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Nephrol Dial Transplant (2001) 16: 939-944
© 2001 European Renal Association-European Dialysis and Transplant Association

Identification of mutations including de novo mutations in Korean patients with hypokalaemic periodic paralysis

Sung Han Kim1, Un Kyung Kim1, Jae Jin Chae2, Dae Joong Kim3,, Ha-Young Oh3, Byoung Joon Kim4 and Chung Choo Lee1

1 Department of Biology and SRC for Cell Differentiation, Seoul National University, 2 Department of Molecular Biology, Seoul National University, 3 Division of Nephrology, Department of Medicine, 4 Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Hypokalaemic periodic paralysis (hypoPP) is an autosomal dominant disorder involving the abnormal function of ion channels and it is characterized by paralysis attacks of varying severity, accompanied by a fall in blood potassium levels. Linkage analysis showed that the candidate locus responsible for hypoPP was localized to chromosome 1q31-32, and this locus encoded the muscle dihydropyridine-sensitive calcium channel {alpha}1-subunit (CACNA1S). So far, three different mutations in CACNA1S gene have been identified in patients with hypoPP: Arg528His, Arg1239His and Arg1239Gly in Caucasian patients. However, there are few reports about the mutations of CACNA1S gene in other races.

Methods. In this study, four Korean families with five hypoPP patients were screened for mutations of CACNA1S gene with polymerase chain reaction-based restriction analysis and single-strand conformation polymorphism analysis. To determine the mode of inheritance, haplotype analysis was done with three microsatellite markers (D1S1726, CACNL1A3, and D1S1723).

Results. Arg528His mutation was detected in three families, and one family had no known mutations. Moreover, for the first time, we detected de novo Arg528His mutations in two out of three families with hypoPP. Haplotype analysis using three microsatellite markers (D1S1726, CACNL1A3, and D1S1723) suggested the occurrence of de novo Arg528His mutations in two of the three families with Arg528His mutation.

Conclusions. Arg528His mutations of CACNA1S, including de novo Arg528His mutations, were found in Korean patients with hypoPP. These results imply that de novo mutation, in addition to non-penetrance, is one of the genetic mechanisms that can explain the previous clinical observation that hypoPP occurs sporadically without family history.

Keywords: de novo; hypokalaemia; Korean; mutation; paralysis; periodic



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 References
 
Familial hypokalaemic periodic paralysis (hypoPP) is an autosomal dominant disorder of the skeletal muscle characterized by episodic attacks of muscle weakness that are associated with a decrease in blood potassium levels. The attacks occur variously from once in a lifetime to several times per week. The prevalence has been estimated at about 1/100000 [1]. The onset of hypoPP usually occurs in the second decade of the life, and one-third of the patients develop the disease before the age of 16 years. [2,3].

Recently, the hypoPP locus was genetically mapped to chromosome 1q31-32, where the {alpha}1-subunit of the dihydropyridine receptor gene (CACNA1S) is known to reside [4–6]. The dihydropyridine receptor belongs to a family of voltage-gated ion channels and, with potassium, sodium, and calcium channels, shares a common structure consisting of six transmembrane segments [7]. The {alpha}1-subunit of the dihydropyridine receptor is composed of four domains [8]. Deleterious mutations have been demonstrated in the voltage-sensor segment S4 of the dihydropyridine receptor, establishing CACNA1S as the causative gene for hypoPP [5,9].

So far, three missense mutations have been identified in the highly conserved putative membrane spanning domains (Arg528His, Arg1239His, and Arg1239Gly). Arg528His and Arg1239His presently account for the majority of identified mutations in hypoPP families [5,9–11]. Incomplete penetrance was observed in families displaying the Arg528His mutations [11,12].

In the present study, we performed mutation analysis regarding the three known mutations of CACNA1S gene in four Korean families with hypoPP. We identified Arg528His mutation in three families, and two of the families showed de novo mutations.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 References
 
Patients
Five hypoPP patients and their family members from four Korean families were analysed. The characteristics and clinical features of the patients are summarized in Table 1Go. The patients presented typical clinical features of hypoPP, i.e. episodic paralysis of limbs with concomitant hypokalaemia that improved with normalizing serum potassium concentration. The paralysis attacks always started at dawn. All patients responded to treatment and were free of paralysis attacks after the treatment. Informed consent was obtained from all patients and the project was approved by the institutional review board.


