Validation of six closely linked STRs located in the chromosome X centromere region

Abstract

We propose that clusters of closely linked markers, which segregate as stable haplotypes, provide a high potential to solve complex kinship cases. It is known that the X-chromosomal centromere region shows an extremely low degree of recombination. Hence, we focused our interest on the region between 56 and 64 Mb distant from the Xp telomere and considered 6 STRs which are now registered in the Genome Data Base as DXS10161, DXS10159, DXS10162, DXS10163, DXS10164, and DXS10165. All of these markers show a tetranucleotide or pentanucleotide structure and exhibit high or medium polymorphic information content. As a peculiarity, DXS10163 is a combination of a pentanucleotide STR and an 18 bp INDEL polymorphism. We report here the primer sequences, the repeat structures, the allele distributions and parameters of forensic interest for a German population sample.

Introduction

In recent years, numerous STRs spread over the whole X chromosome (ChrX) have been detected and established for forensic purposes [2, 3, 7, 13–15]. Typing of male DNA enables direct recognition of the ChrX marker haplotypes. Considering pedigrees often provides a way to deduce haplotypes also for females in an indirect manner. For example, ChrX typing of a mother and her son reveals both the male and the maternal haplotypes. Whereas most of the STRs used in the forensic field exhibit allele frequencies in the range of 0.05–0.40, the majority of haplotypes comprising 2–3 STRs exhibit frequencies between 0.001 and 0.02. Hence, in kinship testing, when two (or more) persons share such a rare STR cluster haplotype, there is a strong indication of kinship. Therefore, our group is attracted to the idea to use tightly linked STR clusters, which segregate as stable haplotypes, in kinship testing in complex cases. As has been demonstrated [6, 14, 15], this approach is indeed successful and can be used for solving complex deficiency cases. The usefulness of such STR clusters, among other things, depends on their stability against recombination. Several ChrX-linked STR clusters have been described during recent years. Information on recombination activity of the chromosomal regions has been known for more than 10 years since Nagaraja et al. [12] published an X chromosome recombination map at 75-kb STS resolution. Information on recombination activity between mapped and unmapped markers can be exactly retrieved from the second generation combined linkage-physical map of the human genome of chromosomes [10]. With respect to the susceptibility to recombination, the established clusters map to quite different regions. However, none of them can be regarded as intrinsically free of recombination. STR clusters for usage in ChrX haplotyping are described for the regions Xp22 [7], Xp11.23 [1], Xq12 [6], Xq21 [15], Xq22 [4], Xq26.2 (Rodig et al., unpublished results) and Xq28 [5].

It is well known that recombination occurs extremely rarely around the centromere, and a further low recombination region lies at Xq13.3–Xq21.3 about 76–84 Mb distant from the Xp telomere [8, 9]. The study presented here establishes six ChrX microsatellites which from the theoretical point of view were the most promising STRs in the contigs NT_011669 and NT_011630. All of these markers are located in the centromere region between 56.0 Mb and 64.0 Mb from Xptel. The newly described STRs were registered in the GDB as DXS10161, DXS10159, DXS10162, DXS10163, DXS10164 and DXS10165 (Table 1). DXS10163 represents a combination of a pentanucleotide STR and an INDEL polymorphism. The latter class of markers are diallelic polymorphisms which are systematically described by Mills et al. [11]. These markers seem to be underestimated for use in forensic science and more attention should be paid to them.

Table 1 Sequences and position of the STR primers used in this study

Materials and methods

In this study, we investigated a German population sample of unrelated individuals for the six markers and the number of X chromosomes investigated was 538 at least. These samples were drawn from cases of routine kinship testing concerning female children. The people involved gave their consent for the investigation of STRs of forensic significance. Additionally, we typed the reference cell line DNA samples K562 and 9947A. For sequencing, we selected two to five amplicons of male DNA specimens for every fragment length of each marker. ChrX typing of 354 male DNA specimens directly provided the haplotype data comprising all of the six STR loci. We investigated 109 female meioses with regard to the possible occurrence of crossing-over.

To check for deviation from the Hardy–Weinberg equilibrium (HWE), we determined the genotypes of at least 202 females.

All primer data are shown in Table 1. Primer sequences were established by checking the contigs NT_011669 and NT_011630 (Genome Systems Human BAC Library) and the exact map positions were retrieved using the UCSC in silico PCR tool (http://www.genome.ucsc.edu/).

