Rare sequence variation in the genome flanking a short tandem repeat locus can lead to a question of "nonmaternity"

Abstract

Typing of STR (short tandem repeat) alleles is used in a variety of applications in clinical molecular pathology, including evaluations for maternal cell contamination. Using a commercially available STR typing assay for maternal cell contamination performed in conjunction with prenatal diagnostic testing, we were posed with apparent nonmaternity when the two fetal samples did not demonstrate the expected maternal allele at one locus. By designing primers external to the region amplified by the primers from the commercial assay and by performing direct sequencing of the resulting amplicon, we were able to determine that a guanine to adenine sequence variation led to primer mismatch and allele dropout. This explained the apparent null allele shared between the maternal and fetal samples. Therefore, although rare, allele dropout must be considered whenever unexplained homozygosity at an STR locus is observed.

Figure 1

Genomic nucleotide sequence (5′->3′) of the D8S1179 STR and flanking genomic sequence with primer sets used for its amplification. The sequence is from chromosome 8q24.13, GenBank sequence AF216671 (the original sequence clone, GenBank sequence G08710, is similar to the sequence listed, differing only in repeat pattern present). Location of the D8S1179 STR locus is indicated by capitalization. Location of publicly available primer sets is indicated by an underline and in gray (Promega PowerPlex 16 primer set; Promega Corp). The location of our sequencing primer set MCCInvF and MCCInvR is indicated in italics.

Figure 2

AMPF/STR Profiler Plus D8S1179 STR electopherograms for mother and two fetal samples. A: The maternal sample appears homozygous for a 10 STR allele. B: Fetal sample A appears homozygous for a 13 repeat allele. C: Fetal sample B appears homozygous for a 14 repeat allele. The x axis indicates elution time units.

Figure 3

A: PCR products generated by MCCInvF and MCCInvR primers run on a 1.7% agarose gel. (1) Fetal sample A. (2) Fetal sample B. (3) The maternal sample. The presence of two bands in the maternal sample lane indicates amplification of two D8S1179 alleles. Only single thick bands are seen in the fetal samples. The DNA ladder used for sizing is a 50-bp ladder. B: Sequencing data for maternal and fetal D8S1179 alleles. (1) The upper band from the maternal sample. (2) The lower band from the maternal sample. (3) Fetus A. (4) Fetus B. An arrow indicates the location of the G to A sequence change identified 56-bp downstream of the D8S1179 STR locus. This G>A change is present in sequence panels 1, 3, and 4.

Figure 4

Sequence variation in the genomic sequence (5′->3′) flanking the D8S1179 allele leads to allele dropout. Location of the D8S1179 STR locus is indicated by capitalization. Location of the AMPF/STR Profiler Plus primer set is indicated by an underline. The G to A sequence variation at bp 143620 of reference sequence AF216771 (position 147 of reference sequence G08710) found in the genomic sequence flanking the D8S1179 allele and causing allele dropout of the 15 STR allele in our patient's samples is indicated with an arrow.