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  		Table 1. Characteristics and clinical features of the patients

 

Restriction analysis for mutations
Genomic DNA was prepared from the peripheral blood of five hypoPP patients and their family members, using a method described by John et al. [13]. As the Arg528His mutation disrupts a BbvI restriction site, it was easily analysed by polymerase chain reaction (PCR) according to the previous protocol that used the forward primer 5'-GGAGATCCTGCTGG TGGAGTCG-3' and reverse primer 5'-TCCTCAGGAGGCGGATGCAG-3' [9,12]. The PCR products were digested overnight with BbvI and electrophoresed on 4% NuSieve® 3 : 1 agarose gels (FMC Bioproducts, Rockland, ME). This analysis was also performed on 50 unaffected and unrelated Koreans. As the Arg1239His mutation creates a NlaIII restriction site, it was also analysed by PCR using the forward primer 5'-CGCATCTCCAGCGCCTTCTTC-3' and reverse primer 5'-CGTCCACAGGAGGGTTCGCAC-3' [9,12]. The PCR products were digested with NlaIII, electrophoresed on 20% polyacrylamide gels, and stained with Syber green (Figure 1Go).



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  		Fig. 1. The wild-type and mutant dihydropyridine receptor gene sequences for the Arg528His and Arg1239His mutations. The restriction-enzyme sites modified by the mutations are indicated—the Arg528His mutation abolishes a BbvI site, and the Arg1239His mutation creates new NlaIII site.

 
Sequencing of the mutation was performed on at least one affected individual per family.

Single-strand conformation polymorphism analysis
For internal labelling, 1 µCi of [{alpha}-32P]dCTP (Amersham Pharmacia Biotech, Uppsala, Sweden) was added to the PCR reaction mixture. Three microlitres of radiolabelled PCR products were mixed with the loading buffer. The samples were denatured by boiling for 5 min and then cooled with ice. Samples were loaded on a 0.7xMDETM gel (FMC Bioproducts, Rockland, ME) in 0.6xTBE buffer. Electrophoresis was carried out with an IBI sequencing apparatus (Eastman Kodak Co., Rochester, NY) at 8 W for 12 h at room temperature. After electrophoresis, each gel was dried and autoradiographed for 1 day.

Haplotype and genetic analysis
Haplotypes of the four hypoPP families were constructed by using three dinucleotide repeat polymorphisms (D1S1273, D1S1276, and CACNL1A3) [6,14]. For internal labelling, 1 µCi of [{alpha}-32P]dCTP (Amersham Pharmacia Biotech, Uppsala, Sweden) was added to the PCR reaction mixture. The PCR consisted of 30 cycles: 30 s at 94°C, 30 s at 55°C, 30 s at 72°C, which were initiated by one cycle of 5 min denaturation and finished with one 5-min extension step. The PCR products were electrophoresed on 6% polyacrylamide gels with 7 M urea, and the gel was dried and autoradiographed overnight. The genetic distance between the markers are 0 cM in 8 CEPH pedigrees [9]. The dinucleotide-repeat alleles were numbered according to the increasing size on the same gel.

To make certain that the above dinucleotide repeats are informative as a marker in Korean people, allele frequencies of dinucleotide-repeat alleles were determined by typing 57 unrelated and unaffected Korean individuals (n=114). Heterozygosities and polymorphism information content (PIC) values were calculated according to the method of Ott et al. [15].



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 References
 
Mutation analysis
Five HypoPP patients and their family members from four Korean families were screened for previously known mutations in CACNA1S gene (Arg528His, Arg1239His, and Arg1239Gly). Restriction analysis with BbvI enzyme and sequence analysis showed that three families had Arg528His mutation (Figure 1Go), while no such mutation was found in 50 unaffected and unrelated Koreans. Interestingly, we observed de novo Arg528His mutations in two of the three families with Arg528His mutation. Parents of probands in families 1 and 2 were normal according to their medical history and clinical criteria, but their offsprings were affected with hypoPP. As shown in Figure 2Go, II-3 of family 1 and II-1 of family 2 displayed the restriction pattern of Arg528His mutation, whereas in I-1 and I-2, the restriction pattern was normal. Restriction analysis with NlaIII enzyme showed that four families did not have the Arg1239His mutation. To detect other mutations surrounding the codons of 528 and 1239 associated with hypoPP, SSCP analysis was performed, but no deleterious mutation was detected.



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  		Fig. 2. Restriction analysis with BbvI for Arg528His mutation in CACNA1S gene and the identified mutant sequence (D). Clinically affected and unaffected individuals in families are represented by filled and open symbols respectively. PCR products were digested with BbvI enzyme, electrophoresed on 4% NuSieve® 3 : 1 agarose gels, and stained by ethidium bromide. A normal individual has two bands of 33 and 44 b.p. An affected heterozygote has an additional undigested 77-b.p. band. Restriction analysis was performed for family 1 (A), family 2 (B), and family 3 (C). De novo Arg528His mutation was detected by restriction analysis for family 1 and 2. M, DNA size marker (øX174/HaeIII).