A special three-primer system was introduced for the amplification of the combined STR-INDEL polymorphism DXS10163. One forward primer was combined with two differently labeled reverse primers: a HEX-labeled primer matching the INDEL deletion allele and a FAM-labeled primer is appropriate for the INDEL insertion allele. Thus, the STR alleles linked to the INDEL short allele occur in the green channel and all STR alleles combined with the INDEL long allele appear in blue.

PCR amplification was carried out in a 15-μl PCR reaction volume containing approximately 0.1–1 ng DNA, 200 μM of each dNTP, 1.5 mM MgCl₂, 0.5 μM of each primer, 1 U Taq polymerase (AmpliTaq-Gold, Applied Biosystems, Foster City, CA) and 1/10 volume of the appropriate Taq polymerase buffer. The following PCR cycle protocol was used: 95°C–10 min soak; 94°C–45 s, 59°C–1 min, 72°C–1 min, 30 cycles, 72°C–10 min final extension in a T3 (Biometra, Göttingen, Germany).

The same conditions were used to generate amplicons for the cycle sequencing procedure.

The resulting PCR products were analyzed in the denaturing polymer POP4 on ABI Prism®310 and ABI Prism®3130 Genetic Analyzers (Applied Biosystems) and the Genotyper and Genemapper software (Applied Biosystems) were used. Amplicon sizing was based on the 550 size standard (Biotype AG, Dresden, Germany).

To analyze the variability of the microsatellite repeat structures and the adjacent regions, we produced appropriate amplicons of male DNA samples using primers given in Table 1 and performed the cycle sequencing procedure. The Big Dye Cycle Sequencing kit (Applied Biosystems) was used as recommended by the manufacturer.

HWE analysis was done using the exact test. Parameters of forensic interest were calculated using formulas as reviewed earlier [14].

Results and discussion

All markers showed robust amplification properties and appear suitable for forensic purposes. Allele sequence structure and nomenclature of the six markers is shown in Tables 2. Five markers exhibited a regular repeat structure. The repeat flanking regions analyzed in this study are in accordance with the GenBank sequences, and no SNPs were detected. DXS10163 is a combined marker consisting of a pentanucleotide STR and an INDEL polymorphism. The INDEL element exhibiting 18 nucleotides (gtttcaaggaatttaccc) begins 16 bp downstream of the repeat region. This situation results in two series of haplotypes. The (long) L-type alleles contain the 18 bp INDEL element and a variable number of STR repeats. The (short) S-type represents the variable STR alleles in combination with the deletion of the INDEL element. As described, a three-primer system using allele specific reverse primers enables a joint analysis of the STR and the INDEL polymorphism revealing the DXS10163 haplotypes.

Table 2 Sequence structure of the ChrX marker alleles

Allele frequencies and statistical parameters of forensic interest are given in Tables 3 and 4. Of the six STRs, five showed high PIC values in the range of 0.628–0.745, solely DXS10164 exhibited a low PIC of 0.421. Nevertheless, this STR may contribute sufficiently to the individualisation of the ChrX in the context of haplotyping the centromere region.

Table 3 Allele frequencies in a German population

Table 4 Statistical parameters

A summary of the features of the STRs and of the cell line DNA typing patterns, which can be used as intralaboratory and interlaboratory standards, is shown in Table 5.

Table 5 STR features and control DNA patterns

Table 6 presents a review of the distribution and the frequencies of haplotypes from our sample of 354 chromosomes of which 72.88% are unique with frequencies lower than 0.003. All haplotypes are given in detail as ESM 1.

Table 6 Distribution of the STR haplotypes DXS10161-DXS10159-DXS10162-DXS10163-DXS10164-DXS10165 in a sample of 354 X chromosomes

In our first family study, we checked 109 female meioses. However, only 61 meioses were informative in DXS10161 and DXS10165, which are the both outer markers of the investigated centromere region. No recombinations were found.

Whether or not this centromere STR cluster is truly free of recombination will become clear in future investigations involving several hundred meioses. With regard to the assumed lack of recombination, this X-chromosomal cluster might provide a counterpart to Y-chromosomal STR haplotypes and may complement the current Y-chromosomal and mitochondrial studies for population genetics and human migration.