TRPM7 is not associated with amyotrophic lateral sclerosis-parkinsonism dementia complex in the Kii peninsula of Japan†
How to Cite this Article: Hara K, Kokubo Y, Ishiura H, Fukuda Y, Miyashita A, Kuwano R, Sasaki R, Goto J, Nishizawa M, Kuzuhara S, Tsuji S. 2010. TRPM7 Is Not Associated With Amyotrophic Lateral Sclerosis-Parkinsonism Dementia Complex in the Kii Peninsula of Japan. Am J Med Genet Part B 153B:310–313.
Abstract
Amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS/PDC) is a distinct neurodegenerative disorder characterized by ALS pathology with neurofibrillary tangles (NFTs) in the spinal cord and brain. Recent clinical studies have revealed a high incidence and a high familial occurrence of ALS/PDC in both Guam and the Kii peninsula of Japan, suggesting a strong genetic predisposition to this disorder. The T1482I variant (rs8042919) of TRPM7 gene which is suggested to play roles in regulating the cellular homeostasis of Ca2+, Mg2+, and trace metals, has recently been reported to be associated with Guamanian patients with ALS/PDC. To investigate whether TRPM7 is associated with Kii ALS/PDC, we conducted parametric linkage analyses of the TRPM7 locus in a large extended family with ALS/PDC. Linkage analysis did not reveal any evidence supporting the linkage to the TRPM7 locus. Resequencing of the entire coding region of TRPM7 did not reveal any pathogenic mutations in an affected individual in this family. The allele frequencies of the T1482I in affected individuals in this family or in those from other families are not significantly different from those in regional controls or those in HapMap-JPT samples. These results indicate that TRPM7 is not associated with ALS/PDC in the Kii peninsula of Japan. © 2009 Wiley-Liss, Inc.
INTRODUCTION
Amyotrophic lateral sclerosis and parkinsonism-dementia complex (ALS/PDC) is a unique form of ALS highly prevalent in the island of Guam, southern West New Guinea, and the Kii peninsula of Japan [Kimura, 1961; Elizan et al., 1966; Gajdusek and Salazar, 1982]. Both Guamanian and Japanese patients with ALS/PDC are pathologically characterized by neurofibrillary tangles (NFTs) in the brain and spinal cord in addition to the ALS pathology affecting the upper and lower motor neurons [Hirano et al., 1961]. High incidences of ALS in Guam and the Kii peninsula of Japan have been reported since the 1950s [Kurland and Mulder, 1954] and 1960s [Kimura, 1961], respectively. Recent studies have indicated that the PDC type is still common, but also that the incidence of the ALS type is decreasing in both Guam and the Kii peninsula of Japan [Plato et al., 1969; Kuzuhara, 2007]. Because the disease focus occurs in a restricted area among three genetically different populations, genetic and/or environmental factors have been proposed as the etiologies of this disorder. A recent epidemiological study of Kii ALS/PDC has revealed that approximately 70% of patients have a family history of ALS/PDC [Kuzuhara et al., 2001; Kuzuhara, 2007]. Furthermore, families with multiple cases of ALS/PDC are common in Guam [Kurland and Mulder, 1955; Morris et al., 2004]. These observations made in both Guam and the Kii peninsula of Japan strongly suggest the involvement of genetic factors in ALS/PDC.
A comprehensive mutational analysis of 19 candidate genes including ALS/FTLD-related genes (SOD2, SOD3, ALS2/alsin, SMN1, PGRN, ANG, VEGF, VCP, VAPB, DCTN1, CHMP2B, and TARDBP/TDP-43), the tauopathy-related gene (GSK3β), and parkinsonism-related genes (α-synuclein, LRRK2, parkin, DJ-1, PINK1, and ATP13A2) did not reveal any mutations in these genes in the patients with ALS/PDC [Tomiyama et al., 2008]. The T1482I variant of transient receptor potential melastatin 7 gene (TRPM7) has recently been reported to be associated with five Guamanian ALS/PDC patients [Hermosura et al., 2005]. TRPM7 is a member of the TRP superfamily of ion channels that has been suggested to play roles in the homeostatic regulation of Ca2+ and Mg2+. The association of the TRPM7 variant with ALS/PDC may support the environmental factor hypothesis that prolonged exposure to low level of Ca2+ and Mg2+ contributes to the high incidence of development of ALS/PDC [Garruto, 1991]. To explore the implication of TRPM7 in Kii ALS/PDC, we have conducted parametric linkage analyses of the TRPM7 locus, and resequenced the entire coding region of TRPM7 of an affected individual using a large extended family with ALS/PDC in the Kii peninsula of Japan. We further compared the frequencies of T14821 in the affected individuals in this family or in those from other families with those in regional controls or HapMap-JPT samples to investigate the potential association of T1482I with ALS/PDC in the Kii penisula.
