Abstract
Objective: To report a Dutch family with autosomal dominant cerebellar ataxia (ADCA) based on a novel mutation in the PRKCG gene.
Methods: The authors studied 13 affected members of the six-generation family. After excluding the known spinocerebellar ataxia (SCA) genes, a combination of the shared haplotype approach, linkage analysis, and genealogic investigations was used. Exons 4 and 5 of the candidate gene, PRKCG, were sequenced.
Results: Affected subjects displayed a relatively uncomplicated, slowly progressive cerebellar syndrome, with a mean age at onset of 40.8 years. A focal dystonia in two subjects with an onset of disease in their early 20s suggests extrapyramidal features in early onset disease. Significant linkage to a locus on chromosome 19q was found, overlapping the SCA-14 region. Based on the recent description of three missense mutations in the PRKCG gene, located within the boundaries of the SCA-14 locus, we sequenced exons 4 and 5 of this gene and detected a novel missense mutation in exon 4, which involves a G→A transition in nucleotide 353 and results in a glycine-to-aspartic acid substitution at residue 118.
Conclusion: A SCA-14-linked Dutch ADCA family with a novel missense mutation in the PRKCG gene was identified.
A major part of the genetic background of autosomal dominant spinocerebellar ataxias (ADCA) has been elucidated in the past decade.1 Twenty-one genetic loci have been detected by linkage studies (spinocerebellar ataxia [SCA]-1–8, SCA-10–19, and SCA-21–23) and 10 of the corresponding genes have been identified.2-22⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓ In approximately 60 to 70% of Dutch ADCA families, a mutation is found in the SCA-1, SCA-2, SCA-3, SCA-6, or SCA-7 gene.23 The other genes and loci seem to be rare or are confined to specific populations.24,25⇓ Although the contribution of these genes to the Dutch ADCA population is unknown, novel mutations are likely present.
We recently identified two novel loci in two Dutch ADCA families, SCA-1919 and SCA-23.22 In addition, a missense mutation in the FGF14 gene on chromosome 13q34 was found in another Dutch family.26 We reasoned that several of the Dutch mutation-negative ADCA families must have common origins and that, by combined linkage, haplotype, and genealogic analyses, we should be able to link some of the small ADCA families in larger clusters.
Here, we report a large six-generation Dutch ADCA family in which linkage to the SCA-14 locus on chromosome 19q13.4-qter was found. Recently, missense mutations in exon 4 of the PRKCG gene that encodes protein kinase Cγ (PKCγ) were reported in two ADCA families, one of which involved a SCA-14-linked family reported previously, and in one sporadic case.27,28⇓ We therefore sequenced exons 4 and 5 of the PRKCG gene and identified a novel missense mutation (353G→A) in exon 4. This new family adds to the clinical and genetic characterization of PRKCG mutations in SCA-14 patients.
Subjects and methods.
After diagnostic screening, 24 Dutch ADCA families with affected individuals in multiple generations and without a mutation in the SCA-1, SCA-2, SCA-3, SCA-6, or SCA-7 gene were identified. By linkage, haplotype, and genealogic analysis (see below), three independently referred families (RF13, RF11, and RF17) were found to be linked and the pedigree of this combined large six-generation family is depicted in figure 1 (only the affected individuals are shown). Of this extended family, 22 family members from three generations were included in the study and all gave informed consent for additional research studies. In the past 18 months, all the affected family members received a full neurologic examination and a comprehensive case history was taken. When appropriate, neurologic symptoms were graded as absent, mild, moderate, or severe. Blood samples (20 mL EDTA) were taken later.
Figure 1. The pedigree of the combined family RF13, RF11, and RF17. Haplotype analysis for seven chromosome 19 markers is shown. The disease haplotype is boxed. Open figures = unaffected; closed figures = affected; dotted figures = obligate carrier; square = male; circle = female; diamond = unknown sex; / = deceased. In order to maintain confidentiality, unaffected family members are not shown.
Exclusion of the known SCA genes.
