Homozygosity for a missense mutation in the 67 kDa isoform of glutamate decarboxylase in a family with autosomal recessive spastic cerebral palsy: parallels with Stiff-Person Syndrome and other movement disorders

Background Cerebral palsy (CP) is an heterogeneous group of neurological disorders of movement and/or posture, with an estimated incidence of 1 in 1000 live births. Non-progressive forms of symmetrical, spastic CP have been identified, which show a Mendelian autosomal recessive pattern of inheritance. We recently described the mapping of a recessive spastic CP locus to a 5 cM chromosomal region located at 2q24-31.1, in rare consanguineous families. Methods Here we present data that refine this locus to a 0.5 cM region, flanked by the microsatellite markers D2S2345 and D2S326. The minimal region contains the candidate gene GAD1, which encodes a glutamate decarboxylase isoform (GAD67), involved in conversion of the amino acid and excitatory neurotransmitter glutamate to the inhibitory neurotransmitter γ-aminobutyric acid (GABA). Results A novel amino acid mis-sense mutation in GAD67 was detected, which segregated with CP in affected individuals. Conclusions This result is interesting because auto-antibodies to GAD67 and the more widely studied GAD65 homologue encoded by the GAD2 gene, are described in patients with Stiff-Person Syndrome (SPS), epilepsy, cerebellar ataxia and Batten disease. Further investigation seems merited of the possibility that variation in the GAD1 sequence, potentially affecting glutamate/GABA ratios, may underlie this form of spastic CP, given the presence of anti-GAD antibodies in SPS and the recognised excitotoxicity of glutamate in various contexts.Table 4 GAD1 single nucleotide substitutions detected on mutation analysis and occurring in sequences submitted to NCBI SNP database and in the literature. This is not a definitive list, but includes those described at the time of the mutational analysis. *Nucleotide positions were not provided by Maestrini et al. [47]. Source SNP position in mRNA, from the translational start site (bp) Gene position of SNP(bp) Amino acid change (A)Lappalainen et al. (2002) A(-478)Del Exon 0 (73) No substitution (B)Lappalainen et al. (2002) G(-147)A Exon 0 (404) No substitution (C)Lappalainen et al. (2002) A(-39)C Exon 1 (25) No substitution (D)Spastic CP patients family B G(36)C Exon 1 (97) Ser(12)Cys (E)NCBI collated resource G(48)C Exon 1 (104) Pro(17)Ala (F)Control samples & family A NCBI collated resource T(110)C Exon 2 (29) No substitution (G)Kure et al. (1998) T(315)C Exon 4 (14) No substitution (H)Bu and Tobin (1994) Kure et al. (1998) A(407)G Exon 4 (105) No substitution (I)Maestrini et al. (2002)* G/C Intron 4 No substitution (J)NCBI collated resource C(696)T Exon 6 (56) No substitution (K)Lappalainen et al. (2002) T/Del Intron 7 (35) No substitution (L)In control samples Lappalainen et al. (2002) T/C Intron 8 (185) No substitution (M)Maestrini et al. (2002)* C/T Intron 9 No substitution


