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Figure 1.
CTGallele distribution at the myotonic dystrophy protein kinase gene in 370 chromosomes in a healthy Kuwaiti population.

CTGallele distribution at the myotonic dystrophy protein kinase gene in 370 chromosomes in a healthy Kuwaiti population.

Figure 2.
CTG allele frequency in healthy (non–myotonic dystrophy) subjects from the following 3 populations: Kuwaiti (present study), black South African, and European. A low prevalence of (CTG)>18can be observed in Kuwaiti and black South African populations.

CTG allele frequency in healthy (non–myotonic dystrophy) subjects from the following 3 populations: Kuwaiti (present study), black South African,20 and European.11 A low prevalence of (CTG)>18can be observed in Kuwaiti and black South African populations.

Comparison of CTG Allele Groups Among Non–Myotonic Dystrophy Subjects
Comparison of CTG Allele Groups Among Non–Myotonic Dystrophy Subjects
1.
Harper  PS The molecular dystrophies.  In: Scriver  CR, Beaudet  AL, Sly  WS, Valle  D, eds. Metabolic Basis of Inherited Disease.6th ed. New York, NY: McGraw-Hill Co; 1989.
2.
Meiner  AWolf  CCarey  N  et al Direct molecular analysis of myotonic dystrophy in the German population: important considerations in genetic counselling. J Med Genet.1995;32:645-649.
PubMed
3.
Harper  PS Myotonic Dystrophy. 2nd ed. Philadelphia, Pa: WB Saunders Co; 1989.
4.
Harley  HGBrook  JDRundle  SA Expansion of unstable DNA region and phenotypic variation in myotonic dystrophy. Nature.1992;355:545-546.
PubMed
5.
Buxton  JShelbourne  PDavies  J  et al Detection of an unstable fragment of DNA specific to individuals with myotonic dystrophy. Nature.1992;355:547-548.
PubMed
6.
Aslanidis  CJansen  GAmemiya  C  et al Cloning of the essential myotonic dystrophy region and mapping of the putative defect. Nature.1992;355:548-551.
PubMed
7.
Brook  JDMcCurrach  MEHarley  HG  et al Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family member. Cell.1992;68:799-808.
PubMed
8.
Mahadevan  MTsilfidis  CSabourin  L  et al Myotonic dystrophy mutation: an unstable CTG repeat in the 3′ untranslated region of the gene. Science.1992;255:1253-1255.
PubMed
9.
Fu  YHPizzuti  AFenwick Jr  RG  et al An unstable triplet repeat in a gene related to myotonic muscular dystrophy. Science.1992;255:1256-1258.
PubMed
10.
Brunner  HGNillesen  Wvan Oost  BA  et al Presymptomatic diagnosis of myotonic dystrophy. J Med Genet.1992;29:780-784.
PubMed
11.
Imbert  GKretz  CJohnson  KMandel  JL Origin of the expansion mutation in myotonic dystrophy. Nat Genet.1993;4:72-76.
PubMed
12.
Chakraborty  RStivers  DNDeka  RYu  LMShriver  MDFerrell  RE Segregation distortion of the CTG repeats at the myotonic dystrophy locus. Am J Hum Genet.1996;59:109-118.
PubMed
13.
Watkins  WSBamshad  MJorde  LB Population genetics of trinucleotide repeat polymorphisms. Hum Mol Genet.1995;4:1485-1491.