Diagnostic screening was performed to exclude a mutation in the SCA-1, SCA-2, SCA-3, SCA-6, and SCA-7 genes in the 24 families. High molecular weight DNA was obtained from leukocytes by a routine salting out procedure. No repeat expansions were found in the affected individuals. Further testing with the more recently identified SCA genes, including SCA-8, SCA-10, SCA-12, and SCA-17, was carried out and proved to be negative (data not shown). These 24 families were subsequently used in a combined linkage, haplotype, and genealogic approach.
Shared haplotype analysis.
Because most of the 24 ADCA families were too small to perform traditional linkage analysis, we used an alternative strategy to localize the disease-causing genes in these families: the shared haplotype analysis (SHA). This method is based on the assumption of a limited number of founder mutations in the Dutch ADCA families, which had already been observed in SCA-3 and SCA-6 families (unpublished data). For all 24 ADCA families, we focused on the remaining reported SCA loci, namely SCA-4, SCA-5, SCA-11, SCA-13, SCA-14, SCA-16, SCA-18, SCA-19, SCA-21, and SCA-23. Polymorphic markers within the candidate regions, with an average spacing of approximately 2.0 cM, were selected from the Marshfield database (see http://research.marshfieldclinic.org/genetics/). The different marker alleles were defined using the CEPH 133101 control sample. Haplotypes were constructed using the program GENEHUNTER (v1.2) and were corrected manually. We searched for identical haplotypes within the reported SCA loci in these small ADCA families. For each SCA locus, we created 100 control haplotypes from the genotypes of unrelated persons from a series of families collected for other purposes (data not shown). The alleles that characterize the SCA-14 haplotype are represented by the following: D19S206: allele 7, 132 base pair (bp); D19S571: allele 1, 198 bp; D19S589: allele 2, 169 bp; D19S924: allele 3, 201 bp; D19S927: allele 2, 142 bp; D19S926: allele 4, 105 bp; and D19S605: allele 2, 117 bp.
Meanwhile, an exhaustive genealogic analysis was initiated to explore possible links between the apparently unrelated ADCA families that were included in the SHA, because other studies have shown that families in the Netherlands are often interrelated several generations back.
Linkage analysis.
Two-point and multipoint linkage analyses were performed with the MLINK and LINKMAP programs of the LINKAGE package (v5.2). In our model, we applied a disease frequency of 1.0 in 100,000 and equal allele frequencies. Both analyses were based on an affected-only strategy to avoid the problem with possible presymptomatic carriers.
Mutation analysis.
Based on the recent report of three missense mutations in exon 4 of the PRKCG gene, we decided to screen exons 4 and 5 of this gene for mutations in affected individuals from all 24 families.27 An additional 85 anonymous control individuals with no history of ataxia were also sequenced. Both exons were amplified using the primers described in the original report.27 The template for the sequence reaction was amplified with Ampli Taq DNA polymerase, using a PCR mixture containing 10X Pol buffer (67 mM Tris-HCl, 6.7 mM MgCl2, 10 mM β-mercaptano-ethanol, 6.7 mM EDTA [pH = 8.0], and 16.6 mM [NH4]2SO4), 1.5 mM dNTP, 0.15 mg/mL bovine serum albumin, and 10% dimethylsulfoxide. The PCR protocol started with 4 minutes of denaturation at 94 °C, followed by 35 cycles of 1 minute denaturation at 94 °C, 1 minute of annealing at 56 °C, and 2 minutes of extension at 72 °C, and ending with a final extension step of 7 minutes at 72 °C. PCR fragments were purified with Manu 30 Multiscreen PCR filter plate (Millipore) and dissolved in 40 μL of water. DNA sequencing was performed using the Big Dye Terminator cycle sequencing ready reaction kit (Applied Biosystems, Warrington, UK). Sequence products were purified with the ManuN45 multiscreen sequence reaction filter plates (Millipore) and then were run on a 3100 ABI automated sequencer (Applied Biosystems, Foster City, CA). Data were analyzed with Sequencing Analysis (v3.7) software.
Results.
Shared haplotype analysis.
