Molecular and Cellular Probes (1998) 12, 181–184

Article No. ll980162

Short Communication

More microsatellite markers around D6S105

Jennifer J. Pointon, Caroline Stone,1 Jeremy D. Shearman,2

Alison T. Merryweather-Clarke,1 William M. Rosenberg2 andKathryn J. H. Robson1∗

1MRC Molecular Haematology Unit and 2Nuffield Department of Clinical Medicine,Institute of Molecular Medicine, Headington, Oxford OX3 9DS, UK

(Received 12 November 1997, Accepted 2 March 1998)

KEYWORDS: D6S105, chromosome b, haemochromatosis microsatellite markers.

SOURCE/DESCRIPTION

Genetic haemochromatosis (GH) is an autosomal disequilibrium studies suggested that the HFE genemapped telomeric to D6S1053 and this proved to berecessive disease resulting in inappropriate absorption

of iron from the gut (for review see Bothwell et al.).1 correct with identification of the HFE gene more than4 Mb telomeric to HLA-A.4 To increase the numberGenetic haemochromatosis was mapped close to the

Major Histocompatibility Complex on 6p.2 Linkage of polymorphic markers in the region around D6S105

Table 1. Primer sequences

Primer sequences Annealingtemperature

(°C)

D6S1260 F(5′-ACTGCTCCTGGGGATTG) 64R(5′-GTACATGCCTTTGTTAACATC)

D6S1261 F(5′-CCTATTTATCTCATTGACACCC) 54R(5′-TTGAGACAGCGTCTTACTG)

D6S2252 F(5′-CTAATCTCCAAATGCCTAAG) 55R(5′-GATTTAGAAATGTAGGCCTAG)

D6S2518 F(5′-ACTTCCAAAGGACACAAAGAA) 54R(5′-GTTGTGATATGTGTCTCTGAA)

D6S2519 F(5′-TCCTGGGAACTGATTTGAGA) 54R(5′-CTTTCTTCCCACTTTCTCAC)

D6S2520 F(5′-TTGCAGTGAGCTGAGATTTT) 56R(5′-AGACTCTATAATGCTGTCATAT)

D6S2521 T(5′-CAAAGTGGGGAATCTAGACTT) 60R(5′-CTCAGCTCCCTGTTCTGCTT)

∗ Author to whom all correspondence should be addressed at: MRC Molecular Haematology Unit, Institute of Molecular Medicine,Headley Way, Headington, Oxford OX3 9DS, UK.

0890–8508/98/030181+04 $30.00/0 1998 Academic Press

J. Pointon et al.182

Table 2. Allele frequencies and heterozygosity values for D6S1260, D6S1261, D6S2518,D6S2519, D6S2520, D6S2521 and D6S2252 in unrelated individuals

CA repeat Allele Size (bp) Frequency CEPH controls Reference codea

allele (bp)

D6S1260 1 144 0·015 152/158 K-1340 GM 70562 146 0·015 148/150 K-1341 GM 70343 148 0·1184 150 0·2355 152 0·1476 145 0·2507 156 0·0898 158 0·1039 160 0·015

10 162 0·015Estimated from 34 unrelated individuals, heterozygosity=0·79

D6S1261 1 245 0·105 247/247 K-1340 GM 70562 247 0·7243 249 0·1454 251 0·0135 253 0·013

Estimated from 38 unrelated individuals, heterozygosity=0·50

D6S2518 1 254 0·011 250/252 K-1331 GM 70502 252 0·351 250/254 K-1331 GM 70163 250 0·298 250/254 K-1340 GM 70564 248 0·276 254/254 K-1340 GM 70225 246 0·0216 244 0·0007 242 0·0218 240 0·021

Estimated from 35 unrelated individuals, heterozygosity=0·657

D6S2519 1 116 0·023 128/132 K-1331 GM 70162 118 0·140 118/120 K-1331 GM 70073 120 0·058 136/140 K-1340 GM 70564 122 0·023 126/140 K-1340 GM 70225 124 0·0816 126 0·1867 128 0·1168 130 0·0819 132 0·023

10 134 0·02311 136 0·07012 138 0·04713 140 0·128

Estimated from 42 unrelated individuals, heterozygosity=0·95

D6S2520 1 189 0·010 193/197 K-1331 GM 70162 191 0·010 197/211 K-1331 GM 70073 193 0·271 195/197 K-1340 GM 70224 195 0·063 201/211 K-1340 GM 70565 197 0·2816 199 0·0637 201 0·0948 203 0·0009 205 0·000

10 207 0·00011 209 0·03112 211 0·12513 213 0·03114 215 0·01015 217 0·00016 219 0·00017 221 0·00018 223 0·010

Estimated from 48 unrelated individuals, heterozygosity=0·875

More microsatellite markers around D6S105 183

Table 2—continued

CA repeat Allele Size (bp) Frequency CEPH controls Reference codea

allele (bp)

