain reaction.

4. A method according to claim 1, wherein the subject is a male subject.

5. A method of screening for autism in a subject, comprising: detecting the presence or absence of a mutation in the first neuronal exon of the human aromatic L-amino acid decarboxylase (AADC) gene; the presence of such mutation indicating said subject is afflicted with, or is at increased risk of developing, autism.

6. A method according to claim 5, wherein said detecting step is carried out by: collecting a biological sample containing nucleic acid from said subject; and determining the presence or absence of said mutation from said nucleic acid.

7. A method according to claim 6, wherein said determining step is carried out by polymerase chain reaction.

8. A method according to claim 5, wherein said mutation is a deletion mutation.

9. A method according to claim 8, wherein said mutation is a delta 26-29 deletion mutation.

10. A method according to claim 5, wherein said subject is a male subject.

11. An isolated DNA encoding human aromatic L-amino acid decarboxylase (AADC), said DNA containing a mutation in the first neuronal exon thereof.

12. An isolated DNA according to claim 11 , wherein said mutation is a deletion mutation.

13. An isolated DNA according to claim 12, wherein said deletion mutation is a delta 26-29 deletion mutation.

14. A vector containing a DNA according to claim 11.

15. A cell containing and expressing a DNA according to claim 11.