Background
Cerebral palsy (CP) is a term used to define a group of disorders [1] characterized by a non-progressive abnormality of posture and movement, resulting from defects in the developing nervous system [2]. Approximately 1 in 250 to 1000 live births presents with CP, making it one the commonest congenital disabilities [3]. Many different aetiological factors have been implicated. Among preterm infants, the incidence of CP generally increases with decreasing gestational age and the origin in most cases may be traced to post/peri-partum periventricular leukomalacia and intraventricular/periventricular haemorrhage [4]. Conversely in term infants perinatal causes can only confidently be attributed where there is documented perinatal hypoxia/acidosis and clinical encephalopathy in the early neonatal period [5]. Prenatal risk factors in the aetiology of CP include low birth-weight, intrauterine infection and exposure to teratogens during pregnancy [6,7]. The cause in a large proportion of cases remains obscure.
A non-progressive, autosomal recessive, symmetrical spastic CP locus has been mapped to a 5 cM region between D2S124 and D2S333, at 2q24-31.1 (LOD score of 5.75) in consanguineous families originating from the Mirpur region of Pakistan (OMIM:603513) [10]. Affected individuals had no identifiable perinatal cause of CP, or underlying diagnosis and presented with developmental delay, mental retardation and sometimes epilepsy as part of the phenotype. We initially performed detailed physical mapping of the 5 cM region, so as to accurately define the marker order and to refine the linkage interval. The positions of a large number of genes and ESTs were defined accordingly, allowing the rapid identification of candidate disease genes. The minimum region of homozygosity was reduced to 0.5 cM by typing large numbers of microsatellite markers in the families. Subsequently, portions of this region have been sequenced in the human genome project. Within the region we have concentrated on the positional candidate GAD1, which codes for the 67 kDa isoform of L-glutamate decarboxylase (GAD EC:4.1.1.15).
GAD requires the cofactor pyridoxal 5'-phosphate (PLP) and catalyses the production of Gamma-aminobutyric acid (GABA) from glutamate [16]. Two separate, independently-regulated genes, GAD1 and GAD2 (at chromosome 10p11, encoding a 65 kDa GAD isoform), have presumably arisen by duplication and been conserved during evolution, as indicated by their sequence homology [17] and the retention of common intron-exon boundary splice sites [18]. Their N-termini demonstratẽ 23% homology, but their C-termini, which contain the catalytic site, have ~73% amino acid sequence identity between the isoforms [19]. GABA and glutamate are the most abundant amino acid neurotransmitters in the brain. GABA, an inhibitory neurotransmitter, and excitatory glutamate, both play important roles in synaptic plasticity and neuroendocrine function [20]. Both isoforms of GAD are also involved in intermediary metabolism, participating in the GABA shunt, which bypasses two steps of the TCA cycle [21]. GAD activity of both isoforms, is ubiquitous, but highest in the brain and pancreatic islets of Langerhans. We therefore performed detailed mutational screening of GAD1 in familial spastic CP probands and unaffected family members.

Features and pedigrees of ascertained families
Family A (4718/4719) The oldest affected male diagnosed with non-progressive, spastic CP, demonstrated global developmental delay, with no associated neurological abnormalities and moderate mental retardation. His affected younger sister was also diagnosed with spastic CP, global developmental delay and moderate mental retardation.

Family B (4578/4579/4581/4679)
The oldest non-progressive, spastic CP female has severe mental retardation, and is confined to a wheelchair. The next oldest spastic CP male has severe mental retardation, mild hypertonia and ataxia of the upper limbs. The next oldest spastic CP male is a dizygotic twin born by Caesarean section. This patient on presentation demonstrated developmental delay, mild hypertonia and ataxia of the upper limbs. The youngest affected female is not able to walk or stand unaided and has severe developmental delay. Details of the clinical picture in these pedigrees have been described previously [10,14].
The YAC chromosomal DNA was purified and separated for sizing by CHEF electrophoresis. The switching angle was 120°, using a CHEF™ electrophoresis tank (Bio Rad), run for 16 hours at 6 V/cm, 10°C with an initial pulse time of 30 sec to a final pulse time of 90 sec. DNA was stained with (20 mg/ml) ethidium bromide solution (BDH) for 2 hours and visualized under ultra-violet illumination. Southern blotting and membrane hybridisation of CHEF gels were performed on Hybond-N™ membranes (Amersham). DNA was immobilised by heating the membrane to 80°C in vacuum for 1 hour in a gel dryer (Bio-Rad). Radio-labelled YAC vector-specific probes were generated using the Megaprime random primer kit (Amersham) as described in the manufacturer's instructions, using PCR products as a template. Sizes were estimated based on comparisons with the known sizes of the native yeast chromosomes.

Single-strand conformational polymorphism analysis
GAD1 exon sequences were amplified by PCR using the primers described in Table 1. SSCP was performed on GeneGel Excel (Amersham), using a 12.5/24 gel (14°C, 600 V, 25 mA, 15 W for 80 min) and the DNA was visualised by silver staining as per the manufacturer's instructions.