After all the markers were typed and haplotypes constructed, we identified a shared haplotype in 3 of the 24 ADCA families. In these families (RF13, RF17, and RF11), this shared haplotype covered almost the complete SCA-14 locus. The genealogic investigations revealed that the RF17 and RF11 families could indeed be linked to the large RF13 family (see figure 1). The closest common ancestor in these three families was identified four generations back. A partially shared haplotype, which corresponded only to the lower part of the SCA-14 region between marker D19S924 and marker D19S605, was found in two other families (data not shown). However, the PRKCG gene turned out to be located outside this part of the haplotype. Therefore, this result was marked as false positive.
The SCA-14 haplotype was not observed in controls, where the allele frequencies were for marker D19S206: allele 7, 0.10; D19S571: allele 1, 0.38; D19S589: allele 2, 0.30; D19S926: allele 4, 0.18; D19S927: allele 2, 0.41; D19S926: allele 4, 0.27; and D19S605; allele 2, 0.32.
Linkage analysis and haplotype analysis in the SCA-14 locus.
The positive results from the SHA were followed up and confirmed by multipoint linkage analyses in the extended six-generation pedigree (see figure 1). An affected-only strategy was used to perform the linkage analysis with seven markers in the SCA-14 region. Significant linkage of 3.61 (two-point lod score) at theta = 0.00 was found with marker D19S924 and a multipoint lod score (Zmax) of 4.56 was obtained. Finally, haplotype analysis determined the upper and lower boundaries of the SCA-14 candidate interval by recombination events for markers D19S206 and D19S926 (see figure 1). This resulted in a SCA-14 candidate interval of approximately 9.54 cM and covering 10.2 Mb genomic DNA.
Mutation analysis.
At the time we obtained our linkage results, three missense mutations in exon 4 of the PRKCG gene, located within the SCA-14 region, were reported.27 Accordingly, we sequenced exons 4 and 5 of the PRKCG gene in affected individuals from all 24 ADCA families. In the extended six-generation family, we identified a G→A transition in nucleotide 353 resulting in a base pair substitution from glycine to aspartic acid, as depicted in figure 2A, that replaces a nonpolar for a charged polar amino acid. The Gly118Asp mutation is present in the C1B domain of the PKCγ protein. This C1B domain is highly conserved during evolution and the original glycine residue is present in many organisms (see figure 2B). Subsequently, all related family members were sequenced and screened for the Gly118Asp mutation. The mutation cosegregated completely with the disease phenotype in the family. In addition, two individuals were identified who also carried the mutation, which was in agreement with the haplotype analysis, but they did not show signs of the disease phenotype. Neither unaffected individual is depicted in figure 1 in order to maintain confidentiality. No mutation was identified in any of the other 21 ADCA families. The Gly118Asp mutation was not detected in 85 control individuals (170 chromosomes), indicating that this change is unlikely to be a polymorphism.
Figure 2. (A) Sequence electropherograms for a part of exon 4 of the PRKCG gene. The upper panel shows the DNA sequence of a normal control. The DNA sequence of an affected individual (lower panel) shows a heterozygous mutation at base pair position 353G→A (indicated by an asterisk). (B) Evolutionary conservation of the Cys2/C1B domain at the glycine residue 118 (boxed) in different isozymes of PRKCG and different organisms.
Clinical characteristics.
Thirteen family members of the extended six-generation family were found to be affected. The clinical features of affected family members are given in the table⇓.
Table Clinical characteristics of affected family members
Table Continued
The mean age at onset was 40.8 ± 10.7 years (range 21 to 59 years). One family member (V-8), with an age in the onset range, was thought not to have any symptoms, but on examination this individual was found to have difficulty in turning, an upper limb action tremor, and slight abnormalities in the heel-to-shin test. Findings were concluded to be too subtle to clearly indicate disease onset.