PubMed
14.
Chung  MYRanum  LPDuvick  LAServadio  AZoghbi  HYOrr  HT Evidence for a mechanism predisposing to intergenerational CAG repeat instability in spinocerebellar ataxia type I. Nat Genet.1993;5:254-258.
PubMed
15.
Hirst  MCGrewal  PKDavies  KE Precursor arrays for triplet repeat expansion at the fragile X locus. Hum Mol Genet.1994;3:1553-1560.
PubMed
16.
Leeflang  EPArnheim  N A novel repeat structure at the myotonic dystrophy locus in a 37 repeat allele with unexpectedly high stability. Hum Mol Genet.1995;4:135-136.
PubMed
17.
Ashizawa  TEpstein  HF Ethnic distribution of the myotonic dystrophy gene. Lancet.1991;338:642-643.
PubMed
18.
Davies  JYamagata  HShelbourne  P  et al Comparison of the myotonic dystrophy associated CTG repeat in European and Japanese populations. J Med Genet.1992;29:766-769.
PubMed
19.
Krahe  REckhart  MOgunniyi  AOOsuntokun  BOSiciliano  MJAshizawa  T De novo myotonic dystrophy mutation in a Nigerian kindred. Am J Hum Genet.1995;56:1067-1074.
PubMed
20.
Goldman  ARamsay  MJenkins  T Absence of myotonic dystrophy in southern African Negroids is associated with a significantly lower number of CTG trinucleotide repeats. J Med Genet.1994;31:37-40.
PubMed
21.
Tishkoff  SAGoldman  ACalafell  F  et al A global haplotype analysis of the myotonic dystrophy locus: implications for the evolution of modern humans and for the origin of myotonic dystrophy mutations. Am J Hum Genet.1998;62:1389-1402.
PubMed
22.
Yamagata  HMiki  TOgihara  T  et al Expansion of unstable DNA region in Japanese myotonic dystrophy patients [letter]. Lancet.1992;339:692.
PubMed
23.
Lavedan  CHofmann-Radvanyi  HBoileau  C  et al French myotonic dystrophy families show expansion of a CTG repeat in complete linkage disequilibrium with an intragenic 1 kb insertion. J Med Genet.1994;31:33-36.
PubMed
24.
Martorell  LMonkton  DGSanchez  ALopez De Munain  ABaiget  M Frequency and stability of the myotonic dystrophy type 1 premutation. Neurology.2001;56:328-335.
PubMed
25.
Meiner  AThamm  BStrenge  SFroster  U Instability in the normal CTG repeat range at the myotonic dystrophy locus [letter]. J Med Genet.1998;35:791.
PubMed
26.
Zerylnick  CTorroni  ASherman  SLWarren  ST Normal variation at the myotonic dystrophy locus in global human populations. Am J Hum Genet.1995;56:123-130.
PubMed
27.
Goldman  ARamsay  MJenkins  T Ethnicity and myotonic dystrophy: a possible explanation for its absence in sub-Saharan Africa. Ann Hum Genet.1996;60:57-65.
PubMed
28.
Mor-Cohen  RMagal  NGadoth  NShohat  TShohat  M Correlation between the incidence of myotonic dystrophy in different groups in Israel and the number of CTG trinucleotide repeats in the myotonin gene. Am J Med Genet.1997;71:156-159.
PubMed
29.
Culjkovic  BStojkovic  OVukosavic  S  et al CTG repeat polymorphism in DMPK gene in health Yugoslav population. Acta Neurol Scand.2002;105:55-58.
PubMed
Original Contribution
June 2004