Sequencing of GAD1 exon sequences
Primers used to amplify the exons of GAD1 were designed from the sequence of BAC RP11-570c16 and obtained from Lifetech (Table 1). PCR products were purified from agarose gels using QIAquick gel extraction kits (Quiagen), sequenced using ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kits (Applied Biosystems) and then analysed using an ABI Prism 377 automated sequencer.

Physical map of the spastic CP locus
An integrated YAC (ICI and CEPH mega YAC libraries) and RP-9 PAC (HGMP Resource Centre) contig for the interval D2S2157 to D2S385 was constructed ( Figure 1). This map provided physical continuity connecting 25 loci from centromere to telomere spanning the entire 2q24.3-31.1 cytogenetic band region. These data were combined with BAC contigs, constructed at Washington University http://genome.wustl.edu/gsc, in an attempt to form an ordered BAC/YAC contig across the minimum region of An integrated physical YAC contig spanning the human chromosome 2 spastic CP locus The size of the region incorporating the three contigs was estimated to be at least 2870 kb. The sizes of the ICI YACs were estimated to encompass a minimum locus of 2940 kb. Microsatellite markers, ESTs and known genes, mapped to this region by NCBI, were then located on the physical contig by PCR and BLAST sequence homology searches of the Washington University BAC contigs. The expression profiles of unidentified EST clusters were determined and used to form EST "bins". These contained groups of ESTs, which mapped to adjacent locations and showed common expression profiles, suggesting that they might represent different exons of the same gene. This placement of known genes and ESTs onto the physical map provided an annotation of the gene content of the region, at the time was constructed before any such facility was available from the HGP, as the focus for candidate disease gene selection.

Genetic mapping data
From the PAC/YAC contig, 20 polymorphic microsatellite markers were identified that span the chromosome 2q24-31.1 CP critical region. These were then used, with informed consent and local research ethics committee approval, in the detailed mapping of previously linked families [10] ( Figure 2). These data did not support the presence of a founder mutation for autosomal recessive spastic CP, in that families did not share a common haplotype across the minimal homozygous region between D2S2345 and D2S326.

Sequence analysis of GAD1
GAD1 was sequenced in affected and unaffected individuals in both families ascertained. In order to differentiate possible disease-causing mutations from polymorphisms, 100 control individuals were screened by SSCP to detect any GAD1 sequence variations in the normal population. SSCP variants were then sequenced to identify the underlying nucleotide substitutions. An homozygous G(36)C ( Figure 3A,3B) nucleotide change was observed in 4 affected patients, which generated a Ser(12)Cys amino acid substitution. No obligate carriers were identified for this mutation. This variant has not been previously described and was not present in 200 normal chromosomes. A number of other sequence changes were detected, but none of these resulted in amino acid changes. These variants and all those recorded previously in the literature are presented in Table 4 and Figure 4. For those rare variants in the databases, which result in amino acid changes, no homozygous or compound heterozygous individuals have yet been described.