 

Haplotype and genetic analysis
To determine how informative each dinucleotide repeat was in the Korean population, we analysed the number of alleles, their size, PIC, and heterozygosity content of the markers in 57 unrelated Korean individuals. The allele frequencies and their standard errors of three markers are summarized in Table 2Go. The allele frequency distributions of these markers showed bimodal or more complex aspects. The heterozygosity values for the markers range from 58 to 76%. D1S1723 and CACNL1A3 showed lower heterozygosity values than those found in other studies (Table 3Go). But the heterozygosity value for D1S1726 was higher than the value reported for Caucasians. All of these markers were highly informative with PIC values over 0.5. The disease haplotype for each family was determined and is detailed in Figure 3Go. All three families have different disease haplotype. Moreover, the haplotype analysis confirmed de novo Arg528His mutations in two of the families. Interestingly, all the family members in family 3 shared the identical haplotype, which suggests the possibility of consanguinity.


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  		Table 2. Allele frequencies of three (CA)n markers in Koreans

 

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  		Table 3. Heterozygosities and PIC values for the three (CA)n markers

 


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  		Fig. 3. Partial pedigrees of hypoPP families with their haplotypes. Clinically affected and unaffected individuals in families are represented by filled and open symbols respectively. Haplotypes are constructed for markers D1S1726, CACNL1A3, and D1S1723. (A) Partial pedigrees of family 1. II-1 and II-2 have the same haplotypes but only II-1 has the disease of hypoPP by de novo Arg528His. II-1 is a case 1. (B) Partial pedigrees of family 2. II-3 is a case 2. (C) Partial pedigrees of family 3. I-1 is a case 3 and II-1 is a case 4.

 



   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
References
 
Recently, a gene linked to hypoPP has been mapped to chromosome 1q31-32 by linkage analysis in hypoPP families from different European populations. The CACNA1S gene has been mapped to the same region. Mutation analysis has shown that the CACNA1S gene is a major locus for hypoPP. Two point mutations, Arg528His and Arg1239His, are predominant mutations in Caucasian populations [5,9,11] and a Japanese family with Arg528His mutation has been reported [10]. Plassart et al. [16] has also shown the genetic heterogeneity for hypoPP for the first time.

The mutations identified previously (Arg528His and Arg1239His) can be easily detected by PCR amplification and restriction analysis. We performed restriction analysis on four hypoPP families and on 50 unaffected and unrelated Koreans. We detected Arg528His mutations in three families, whereas no such mutation was found in the 50 unaffected and unrelated Koreans. This indicates that the Arg528His mutation caused hypoPP in the Korean population that is of different ethnic background from the Caucasian. Moreover, Arg528His mutations in two of the families (family 1, and 2) were de novo. Two cases of de novo Arg1239His mutation have already been reported [5,11] but a de novo Arg528His mutation has not been reported anywhere.

The choice of genetic markers for human linkage analysis is based on both the location of the marker with respect to a putative disease gene and the degree of polymorphism of the marker. The latter may vary significantly between populations, resulting in a different PIC, which may affect the choice of a particular marker for linkage study. In this study, allele distributions of three markers for hypoPP in the Korean population were bimodal or more complex, and revealed a high degree of polymorphism. Thus, these highly informative polymorphisms will be useful for population-associated and family-based linkage studies in Koreans. The haplotype analysis with the above markers confirmed the de novo Arg528His mutations in two families.

It is well known from clinical observation that hypoPP can occur sporadically without family history [17]. Concerning the genetic mechanism for these clinical observations, non-penetrances has been reported in families with Arg528His mutation [11,12] and a de novo Arg1239 mutation has been reported [5,11]. Our finding of de novo Arg528His mutations provides substantial evidence to support de novo mutation as one of the genetic mechanisms that cause sporadic cases of hypoPP.

No common haplotypes were found in our population of patients with hypoPP. This variety of haplotypes may be due to de novo mutation occurring at two sites (Arg528 and Arg1239). Both 528 and 1239 codons have a CpG dinucleotide and a G to A transition in both sites, resulting in an Arg to His amino acid change. The C to T, or G to A transition in CpG dinucleotides are the most common point mutations within the gene coding regions that cause human genetic diseases [18,19]. In addition to the de novo Arg1239His mutation previously reported, identification of de novo Arg528His mutation in this study shows that the recurrent mutations at 528 and 1239 codons may occur more frequently compared with other mutations that cause genetic diseases. It may be that these two sites are intrinsically unstable, causing point mutation. These recurrent mutations may have also resulted in a variety of haplotypes for hypoPP in many populations. To confirm the above possibility, extensive genetic studies for hypoPP must be carried out in many populations of different ethnic backgrounds.



   Acknowledgments
 
This work was supported in part by research grants from the Korean Sciences and Engineering Foundation through the Research Center for Cell Differentiation at Seoul National University.



   Notes
 
Correspondence and offprint requests to: Dae Joong Kim MD, Division of Nephrology, Samsung Medical Center, 50 Ilwondong, Kangnamgoo, Seoul, Korea 135-710. Back



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 14. 3.00
Revision received 8.11.00.
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