CTG Repeat Number at the Myotonic Dystrophy Locus in Healthy Kuwaiti IndividualsPossible Explanation of Why Myotonic Dystrophy Is Rare in Kuwait

Author Affiliations

From the Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences and Nursing, Kuwait University (Dr Alfadhli), and the Divisions of Molecular Genetics (Dr Elshafey) and Clinical Genetics (Drs Bastaki and Al-Awadi), Kuwait Medical Genetic Center, Sulaibekhat, Kuwait.

Arch Neurol. 2004;61(6):895-898. doi:10.1001/archneur.61.6.895
Abstract

Background  Myotonic dystrophy is caused by unstable (CTG)n repeat expansion. On normal chromosomes, this repeat is highly polymorphic, with a copy number ranging from 4 to 38. Myotonic dystrophy is considered more prevalent in Western European and Japanese populations but less prevalent, rare, or even absent in others. It has been proposed that the expanded (CTG)n alleles originated from the group of the large normal alleles.

Objective  To determine whether there is a lower prevalence of the large alleles in the Kuwaiti population.

Design and Participants  We determined the size distribution of the CTG repeats by means of polymerase chain reaction in blood DNA derived from 185 healthy Kuwaiti individuals representing the 5 Kuwaiti provinces.

Results  We found a total of 17 (CTG)n alleles, with a range of 5 to 37 repeats. The (CTG)5 allele was the most frequent single allele (100/370 [27.0%]), whereas the (CTG)10-13 was the most frequent class of alleles (161/370 [43.5%]). Using 18 repeats as the cutoff point, χ2 analysis showed a statistically significant lower frequency of greater than 18 alleles in the Kuwaiti population compared with the European population (χ2 = 12.7; P<.001).

Conclusions  These data may explain the rare occurrence of myotonic dystrophy in the Kuwaiti population. Further study of healthy families within the high-normal repeat range is in progress to investigate the possible instability of the (CTG)>18 alleles in our area.

Myotonic dystrophy (DM) is an autosomal dominant disorder with high penetrance and rare new mutations.1,2 It is the most common adult form of muscular dystrophy. The clinical features of this disease are myotonia, weakness, muscle wasting, frontal baldness, cataract, hypogonadism, and electrocardiographic changes.3 The causative mutation in DM is an expansion of an unstable tandem repeat of the CTG sequence located in the 3′ untranslated region of a gene, with strong homology to the protein kinase family, on chromosome 19q13.3.49 In unaffected individuals, the (CTG)n repeat number is polymorphic, ranges from 5 to 38 repeats, and is stably inherited.710 In DM, at least 50 copies are present in the minimally affected patients7 and dramatically increase to an estimated 2000 copies in severely affected individuals.8,9

Factors that affect trinucleotide repeat stability, normal allelic variation, and the generation of new disease alleles are not fully understood. It has been proposed that expanded alleles originated from the group of large normal alleles.11,12 The heterogeneous class of (CTG)≥19alleles may constitute a reservoir for recurrent DM mutations. This model has been supported by the finding that differences between the frequency of large alleles in the high-normal range of allele size distribution ([CTG]≥19) in the global human population are congruent with observed variation in the prevalence rate of the disease.13 Moreover, at the higher allele range, loss of interrupting motifs within tracts of trinucleotide repeats leads to greater instability and predisposes alleles to expansion.1416

At present, disease incidence is globally quite variable. Myotonic dystrophy is considered to be most prevalent in western European and Japanese populations, less prevalent in Southeast Asian populations, and rare or absent in southern and central African populations.3,1719 In the state of Kuwait, the Ministry of Planning estimates the population at 2243 million, of which about 38% are Kuwaiti. Because DM is less frequently observed in native Kuwaitis (only 2 families have been identified with the disease [unpublished data, S.A., L.B., and S. Al-Awadi, MD, January 2004]), we conducted a study of CTG–repeat length polymorphism in healthy Kuwaiti individuals in an attempt to explain the paucity of DM-affected families in this population.

METHODS
SUBJECTS

One hundred eighty-five healthy native Kuwaiti individuals participated in this study. Samples were collected from Kuwaiti individuals coming to the major hospitals in the 5 provinces of Kuwait for checkup. All individuals belonged to known Kuwaiti tribes with minimal non-Kuwaiti admixture. Consent agreement was obtained from all volunteer subjects in this study.

POLYMERASE CHAIN REACTION ANALYSIS

We extracted DNA from peripheral blood leukocytes and used polymerase chain reaction (PCR), with the primer set 409/406,7 to amplify a DNA segment that contains the CTG repeats at the 3′ end of the DM-kinase gene. Nested PCR using the primer set 409/4107 was performed to create a shorter product for proper sizing. The PCR analyses were performed in a 30-µL PCR mix containing 1× GeneAmp PCR buffer (Applied Biosystems, Foster City, Calif), 200 µmol/L of each dNTP (deoxyribonucleic acid), 25 pmol of each primer, 1 µg of genomic DNA, and 2 U of AmpliTaq DNA polymerase (Applied Biosystems). Tubes were heated to 95°C for 5 minutes, and 32 PCR cycles were started as follows: 95°C for 1 minute, 64°C for 1 minute, and 72°C for 1.5 minutes, followed by a final extension step of 72°C for 5 minutes. For nested PCR, we used 1 µL of the first-round PCR product in a reaction with the same previous conditions. The nested PCR products were resolved on 2% Nusieve (Karlan Research Products Corp, Santa Rosa, Calif) agarose gel electrophoresis alongside a 25–base pair ladder and PCR products that contain known CTG repeat lengths detected by sequencing. For proper sizing of the PCR products, we analyzed gel photographs by means of the GeneLine GeneTools software program (version 3.00.22; Spectronics Corporation, Westbury, NY).