Discussion
CP is a term used as a collective definition for a group of neurological disorders [3]. The pathophysiology in most cases is poorly understood, but includes genetic syndromes, congenital malformation, infective intra-uterine encephalitis, cerebral haemorrhage or infarction, ischemic damage, periventricular leukomalacia (PVL), and non-infarctive telencephalic leukomalacia [11]. The contribution of Mendelian inherited cases of CP accounts for approximately 2% of the total number [2,22]. A non-progressive form of autosomal recessive spastic CP has been identified [13]. McHale et al. [10] succeeded in identifying a 5 cM region on chromosome 2q24-31.1, which segregated with disease in consanguineous families. Linkage analysis identified a locus between markers D2S124 and D2S333, which produced a LOD score of 5.75, sufficient to warrant the further investigation described herein.
To refine and confirm the genetic marker order across a region, which was incompletely sequenced at the time, we used YAC and PAC clones to construct a physical framework and performed PCR to map ESTs and microsatellite markers to clones within the partial contig ( Figure 1). When this contig was integrated with the BAC sequence contigs, rearrangement of the BAC order was necessary.
With each subsequent DNA sequence update, the degree of inconsistency was reduced and this led to revision of microsatellite order compared with that used previously [10]. Using the YAC sizes, the sizes of gaps in the BAC contig could be estimated. Having generated a detailed map Annotation of two pedigrees of spastic autosomal recessive CP families and corresponding linkage mapping data Figure 2 Annotation of two pedigrees of spastic autosomal recessive CP families and corresponding linkage mapping data. The markers shown are those that demonstrate the minimal homozygous region between the affected individuals of both families.
spanning the region, we selected microsatellite markers at evenly spaced intervals across the locus. These markers were used to refine the minimum region homozygous by descent in linked families, to between the markers D2S2345 and D2S326. The physical size of the region between these markers is approximately 0.5 cM. There was no suggestion of a founder haplotype common to the two families ( Figure 2).
The Goldenpath Human Genome Working Draft Assembly 2001, is an annotation of the Washington BAC contig, combining sequence data of BACs, ESTs, known genes and hypothetical genes. We mapped ESTs and known, uncharacterised or hypothetical genes on the basis of sequence homology (NCBI http:// www.ncbi.nlm.nih.gov, Whitehead http:// www.genome.wi.mit.edu and Goldenpath databases), onto our YAC/PAC contig. ESTs were then collated according to their expression profiles to produce a "binned" EST map, on which candidate gene selection could be based. This reduced the number of hypothetical genes in the region and allowed the combination of genetic, physical mapping and expression data, into a single comprehensive map.
Electropherograms of the sequence of the exon 1 SNP of GAD1 identified in the process of mutational analysis

(A) (B) (C) (D)
One interesting candidate within the minimal region was GAD1, which encodes GAD 67 . Expression of its transcript is ubiquitous, including the CNS. The main function of GAD 67 is to catalyze the conversion of the excitatory amino acid and neurotransmitter glutamate to GABA, the main inhibitory neurotransmitter in the CNS [23]. In the   developing CNS, GABA has an important role in neuronal differentiation and the control of plasticity [21]. GABA has also been implicated in the pathogenesis of various seizure and movement disorders [20].
Vertebrates have two separate genes coding for GAD, which produce distinct forms of the enzyme. GAD1 and GAD2 have diverged relatively recently in evolution, as indicated by their degree of sequence homology and the retention of common intron-exon boundary splice sites [17] ( Figure 5A). The variants of GAD differ in molecular weight, cellular and sub-cellular localisation, and their interaction with the cofactor PLP [18,20,24].
GAD2, located at 10p13-p11.2, is transcribed to produce a 5.6 kb mRNA in islets and brain, encoding a 65 kDa protein (585 AA residues). The 67 kDa (594 AA residues) form [17] is localised to 2q25-26 and encoded by a 3.7 kb transcript (GAD1) [23]. There is also evidence for a 25 kDa inactive protein (GAD 25 ) produced from an alternatively spliced GAD1 transcript of 2 kb that contains an inframe stop codon. This GAD1 splice variant has only been found in human islets, testis and adrenal cortex, although the homologue is present in fetal mouse brain [25].
In the CNS, GAD 65 appears to be preferentially distributed in axon terminals and the associated synaptic vesicles, whereas GAD 67 is also located in the cell bodies and more uniformly distributed throughout the neuron [24]. This suggests that each GAD isoform is involved in the synthesis of GABA in different sub-cellular compartments [21]. This is supported by the discovery that GAD 65 is the main source of apoGAD (an inactive reservoir), which responds to short-term changes in neuronal activity and is more responsive to levels of PLP [18]. On the other hand, GAD 67 predominantly exists bound to the PLP cofactor (holoGAD), providing a constitutive level of GABA production [20]. Bond et al. [27] showed that GAD 25 is expressed in a temporally controlled manner, in the devel-