MATERIALS AND METHODS
Samples
Genomic DNA was extracted from peripheral leukocytes according to standard protocol after obtaining informed written consent from patients. The clinical and pathological evaluations of the family members are described elsewhere [Tomiyama et al., 2008]. The research project was approved by the ethics committee of Niigata University, Mie University School of Medicine, and the University of Tokyo.
Linkage Analysis of the TRPM7 Locus on Chromosome 15q21.2
Parametric pair-wise linkage analysis of the TRPM7 locus was performed using the Superlink program (http://bioinfo.cs.technion.ac.il/superlink/) ‘Fishelson and Geiger, 2002’ with the 23 family members including the 8 affected individuals (Supplementary Figure, A family). The pedigree information was updated based on information obtained after publication of our previous study [Tomiyama et al., 2008]. Pair-wise lod scores at D15S978 and D15S1016 flanking the TRPM7 locus at 1.6 Mb upstream and 2.6 Mb downstream, respectively, were obtained using autosomal dominant (AD) and autosomal recessive (AR) models. A disease gene frequency of 0.01 and penetrance rates of 0.9 and 1.0 were used.
Resequencing of Entire Coding Regions of TRPM7
Coding regions of TRPM7 were amplified by polymerase chain reaction (PCR) using TaKaRa LA Taq (TaKaRa, Tokyo, Japan) for one patient (V-10) clinically diagnosed with ALS/PDC. The primers were designed using ExonPrimer (http://ihg2.helmholtz-muenchen.de/ihg/ExonPrimer.html; see Supplementary Data). The PCR products were purified using ExoSAP-IT (USB), and subjected to direct nucleotide sequence analysis using a BigDye terminator Cycle Sequencing kit v3.1 and an ABI3100 sequencer (Applied Biosystems, Foster City, CA). The obtained sequence data were analyzed by Variant ReporterTM Software v1.0 (Applied Biosystems).
Association Analysis of T1482I
The allele frequencies of T1482I was investigated in other 7 patients with ALS/PDC included in the linkage study (VI-2, VI-7, VI-9, VI-17, VI-18, VI-21, and VII-4) and another 1 recently diagnosed patient (VI-20) in the A family (Supplementary Figure), 16 Kii-ALS/PDC index patients from other families (6 multiplex families and 10 apparently sporadic patients), and 27 control subjects living in the same region by resequencing exon 28 in TRPM7 using a primer pair of 5′-TGGTGTCCAGGTAGAATAAAG-3′ and 5′-TTCACTGCTCATGTGTTTGAC-3′, or that described in the Supplementary Table (Exon28F and Exon28R).
Association analysis of T1482I variant was conducted with χ2 analysis of the allele frequencies of 9 patients in family A, or of 16 index patients in other families, and those in 27 regional controls, or HapMap-JPT samples.
RESULTS
Pair-wise LOD scores were −infinity at both D15S978 and D15S1016 for AD and AR models with complete penetrance. We obtained LOD scores of 0.64/−0.62 and 0.51/−1.65 for AD and AR models with incomplete penetrance (0.9), respectively (Table I). These results suggest that the linkage of Kii ALS/PDC to the TRPM7 locus is unlikely in this family.
| Mode of inheritance | AD model | AR model | ||
|---|---|---|---|---|
| Penetrance | 0.9 | 1.0 | 0.9 | 1.0 |
| D15S978 | 0.64 | −infinity | 0.51 | −infinity |
| D15S1016 | −0.62 | −infinity | −1.65 | −infinity |
Resequencing of the entire coding regions of TRPM7 of a patient (V-10) from family A revealed two homozygous SNPs in introns (IVS3-26G>C [rs2063011] and IVS22-41T>A [rs675011]). Because both are present in the majority of the HapMap samples, they are unlikely to be pathogenic for Kii ALS/PDC.