RESULTS

We analyzed 370 chromosomes for CTG repeat variability. The frequencies of each (CTG)n allele size in Kuwaiti population are shown in Figure 1. A total of 17 (CTG)n alleles were found, with a range of 5 to 37 repeats. A bimodal distribution of allele sizes with peaks at 5 and at 10 to 13 repeats is shown. The (CTG)5 allele was the most frequent single allele (100 of 370 [27.0%]), whereas the (CTG)10-13 was the most frequent class of alleles (161 [43.5%]). No chromosome was found with 9, 18, or 19 repeats. Except for the occurrence of 1 chromosome with 37 repeats, very few chromosomes (13 [3.5%]) were found with a repeat size of greater than 18.

Figure 2 shows the frequency of various (CTG)nrepeats of this study compared with that of European11 and black South African populations.20 Results of χ2 analysis of the overall distribution of the alleles showed highly significant differences between a Kuwaiti population and black South African (P<.01) and European populations (P<.001).

With 18 repeats designated as the cutoff point, the (CTG)n repeats were grouped into 3 classes (5, 10-18, and >18) (Table 1). Results of χ2 analysis of these data showed a statistically significant lower frequency of greater than 18 alleles in the Kuwaiti compared with the European population (χ2 = 12.7; P<.001). The (CTG)>18 alleles were significantly more frequent in the Kuwaiti compared with the black South African population (χ2 = 8.8; P = .003). Regarding the (CTG)>18 alleles, no significant difference was observed between the Kuwaiti and European populations (χ2 = 0.83; P = .36), whereas a statistically significant lower frequency of this allele group was seen in the Kuwaiti population compared with the black South African population (χ2 = 8.2; P = .004). In the studied Kuwaiti population, the prevalence of the (CTG)5 allele was shown to be not statistically different from that seen in the black South African population (χ2 = 0.01; P = .90), but was significantly lower than that seen in the European population (χ2 = 6.08; P = .01).

COMMENT

Myotonic dystrophy is an autosomal dominant neurological disease with a globally variable disease incidence. This disease is very rare in the Kuwaiti population, and its clinical and genetic features have not yet been documented in this population. Therefore, we undertook a study of the CTG trinucleotide repeat in healthy individuals of the Kuwaiti population in an attempt to explain the apparently rare incidence of the disease and as a step toward more extensive study of the disease in our population.

The CTG allele distribution in the Kuwaiti population showed a bimodal distribution of allele sizes, with the (CTG)5 allele as the most frequent (27%), whereas (CTG)10-13 constituted the most frequent class of alleles (43.5%). These data are in accordance with those observed in European,11 Japanese,18 black South African,20 and most human populations as concluded by Tishkoff et al.21