Life span appears to be unaffected, witnessed by the fact that three affected men are 80 years or older. Striking age at onset variability was observed in two of the four parent-child groups studied. In one pair (V-2 and VI-1), the disease manifested 25 years earlier in the child. The other pair involves an asymptomatic male family member (IV-11) who died at the age of 56 years. His family did not recall any sign or symptom that suggested the presence of a cerebellar dysfunction at that age. However, the age at onset in his two sons (V-10 and V-11) was 21 and 24 years. All subjects displayed cerebellar ataxia with a slowly progressive course, with a disease onset after the age of 30 years in all but two subjects. The markedly earlier onset of the disease in subjects V-10 and V-11 was not accompanied by an increase in the rate of disease progression. A gait disorder was the presenting feature in all affected individuals. In addition to gait and limb ataxia, the neurologic examination revealed cerebellar dysarthria in 11 subjects, slowing of saccadic eye movements in 6 and dysmetric saccades in 7 subjects, and hyperreflexia in 8 individuals. Two older subjects (IV-5 and IV-7) showed marked hyporeflexia (with absent Achilles tendon reflexes), as well as absent sense of vibration below the knees, which suggest the coexistence of a peripheral neuropathy, either age-related or disease-related. Although hypotonia was not observed, individual V-7 showed rigidity of the upper limbs. A focal task-induced dystonia of the right hand (writer’s cramp) was observed in the two brothers with relatively early onset disease (V-10 and V-11). Individual IV-9 was very severely affected and completely immobilized, which prohibited a thorough neurologic examination. However, bradyphrenia and the presence of frontal release signs could indicate additional diffuse involvement of the cerebral cortex. Cognitive decline or mental retardation was not encountered in other family members and no family member showed signs of autonomic disturbances, axial myoclonus, parkinsonism, tremor, or seizures.
MRI had been performed in three family members (V-2, V-6, and VI-1) and showed marked atrophy of cerebellar vermis and hemispheres (figure 3); no cortical atrophy, basal ganglia abnormalities, or white matter changes were observed.
Figure 3. T1-weighted sagittal MRI scan of brain, showing marked atrophy of cerebellar midline structures.
Discussion.
The Dutch SCA-14 family reported here displayed a slowly progressive, relatively isolated SCA, with a mean age at onset of approximately 40 years. In addition to the cerebellar syndrome, hyperreflexia was frequently observed. In two older subjects, the clinical examination was suggestive of a peripheral neuropathy, but whether this represents a true disease characteristic or an age-related feature remains unclear. In two other subjects, the onset of disease in their early 20s and the presence of a focal task-induced dystonia of the right hand in both of them is noteworthy. Whether the dystonia is truly attributable to the disease itself remains to be established, but in this light it should be recalled that in the first SCA-14 family reported by Yamashita et al., axial myoclonus was observed in five early onset subjects.14 Taking the upper limb rigidity in one of the family members reported here also into account suggests that extrapyramidal features (in early onset disease) could indeed be part of the phenotypic spectrum of SCA-14. In addition, agu rats, which carry a homozygous nonsense mutation in the PRKCG gene that results in the complete absence of the catalytic domain of the PKCγ protein, display a parkinsonian phenotype.29 Thus, PRKCG gene mutations appear to lead to pathology in both the cerebellar and the extrapyramidal pathways.
In this extended family, clinical anticipation was suggested in two of the four parent-child combinations. In addition, the family pedigree details of one of the other SCA-14 families also suggest anticipation.14,27⇓ However, caution is needed, because the number of patients for studying the anticipation phenomenon is too small, the recorded age at onset is known to be affected by a recall bias in older individuals, and, regarding this specific missense mutation in the PRKCG gene, the biologic correlate of anticipation, commonly an expanded trinucleotide repeat, is lacking. The absence of the usual indicator of anticipation also casts further doubts on the anticipation being present. We will therefore use the term onset age variability, which is remarkable in this SCA-14 family, where the age at onset ranges from 21 to 59 years. The descriptive characteristics of the other SCA-14 families reported also reveal onset ranges of 10 to 51 years and 12 to 42 years.14,28⇓ This clinical variability may be a reflection of true (but unexplained) anticipation, or of problems in assessing the age at onset, or it may reflect modifying genes in a common genetic background instead of anticipation via the SCA-14 locus.
