Noninvasive prenatal paternity determination using microhaplotypes: a pilot study.

BACKGROUND: The use of noninvasive techniques to determine paternity prenatally is increasing because it reduces the risks associated with invasive procedures. Current methods, based on SNPs, use the analysis of at least 148 markers, on average. METHODS: To reduce the number of regions, we used microhaplotypes, which are chromosomal segments smaller than 200 bp containing two or more SNPs. Our method employs massively parallel sequencing and analysis of microhaplotypes as genetic markers. We tested 20 microhaplotypes and ascertained that 19 obey Hardy-Weinberg equilibrium and are independent, and data from the 1000 Genomes Project were used for population frequency and simulations. RESULTS: We performed simulations of true and false paternity, using the 1000 Genomes Project data, to confirm if the microhaplotypes could be used as genetic markers. We observed that at least 13 microhaplotypes should be used to decrease the chances of false positives. Then, we applied the method in 31 trios, and it was able to correctly assign the fatherhood in cases where the alleged father was the real father, excluding the inconclusive results. We also cross evaluated the mother-plasma duos with the alleged fathers for false inclusions within our data, and we observed that the use of at least 15 microhaplotypes in real data also decreases the false inclusions. CONCLUSIONS: In this work, we demonstrated that microhaplotypes can be used to determine prenatal paternity by using only 15 regions and with admixtures of DNA.

Comparison of southern Chinese Han and Brazilian Caucasian mutation rates at autosomal short tandem repeat loci used in human forensic genetics.

The short tandem repeat (STR) loci used in human genetic studies are characterized by having relatively high mutation rates. In particular, to ensure an appropriate evaluation of genetic evidence in parentage and forensic analyses, it is essential to have accurate estimates of the mutation rates associated with the commonly used autosomal and sex chromosome STR loci. Differences in STR mutation rates between different ethnic groups should also be determined. Mutation data from two laboratories working with different ethnic groups were extracted from many meiotic transmissions ascertained for 15 autosomal STR loci currently used in forensic routine. Forty-five thousand and eighty-five trios were checked for the biological consistency of maternity and paternity through the analysis of a minimum of 15 loci. Mutations were scored as paternal, maternal, or ambiguous according to the most parsimonious explanation for the inconsistency, using always the least requiring hypothesis in terms of number of repeat differences. The main findings are: (a) the overall mutation rate across the 15 loci was 9.78 × 10(-4) per gamete per generation (95% CI = 9.30 × 10(-4)-1.03 × 10(-3)), and with just 48 (out of 1,587) exceptions, all of the mutations were single-step; (b) repeat gains were more frequent than losses; (c) longer alleles were found to be more mutable; and (d) the mutation rates differ at some loci between the two ethnic groups. Large worldwide meiotic transmission datasets are still needed to measure allele-specific mutation rates at the STR loci consensually used in forensic genetics.

Use of X-linked short tandem repeat loci in routine parentage casework.

BACKGROUND: Dealing with genetic inconsistencies in parentage testing, especially in motherless cases, remains a continual difficulty. STUDY DESIGN AND METHODS: Four difficult cases, comprising two trios and two duos, were selected from routine parentage testing casework. In these, relatively low combined paternity indices were observed as a result of few discrepant loci that were treated as being due to paternal mutations. An additional eight short tandem repeat (STR) loci along the X chromosome were studied in the alleged father and female child to try and help resolve these cases. RESULTS: In all four cases, the X chromosome haplotypes in the alleged father were different from those in the child, showing decisively that the alleged father could be excluded from being the biologic father of the child. CONCLUSION: In recent times the study of X chromosome haplotypes has been shown to be useful in parentage testing where the alleged father is absent and where only his close relatives are available for testing. This work demonstrates that such studies can also prove valuable in the testing of standard trios and duos in cases where there only a few genetic inconsistencies amongst the loci tested, making it difficult to distinguish between paternal mutations and a close relative of the alleged father being the biologic father.

Efficient mispriming during apolipoprotein E genotyping

A determinação do genótipo da apolipoproteína E (ApoE) é um procedimento clinicolaboratorial comum. O método mais freqüentemente utilizado envolve a amplificação por reação em cadeia de polimerase (PCR) de uma região alvo, seguida pela digestão com enzima de restrição do fragmento resultante, obtendo-se, assim, um modelo de fragmentos de restrição genótipo-específico. Nós descrevemos uma dificuldade inesperada que encontramos durante nossa rotina laboratorial.