The factors that affect trinculeotide repeat stability, normal allelic variation, and the generation of new disease alleles are not fully understood. Haplotype analysis of DM chromosomes suggested a single origin (founder chromosome) for the trinucleotide repeat expansion.4,8,22,23 This finding was unexpected for DM, a dominant disease that in its severe forms diminishes or abolishes reproductive fitness. Such a disease in general would be characterized by a high level of new mutations that compensate for the loss of abnormal alleles due to the decreased fitness. The most accepted hypothesis to explain this finding was suggested by Imbert et al.11 They proposed that the initial predisposing event(s) leading to the formation of the DM chromosomes consisted of a limited number of duplication steps of a (CTG)5 allele that resulted in generation of (CTG)≥19. The heterogeneous class of (CTG)19-30 alleles, which was found to have an overall frequency of about 10%, may constitute a reservoir for recurrent DM mutations. This hypothesis was supported by the extensive study of Martorell et al.24 They concluded that premutation alleles could not be the long-term source of new DM families, which must ultimately arise from mutations of alleles within the upper-normal size range. The hypothesis was also supported by the finding of Meiner et al,25 who observed an increase of 11 repeats on a paternally transmitted 37 repeats in a non-DM family. Using the 18 repeats as the cutoff point, our results showed a statistically significant lower frequency of (CTG)>18alleles in the Kuwaiti population compared with the European population (χ2 = 12.7; P<.001). This result agrees with those of previous studies in different ethnic groups, which demonstrated a correlation between the frequency of large normal CTG alleles (>18 repeats) and the prevalence of DM in a population.20,21,2629

In conclusion, the low frequency of DM among Kuwaiti individuals is mostly due to the lower prevalence of the (CTG)>18 alleles in this population. Further study of healthy families within the high-normal repeat range is in progress to investigate the possible instability of the (CTG)>18 alleles in our area.

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Article Information

Corresponding author and reprints: Suad Alfadhli, Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences, Kuwait University, PO Box 31470, Sulaibekhat, Kuwait (s.alfadhli@hsc.kuniv.edu.kw).

Accepted for publication January 28, 2004.

Author contributions: Study concept and design (Drs Alfadhli and Elshafey); acquisition of data (Drs Alfadhli and Al-Awadi); analysis and interpretation of data (Drs Alfadhli, Elshafey, and Bastaki); drafting of the manuscript (Dr Alfadhli); critical revision of the manuscript for important intellectual content (Drs Alfadhli, Elshafey, Bastaki, and Al-Awadi); statistical expertise (Drs Alfadhli and Elshafey); obtained funding (Dr Alfadhli); administrative, technical, and material support (Dr Alfadhli); study supervision (Drs Alfadhli and Al-Awadi).

This study was supported by grant NM00/01 from Kuwait University, Sulaibekhat.