Three ADCA families, originally believed to be independent, were found to be linked to a common ancestor four generations back and, with the shared haplotype analysis, a common haplotype covering the SCA-14 locus was identified in these three families. Significant linkage in the combined family to the SCA-14 locus was confirmed by multipoint linkage (Zmax = 4.56) and haplotype analysis. The boundaries of the SCA-14 region were determined by recombinant events with the markers D19S206 and D19S926, providing a candidate interval of approximately 9.54 cM. At that time, mutations in the PRKCG gene were reported to be found in two SCA families and in one sporadic case.27 Interestingly, one of these families is partly of Dutch ethnicity. Based on these results, we screened exons 4 and 5 of the PRKCG gene and identified a novel missense mutation in exon 4 that involves a G→A transition in nucleotide 353, which predicts a glycine-to-aspartic acid substitution (Gly118Asp) and replaces a nonpolar for a charged polar amino acid. The mutation cosegregated with disease in all affected family members. The subject who showed too subtle cerebellar signs on examination to clearly indicate disease onset also carried the mutation. In addition, the mutation was detected in two individuals in whom the presence or onset of cerebellar disease was not or could not be established.
The PRKCG gene encodes PKCγ, an isoform of protein kinase C, which is a member of the family of serine/threonine kinases. These kinases are intermediates in second messenger signaling pathways and are involved in various cellular processes. PKCγ, one of the classic isoforms, is built up of a regulatory and a catalytic domain. The regulatory domain comprises a C1 subdomain, containing two tandem repeat cysteine-rich regions (C1A and C1B), and a C2 subdomain.30 The three missense mutations reported (His101Tyr, Ser119Pro, and Gly128Asp), as well as the Gly118Asp mutation we report here, are all located in the evolutionary conserved C1B region of the C1 regulatory domain, illustrating the important role that mutations in this specific region must play in cerebellar degeneration.27 Two mutations seem to directly alter PKCγ function, as shown by protein-structure modeling studies, mainly by affecting zinc ion interaction and phorbol ester binding affinity.27,31⇓ How this eventually results in the spinocerebellar degeneration remains to be elucidated, but the fact that PKCγ protein levels are reduced in Purkinje cells of SCA-1 transgenic mice and that in one patient with the PRKCG mutation the majority of Purkinje cells did not show ataxin-1 staining points to a potential role of PKCγ in the ataxin-1 pathway.27,32⇓ To address this issue, we investigated whether the age at onset in the affected family members correlated with the length of the CAG repeat on both alleles of the SCA-1 gene, but no significant correlation was found (data not shown).
A mutation in the SCA-1, SCA-2, SCA-3, SCA-6, or SCA-7 gene is found in 60 to 70% of Dutch ADCA families.23 The identification of this SCA-14 family will further contribute to the clarification of the genetic background of the remaining ADCA families.
It is of particular interest that we were able to link three originally independent ADCA families by a combined linkage, haplotype, and genealogy analysis. This confirms our suspicion that there may only be a limited number of independent SCA mutations within the Dutch ADCA families. This assumption is reinforced by our unpublished results, which show a clear geographic distribution of given SCA mutations confirming close temporal and geographic links for many families. Although we were fortunate to be able to reconstruct such a large family as in this example of SCA-14, the future challenge will be to obtain similar results with much smaller ADCA families.
Whereas the mutations in the first SCA genes identified are characterized by a tri- or pentanucleotide repeat expansion that probably leads to a toxic gain of protein function, it is missense mutations that have been found in the two most recent SCA genes. This opens up a new perspective for neuroscientists in the further unraveling of the complicated disease mechanisms of dominantly inherited spinocerebellar degenerations.
Acknowledgments
Supported by a research grant (97252) from the University Medical Center Nijmegen, the Netherlands (B.P.C.v.d.W.), and by a grant (MAR00–107) from the Prinses Beatrix Fonds, the Netherlands (D.S.V.).
The authors thank the participating family members, Dr. J. van Rossum (Leiden) for providing RF11, and Jackie Senior for revising the manuscript. They also thank the members of SCAN (SpinoCerebellar Ataxias in the Netherlands) for their contribution.
Footnotes
-
↵*Both authors contributed equally.
- Received May 5, 2003.
- Accepted August 20, 2003.
References
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