N-terminal catalytic domain
oping striatum and cortex in rodents, suggesting this may provide a mechanism of regulating GABA production in differentiating neurons.
Asada et al. [28] undertook the selective elimination of each GAD isoform in order to determine their respective roles. Gad2-/-mice are slightly more susceptible to seizures, consistent with an excitatory increase in the relative ratio of glutamate/GABA. However, they showed no obvious overall change in neuronal GABA content. Therefore GAD 67 alone appears to produce sufficient GABA for effective neurotransmission [21]. Gad1-/-mice demonstrated a decrease of ~20% in total glutamate decarboxylase activity at birth. This was assayed by the conversion of 14 Clabelled glutamate to 14 CO 2 in the presence of PLP. There was also a marked (7%) reduction in total GABA content in cerebral cortex homogenate measured by liquid chromatography [28]. Unfortunately, these mice died neonatally of severe cleft palate, masking any potential neurological dysfunction and also illustrating a role for Gad 67 in non-neural tissues [21]. It is of interest that mice with mutations in the β-3 GABA receptor (GABRB3) at the Angelman syndrome (OMIM:105830) locus, also display cleft palate, implying a key role for GABA signalling in normal palate development [29,30].
Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disorder (OMIM:266100), characterized by generalized seizures during the first hours of life. The associated pathology may result from an alteration in the binding of the co-factor PLP to GAD. Interestingly epilepsy is commonly associated with CP and grand mal epilepsy developed at age six months in the two linked pedigrees described here [10]. GAD1 mutation was previously suspected of being the cause of PDE. Linkage of pyridoxine-dependent epilepsy has however been reported to 5q31.2-31.3, with GAD1 and GAD2 excluded [31]. Decreased levels of brain and CSF GABA, increased levels of CSF and cortical glutamate, and decreased levels of PLP in the frontal cortex, have been described in this condition.
GAD 65 and GAD 67 have been identified as auto-antigens in "Stiff Person Syndrome" (SPS, OMIM:184850), and in cerebellar ataxia [32][33][34]. GABA-mediated synaptic transmission is thought to be functionally impaired by the production of autoantibodies to GAD 65 and GAD 67 [35][36][37]. This results in a reduction in brain levels of GABA, prominent in the motor cortex, which can be demonstrated by Magnetic Resonance Imaging (MRI) in SPS patients. SPS is a disabling disorder characterised by muscle rigidity and episodic spasms of the musculature, thought to be due to autoimmune-mediated dysfunction of supraspinal GABAergic inhibitory neurons [38]. Hyper-excitability of the motor cortex in SPS has been demonstrated by transcranial magnetic stimulation [39].
Anti-GAD 65 auto-antibodies in the CSF of ataxic and SPS patients selectively suppress GABA-mediated transmission in cerebellar Purkinje cells, without affecting glutamate-mediated transmission [37,40]. Low CSF levels of GABA have been reported in patients with Kok disease (OMIM:149400 also known as hyperexplexia/exaggerated startle reaction/startle disease) [41]. The exact mechanism by which autoantibodies target these intracellular GAD antigens is not clear. However, it is interesting that SPS may also arise in individuals with autoantibodies to gephyrin, a cytosolic protein concentrated at the postsynaptic membrane of inhibitory synapses where it is associated with GABA A receptors [42]. This provides a further example of chronic rigidity and spasm possibly secondary to disruption of the inhibitory synapses.
Mutations in the CLN3 gene are thought to be responsible for the neurodegenerative disorder Batten disease (OMIM:204200). In cln3-knockout mice autoantibodies to GAD 65 have been reported to be associated with brain tissue and result in inhibition of GAD activity [43]. These mice also demonstrate elevated brain glutamate levels as compared with controls, which may have a causative role in the astrocytic hypertrophy evident in cln3-knockout mice and along with anti-GAD 65 autoantibodies in Batten disease patients may contribute to the associated preferential loss of GABAergic neurons.
Drugs, which potentiate the action of GABA, such as benzodiazepines and baclofen, ameliorate muscle rigidity and spasticity. These GABA agonists are thought to counter disinhibition of the velocity-dependent increase in skeletal muscle during stretch reflexes, observed in spasticity, which is the result of inadequate presynaptic inhibition of the muscle spindles [44,35,37]. γ-Vinyl-γaminobutyric acid (GVG) is used to treat neurological disorders including epilepsy, tardive dyskinesia and spasticity. It has been reported that it is the GABA-elevating effect of this compound that is responsible for its anti-convulsive properties [20].
We have identified a GAD1 sequence change G(36)C, which segregates with autosomal recessive spastic CP in 4 affected siblings. This nucleotide substitution causes a missense mutation, changing serine (12) to a cysteine in the N-terminal domain. This serine residue is conserved between all mammals (human/mouse/rabbit/pig) for which data are available. The association of GAD 67 with membranes requires formation of heteromeric links with GAD 65 , which are mediated via their N-terminal domains. The N-terminus of GAD 65 is palmitoylated and binds to the cellular membrane. The first 27 amino acids appear to be essential in this function [32]. The palmitoylation of cysteines 30 and 45 of GAD 65 , and the inability of residues 1-29 of GAD 67 , to substitute for this region, highlights the potential impact on cellular localisation of a nucleotide substitution in this domain [20]. GAD 65 also undergoes phosphorylation of the first four serine residues in the Nterminal domain. These post-translational modifications highlight the importance of the flexibility and accessibility of this domain. N-terminal epitopes of GAD 65 , in the region corresponding to the residue, which undergoes mutation in GAD 67 , are particularly prominent autoantigens [45]. S(12)C amino acid substitution may thus produce subtle effects on cellular localisation, protein-protein interactions and/or protein processing, with a subsequent effect on GABA production. This is not inconsistent with the mouse Gad1 knockout where complete loss of Gad1 enzymatic function (~20% reduction of total Gad activity in the cerebral cortex) resulted in a cleft palate phenotype and neonatal death [28,30]. There is redundancy of GABA production as a result of the presence of two GAD proteins, and the precise function of each isoform may differ between man and mouse. It is interesting to note that the GAD 25 splice variant of GAD 67 , also contains the S(12)C amino acid substitution in affected CP patients. This truncated variant is identical to the first 213 amino acids of GAD 67 , with the addition of an extra 11 C-terminal residues. It lacks the binding site for the cofactor PLP and is believed to lack any GAD activity [25]. The function of GAD 25 is not known, but it may compete with GAD 67 for incorporation into protein complexes. Therefore the presence of an N-terminal mutation would affect both GAD 25 and GAD 67 , and may disrupt a complex regulatory mechanism for GAD 67 .