References
1.
Harper  PS The molecular dystrophies.  In: Scriver  CR, Beaudet  AL, Sly  WS, Valle  D, eds. Metabolic Basis of Inherited Disease.6th ed. New York, NY: McGraw-Hill Co; 1989.
2.
Meiner  AWolf  CCarey  N  et al Direct molecular analysis of myotonic dystrophy in the German population: important considerations in genetic counselling. J Med Genet.1995;32:645-649.
PubMed
3.
Harper  PS Myotonic Dystrophy. 2nd ed. Philadelphia, Pa: WB Saunders Co; 1989.
4.
Harley  HGBrook  JDRundle  SA Expansion of unstable DNA region and phenotypic variation in myotonic dystrophy. Nature.1992;355:545-546.
PubMed
5.
Buxton  JShelbourne  PDavies  J  et al Detection of an unstable fragment of DNA specific to individuals with myotonic dystrophy. Nature.1992;355:547-548.
PubMed
6.
Aslanidis  CJansen  GAmemiya  C  et al Cloning of the essential myotonic dystrophy region and mapping of the putative defect. Nature.1992;355:548-551.
PubMed
7.
Brook  JDMcCurrach  MEHarley  HG  et al Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family member. Cell.1992;68:799-808.
PubMed
8.
Mahadevan  MTsilfidis  CSabourin  L  et al Myotonic dystrophy mutation: an unstable CTG repeat in the 3′ untranslated region of the gene. Science.1992;255:1253-1255.
PubMed
9.
Fu  YHPizzuti  AFenwick Jr  RG  et al An unstable triplet repeat in a gene related to myotonic muscular dystrophy. Science.1992;255:1256-1258.
PubMed
10.
Brunner  HGNillesen  Wvan Oost  BA  et al Presymptomatic diagnosis of myotonic dystrophy. J Med Genet.1992;29:780-784.
PubMed
11.
Imbert  GKretz  CJohnson  KMandel  JL Origin of the expansion mutation in myotonic dystrophy. Nat Genet.1993;4:72-76.
PubMed
12.
Chakraborty  RStivers  DNDeka  RYu  LMShriver  MDFerrell  RE Segregation distortion of the CTG repeats at the myotonic dystrophy locus. Am J Hum Genet.1996;59:109-118.
PubMed
13.
Watkins  WSBamshad  MJorde  LB Population genetics of trinucleotide repeat polymorphisms. Hum Mol Genet.1995;4:1485-1491.
PubMed
14.
Chung  MYRanum  LPDuvick  LAServadio  AZoghbi  HYOrr  HT Evidence for a mechanism predisposing to intergenerational CAG repeat instability in spinocerebellar ataxia type I. Nat Genet.1993;5:254-258.
PubMed
15.
Hirst  MCGrewal  PKDavies  KE Precursor arrays for triplet repeat expansion at the fragile X locus. Hum Mol Genet.1994;3:1553-1560.
PubMed
16.
Leeflang  EPArnheim  N A novel repeat structure at the myotonic dystrophy locus in a 37 repeat allele with unexpectedly high stability. Hum Mol Genet.1995;4:135-136.
PubMed
17.
Ashizawa  TEpstein  HF Ethnic distribution of the myotonic dystrophy gene. Lancet.1991;338:642-643.
PubMed
18.
Davies  JYamagata  HShelbourne  P  et al Comparison of the myotonic dystrophy associated CTG repeat in European and Japanese populations. J Med Genet.1992;29:766-769.
PubMed
19.
Krahe  REckhart  MOgunniyi  AOOsuntokun  BOSiciliano  MJAshizawa  T De novo myotonic dystrophy mutation in a Nigerian kindred. Am J Hum Genet.1995;56:1067-1074.
PubMed
20.
Goldman  ARamsay  MJenkins  T Absence of myotonic dystrophy in southern African Negroids is associated with a significantly lower number of CTG trinucleotide repeats. J Med Genet.1994;31:37-40.
PubMed
21.
Tishkoff  SAGoldman  ACalafell  F  et al A global haplotype analysis of the myotonic dystrophy locus: implications for the evolution of modern humans and for the origin of myotonic dystrophy mutations. Am J Hum Genet.1998;62:1389-1402.
PubMed
22.
Yamagata  HMiki  TOgihara  T  et al Expansion of unstable DNA region in Japanese myotonic dystrophy patients [letter]. Lancet.1992;339:692.
PubMed
23.
Lavedan  CHofmann-Radvanyi  HBoileau  C  et al French myotonic dystrophy families show expansion of a CTG repeat in complete linkage disequilibrium with an intragenic 1 kb insertion. J Med Genet.1994;31:33-36.
PubMed
24.
Martorell  LMonkton  DGSanchez  ALopez De Munain  ABaiget  M Frequency and stability of the myotonic dystrophy type 1 premutation. Neurology.2001;56:328-335.
PubMed
25.
Meiner  AThamm  BStrenge  SFroster  U Instability in the normal CTG repeat range at the myotonic dystrophy locus [letter]. J Med Genet.1998;35:791.
PubMed
26.
Zerylnick  CTorroni  ASherman  SLWarren  ST Normal variation at the myotonic dystrophy locus in global human populations. Am J Hum Genet.1995;56:123-130.
PubMed
27.
Goldman  ARamsay  MJenkins  T Ethnicity and myotonic dystrophy: a possible explanation for its absence in sub-Saharan Africa. Ann Hum Genet.1996;60:57-65.
PubMed
28.
Mor-Cohen  RMagal  NGadoth  NShohat  TShohat  M Correlation between the incidence of myotonic dystrophy in different groups in Israel and the number of CTG trinucleotide repeats in the myotonin gene. Am J Med Genet.1997;71:156-159.
PubMed
29.
Culjkovic  BStojkovic  OVukosavic  S  et al CTG repeat polymorphism in DMPK gene in health Yugoslav population. Acta Neurol Scand.2002;105:55-58.
PubMed
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