D6S2521 1 111 0·0372 113 0·0003 115 0·1114 117 0·0185 119 0·0186 121 0·4077 123 0·0568 125 0·2969 127 0·037

10 129 0·018Estimated from 27 unrelated individuals, heterozygosity=0·74

D6S2252 1 138 0·011 154/156 K-1331 GM 70502 140 0·021 150/150 K-1331 GM 70163 142 0·021 146/148 K-1331 GM 73404 144 0·106 142/150 K-1331 GM 70075 146 0·0436 148 0·2877 150 0·1918 152 0·0859 154 0·223

10 156 0·011Estimated from 57 unrelated individuals, heterozygosity=0·789

a The reference code is the Utah family ID code.CEPH, Centre d’Etude du Polymorphism Humaine.

sub-libraries were made from the yeast artificial chro- products were resolved by denaturing gel electro-phoresis.mosomes (YACs) A116C8, K43B3, Q68B5 in Super-

Cos (Stratagene) as described previously5 and fromB117C7 in Lambda Dash (Stratagene). Details re-garding these YACs and the relative positions of the FREQUENCYCA repeats have been described.6 These YACs span theregion containing the markers D6S105 and D6S1558. A total of 11 unique dinucleotide repeat micro-Subclones containing CA repeat microsatellites were satellites were identified and sequenced. Seven of theidentified by hybridization with a (CA)n probe and microsatellites proved to be both potentially in-sequenced after a further round of subcloning into formative and robust in that they amplified well andpUC18. The sequences flanking the CA repeats were could be sized easily. Levels of polymorphism andused to design primers for amplification by the poly- allele frequencies for these loci were tested in un-merase chain reaction (PCR). related individuals of Caucasian and Jamaican des-

cent (Table 2). The majority of the repeat sequencescontain uninterrupted CA repeats with a minimum of

PCR CONDITIONS nine and a maximum of 26 repeat units. Five of themarkers have observed heterozygosity values of above0·7. The microsatellite D6S1261 contains an in-One of each primer pair was end-labelled using

T4 polynucleotide kinase and 33P c ATP (Amersham, terrupted and complex repeat. Alleles of all sevenmarkers were inherited in a Mendelian fashion (dataUK). Amplification by PCR was carried out using

radioactively labelled primers using a Hybaid Om- not shown). Grandparents from CEPH (Centre d’Etudedu Polymorphism Humaine) families were used asnigene thermal cycler for 1 min at 93°C followed

by 30 cycles of annealing temperature×1 min, controls,7 reference numbers 982 (Utah ped. K-1331),984 (Utah ped. K-1340) and 985 (Utah ped. K-1341).72°C×0·1 min and 93°C×0·5 min, with a final

cycle of annealing temperature×1 min and ex- The microsatellite D6S1261 was mapped by Southernblotting and found to lie proximal to D6S105. Thetension at 72°C for 10 min. Radioactively labelled

J. Pointon et al.184

2. Simon, M., Pawlotsky, Y., Bourel, M., Fauchet, R. &remaining microsatellite markers were mapped byGenetet, B. (1975). Hemochromatose idiopathique:YAC fragmentation.6

maladie associee a l’antigene tissulaire HLA–A3?Nouvelle Presse Medicine 4, 1432.

3. Raha-Chowdhury, R., Bowen, D. J., Stone, C. et al.CHROMOSOMAL LOCALIZATION (1995). New polymorphic microsatellite markers place

the haemochromatosis gene telomeric to D6S105.Human Molecular Genetics 4, 1869–74.The localization of these repeats is between D6S306

4. Feder, J. N., Gnirke, A., Thomas, W. et al. (1996). Aand D6S1558 at 6p21.3-p22.1.novel MHC class l-like gene is mutated in patientswith hereditary haemochromatosis. Nature Genetics 13,399–408.ACKNOWLEDGEMENTS 5. Stone, C., Pointon, J. J., Jazwinska, E. C. et al. (1994).Isolation of CA dinucleotide repeats close to D6S105:

The authors would like to acknowledge the inspiration linkage disequilibrium with haemochromatosis. Humanand guidance of Professor Sir David Weatherall. JDS was Molecular Genetics 3, 2043–6.a Wellcome Trust Research Training Fellow. 6. Shearman, J. D., Pointon, J. J., Merryweather-Clarke, A.

T. et al. (1998). Rapid mapping of markers applyingvectorette technology to YAC fragmentation allows easy

REFERENCES assembly of a high density bacterial clone contig span-ning the markers D6S120–D6S1918. Mammalian Gen-ome 9, 220–225.1. Bothwell, T. H., Charlton, R. W. & Motulsky, A. G.

7. Spurr, N. K. (1992). Standards for reporting alleles at(1995). Hemochromatosis. In Secondary Hemo-high polymorphic loci: a proposal. Human Molecularchromatosis (Scriver, C. R., Beaudet, A. L., Sly, W. S. &

Valle, D., eds. Pp. 2237–69. Genetics 1, 211–2.