Microcephaly

Abstract

The 657del5 mutation of the NBS1 gene has been demonstrated in most patients with Nijmegen breakage syndrome (NBS). We identified four Turkish families in which probands were diagnosed as having NBS and found to be homozygous for the 657del5 mutation. The 657del5 allele in the four Turkish families had a single origin.

KEY WORDS: FOUNDER EFFECT, NIJMEGEN BREAKAGE SYNDROME, NBS1, SLAVS, TURKS.

Nijmegen breakage syndrome (NBS) (OMIM 251260) is a rare autosomal recessive disorder characterized by growth retardation, microcephaly, developmental delay, distinctive facial appearance, immunodeficiency, and predisposition to malignancies. Since its first description in 1981 (Weemaes et al. 1981), more than 130 patients have been reported from all continents (Chrzanowska and Janniger 2001). Most previously identified patients have belonged to Slavic populations.

Mutations in the NBS1 gene were first found to be associated with NBS in 1998 (Matsuura et al. 1998; Varon et al. 1998). Almost all the patients of Slavic origin were found to carry a homozygous 5-bp deletion (657del5) in the sixth exon of the NBSl gene (Matsuura et al. 1998; Varon et al. 1998; Chrzanowska and Janniger 2001). A conserved haplotype associated with this deletion was demonstrated, suggesting that this mutation had a single origin in the Slavic populations (Varon et al. 1998). A combined carrier frequency of this deletion in three Slavic countries (Czech Republic, Poland, and Ukraine) was reported to be 1/177 (Varon et al. 2000).

We have recently reported the first Turkish patient with NBS, who was found to be homozygous for the 657del5 mutation (Tekin et al. 2002). In this report we describe the results of mutation screening in more Turkish patients and demonstrate that the mutation has a single origin.

Materials and Methods

Four families in which probands were diagnosed as having NBS were included in this study. Clinical characteristics and the mutation results of one family have been reported previously (Tekin et al. 2002). The other three families originated from two Central Anatolian cities (one each from Konya and Cankiri) and from an Aegean city (Izmir). They were unrelated and were unaware of any ancestral ties to Slavic populations. All probands in these families were phenotypically diagnosed as having NBS on the basis of growth retardation, microcephaly, developmental delay, and facial features in addition to lymphoreticular malignancies. Cytogenetic and immunological investigations also supported the diagnosis.

We initially used a simple PCR with previously described primers (Matsuura et al. 1998) and ran the PCR products on a 7% polyacrylamide gel (Figure 1). We later directly sequenced the samples (with a Beckman Coulter CEQ 2000XL automated sequencer) and found the homozygous deletion using previously described primer pairs for exon 6 (Varon et al. 1998).

We also screened for the 657del5 mutation in 402 Turkish individuals who did not have any health problem related to the NBS1 gene.

To determine whether the four Turkish families with the 657del5 mutation share the same origin for the mutation, we genotyped four flanking microsatellite markers [D8S1800 (1,288,563 bp centromeric to the mutation), D8S88 (134,722 bp centromeric to the mutation), D8S1811 (249,152 bp telomeric to the mutation), and D8S1724 (436,300 bp telomeric to the mutation)]. Standard primers and PCR reactions were used for amplification. PCR products were run in a vertical gel electrophoresis system (Model S2001, Life Technologies) and visualized using silver staining. CEPH family DNAs (1331-01 and 1331-02) were used for sizing. Because we did not have enough DNA for the proband in one family, we typed only the two parents, who were heterozygous, and took only the shared alleles into account because the parents were related.

Fifteen Turkish families with wild-type NBS alleles were also genotyped for the mentioned microsatellite markers.

To compare the haplotype associated with the 657del5 mutation found in the Turkish population with the haplotype detected in the Slavic populations, we used two homozygous DNA samples from Slavic countries. These samples were kindly provided by Raymonda Varon (Institute of Human Genetics, Charité Humboldt University, Germany).

Results

Mutation screening in all four families showed that the affected probands were homozygous and that their parents were heterozygous for the 657del5 mutation (Figure 1).

Results of investigations for the origin of the 657del5 mutation revealed a founder haplotype, 146 bp-90 bp-108 bp-144 bp, for the markers D8S1800, D8S88, D8S1811, and D8S1724, respectively, in all four families. The same haplotype was not detected in 15 Turkish families without the 657del5 mutation. The most similar haplotype observed in only one allele of the 15 control families was 146 bp-90 bp-108 bp-142 bp for the mentioned markers.