Allele frequencies of T1482I variant (rs8042919) in the nine affected individuals in the A family are similar to those in regional controls (P = 0.38; Table II). We further extended the analysis to the 16 index patients from other families, but we did not observe any significant association of T1482I with ALS/PDC (P = 0.86). Allele frequencies of T1482I in the regional controls are similar to those in the HapMap-JPT samples (P = 0.79; dbSNP: http://www.ncbi.nlm.nih.gov/SNP/). Resequencing of exon 28 of TRPM7 revealed a previously described polymorphism (IVS28+15 C/T, rs3109894), which also did not show any significant association with ALS/PDC. Taken together, we conclude that the T1482I variant is not associated with Kii ALS/PDC.
| No. of subjects (frequency) | ||||
|---|---|---|---|---|
| Affected in A family | Affected in other families | Control in the region | HapMap-JPT | |
| Genotype | ||||
| T/T | 0 (0.0%) | 1 (6.3%) | 0 (0.0%) | 2 (4.4%) |
| C/T | 2 (22.2%) | 4 (25.0%) | 11 (40.7%) | 16 (35.6%) |
| C/C | 7 (77.8%) | 11 (68.8%) | 16 (59.3%) | 27 (60.0%) |
| No. of alleles (frequency) | ||||
|---|---|---|---|---|
| Affected in A family | Affected in other families | Control in the region | HapMap-JPT | |
| Allele | ||||
| T | 2 (11.1%) | 6 (18.8%) | 11 (20.4%) | 20 (22.2%) |
| C | 16 (88.9%) | 26 (81.3%) | 43 (79.6%) | 70 (77.8%) |
| P-value | 0.38a [0.29]b | 0.86a [0.68]b | ||
- a P values of χ2 tests obtained by comparison of allele frequencies of T1482I between affected individuals and the regional controls are shown.
- b P values of χ2 tests obtained by comparison of allele frequencies of T14821 between affected individuals and HapMap-JPT samples are shown in parenthesis. There was no significant difference in the allele frequencies of T1482I between the regional controls and HapMap-JPT samples (P = 0.79).
DISCUSSION
In this study, we did not obtain any supportive evidence for the genetic linkage of Kii ALS/PDC to the TRPM7 locus and resequencing analysis of the entire TRPM7 of the affected individual did not reveal any causative mutations. Furthermore, the allele frequencies of T1482I in the affected individuals are not significantly different from those in regional controls or those in HapMap-JPT samples. Thus, it is unlikely that T1482I or TRPM7 is associated with the Kii-ALS/PDC.
The structure of a large extended family with ALS/PDC in the Kii of Japan is complex for explicitly determining the inheritance mode [Tomiyama et al., 2008]. There are three consanguineous marriages in this pedigree, suggesting an AR pattern, whereas the disease occurs partially in the successive generations, suggesting an AD pattern with reduced penetrance. The complexity of the inheritance pattern has also been discussed with regard to ALS/PDC families in Guam. Formal segregation analysis of Guamanian ALS/PDC families rejected both dominant and recessive models, but were consistent with a 2-allele major locus model [Bailey-Wilson et al., 1993]. Indeed, a genome-wide association study of Guamanian ALS/PDC using 834 microsatellite markers did not provide any associated markers with a genome-wide significant level (P < 0.0001). Furthermore, pair-wise linkage analysis of 17 microsatellite markers in which they determined the threshold for further study (P < 0.015) has shown some interesting loci such as D3S2406 (LOD score = 0.78) and D20S103 (LOD score = 1.82) but failed to identify a convincing single locus [Morris et al., 2004]. These results suggest that familial ALS/PDC is not caused by a mutation of a single gene but is a complex disease involving genetic and environmental factors. Further extended association and linkage studies employing high-density single nucleotide polymorphisms (SNPs) and a larger sample size may be useful to identify susceptibility genes for the Kii ALS/PDC.
Acknowledgements
We thank the family members who contributed immensely to this study. This study was supported in part by KAKENHI (Grant-in-Aid for Scientific Research) on Priority Areas, Applied Genomics, the 21st Century COE Program, Center for Integrated Brain Medical Science, and Scientific Research (A) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a Grant-in-Aid for “the Research Committee for Ataxic Diseases” of the Research on Measures for Intractable Diseases from the Ministry of Health, Labour and Welfare, Japan.