Conclusions
This study illustrates the difficulty of gene cloning in rare autosomal recessive diseases mapped in small, consanguineous pedigrees. Identification of an ancestral haplotype allows refinement of the locus, but this has not been possible in this example. Within the minimal linkage region, any sequence change will segregate with the disease phenotype. Detection of a nonsense mutation leading to a protein truncation would provide compelling support for a mutation as causative. However, in the present example we have not seen such a mutation in the candidate gene so far examined. We are now expressing the variant forms of GAD 67 (S12C) and GAD 25 (S12C), as recombinant proteins, to assess catalytic activity and binding properties with respect to their normal counterparts. However, it may well prove difficult to detect subtle effects based on sub-cellular localisation changes in the mutant proteins, or changes in post-translational modification patterns.
Stability of the mRNA transcripts from these variants will be assessed by transfection studies in neuronal cells. Eventually, it would be of interest to attempt knock-in experiments with GAD1 (G36C), into the Gad1-/-mouse to see if this can rescue the cleft palate phenotype and reveal a CP-like picture. These experiments will be reported elsewhere. However, the possibility that reduced GAD 67 activity may cause CP in the patients studied herein, in a manner reminiscent of that seen in SPS, leads us to report our findings at this stage. The success reported in treating SPS with intravenous immunoglobulin [40,46], suggests further evaluation of GABA agonists in the management of this difficult clinical problem.