When we ran the samples from Slavic populations side by side with Turkish homozygotes for microsatellite markers, we observed that the alleles associated with the 657del5 mutation in Turks were identical with those found in two Slavs (Figure 1).

Results of screening for the 657del5 mutation in 402 healthy Turks did not reveal any heterozygotes.

Discussion

Based on these results, it is clear that the 657del5 mutation, reported to be common in Slavic populations, is present in a group of individuals in Turkey. Predominantly lymphoreticular malignancies have been reported in 40-75% of patients with NBS (Chrzanowska and Janniger 2001; International Nijmegen Breakage Syndrome Study Group 2000). Our detection of homozygotes in four unrelated families presenting with a malignancy implies that NBS is still underdiagnosed in Turkey. Absence of a heterozygote among 402 Turks, however, suggests that the mutation is not common in this population.

More interestingly, the 657del5 mutation in Turks shows the same origin as that described in Slavs. This result suggests the presence of population admixture in modern Turkey. Although it is possible that Turks and Slavs had relations in ancient times, because both populations' histories can be traced back to Asia, they had much closer contact during recent centuries when the Ottoman Empire ruled over most of southeastern Europe to the gates of Vienna. All three branches of Slavic populations-eastern (chiefly Russians, Ukrainians, and Belarusians), western (chiefly Poles, Czechs, Slovaks, and Wends, or Sorbs), and southern (chiefly Serbs, Croats, Slovenes, Macedonians, and perhaps Bulgarians)-might have had contacts with Turks during the Ottoman period. Conservation of an approximately 1.7-Mb DNA region in the NBS1 locus in Turks and Slavs further supports the idea that the 657del5 mutation was introduced into the gene pool of the Turkish population relatively recently. Evaluation of more markers in all populations with this mutation will be necessary to make a correct estimation for the age of the 657del5 mutation.

Acknowledgments We are grateful to Raymonda Varon and Andre Reis of the Institute of Human Genetics, Charite Humboldt University, Germany, for providing two homozygous DNA samples. This study was supported by a grant from the Ankara University Biotechnology Institute.

Received 18 November 2004; revision received 21 February 2005.

Literature Cited

Chrzanowska, K. H., and C. K. Janniger. 2001. Nijmegen breakage syndrome. E-Medicine Dermatol. 2:10. Available at http://www.eraedicine.com

International Nijmegen Breakage Syndrome Study Group. 2000. Nijmegen breakage syndrome. Arch. Dis. Child. 82:400-406.

Matsuura, S., H. Tauchi, A. Nakamura et al. 1998. Positional cloning of the gene for Nijmegen breakage syndrome. Nat. Genet. 19:179-181.

Tekin, M., F. Dogu, N. Tacyildiz et al. 2002. 657del5 mutation in the NBS1 gene is associated with Nijmegen breakage syndrome in a Turkish family. Clin. Genet. 62:84-88.

Varon, R., E. Seemanova, K. Chrzanowska et al. 2000. Clinical ascertainment of Nijmegen breakage syndrome (NBS) and prevalence of the major mutation, 657del5, in three Slav populations. Eur. J. Hum. Genet. 8:900-902.

Varon, R., C. Vissinga, M. Platzer et al. 1998. Nibrin, a novel DNA double-strand break repair protein, is mutated in Nijmegen breakage syndrome. Cell 93:467-476.

Weemaes, C. M., T. W. Hustinx, J. M. Scheres et al. 1981. A new chromosomal instability disorder: The Nijmegen breakage syndrome. Acta Paediatr. Scand. 70:557-564.

MUSTAFA TEKIN,1,2 DUYGU AKCAYOZ,1 CANAN UCAR,3 HUSEYIN GULEN,4 AND NEJAT AKAR1,2

1 Division of Pediatric Molecular Genetics, Ankara University School of Medicine, Ankara, Turkey.

2 Biotechnology Institute of Ankara University, Ankara, Turkey.

3 Division of Pediatric Hematology, Selcuk University School of Medicine, Konya, Turkey.

4 Hematology-Oncology Clinics, Dr, Behcet Uz Children's Hospital, Izmir, Turkey.

Human Biology, June 2005, v. 77, no. 3, pp. 393-397.

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