Alexis Hernandez

Multiplex PCR and minisequencing of SNPs— a model with 35 Y chromosome SNPs

We have developed a robust single nucleotide polymorphism (SNPs) typing assay with co-amplification of 25 DNA-fragments and the detection of 35 human Y chromosome SNPs. The sizes of the PCR products ranged from 79 to 186 base pairs. PCR primers were designed to have a theoretical Tm of 60 +/- 5 degrees C at a salt concentration of 180 mM. The sizes of the primers ranged from 19 to 34 nucleotides. The concentration of amplification primers was adjusted to obtain balanced amounts of PCR products in 8mM MgCl2. For routine purposes, 1 ng of genomic DNA was amplified and the lower limit was approximately 100 pg DNA. The minisequencing reactions were performed simultaneously for all 35 SNPs with fluorescently labelled dideoxynucleotides. The size of the minisequencing primers ranged from 19 to 106 nucleotides. The minisequencing reactions were analysed by capillary electrophoresis and multicolour fluorescence detection. Female DNA did not influence the results of Y chromosome SNP typing when added in concentrations more than 300 times the concentrations of male DNA. The frequencies of the 35 SNPs were determined in 194 male Danes. The gene diversity of the SNPs ranged from 0.01 to 0.5.

High frequencies of Y chromosome lineages characterized by E3b1, DYS19-11, DYS392-12 in Somali males

We genotyped 45 biallelic markers and 11 STR systems on the Y chromosome in 201 male Somalis. In addition, 65 sub-Saharan Western Africans, 59 Turks and 64 Iraqis were typed for the biallelic Y chromosome markers. In Somalis, 14 Y chromosome haplogroups were identified including E3b1 (77.6%) and K2 (10.4%). The haplogroup E3b1 with the rare DYS19-11 allele (also called the E3b1 cluster γ) was found in 75.1% of male Somalis, and 70.6% of Somali Y chromosomes were E3b1, DYS19-11, DYS392-12, DYS437-14, DYS438-11 and DYS393-13. The haplotype diversity of eight Y-STRs (‘minimal haplotype’) was 0.9575 compared to an average of 0.9974 and 0.9996 in European and Asian populations. In sub-Saharan Western Africans, only four haplogroups were identified. The West African clade E3a was found in 89.2% of the samples and the haplogroup E3b1 was not observed. In Turks, 12 haplogroups were found including J2*(xJ2f2) (27.1%), R1b3*(xR1b3d, R1b3f) (20.3%), E3b3 and R1a1*(xR1a1b) (both 11.9%). In Iraqis, 12 haplogroups were identified including J2*(xJ2f2) (29.7%) and J*(xJ2) (26.6%). The data suggest that the male Somali population is a branch of the East African population – closely related to the Oromos in Ethiopia and North Kenya – with predominant E3b1 cluster γ lineages that were introduced into the Somali population 4000–5000 years ago, and that the Somali male population has approximately 15% Y chromosomes from Eurasia and approximately 5% from sub-Saharan Africa.

X-chromosome SNP analyses in 11 human Mediterranean populations show a high overall genetic homogeneity except in North-west Africans (Moroccans)

BackgroundDue to its history, with a high number of migration events, the Mediterranean basin represents a challenging area for population genetic studies. A large number of genetic studies have been carried out in the Mediterranean area using different markers but no consensus has been reached on the genetic landscape of the Mediterranean populations. In order to further investigate the genetics of the human Mediterranean populations, we typed 894 individuals from 11 Mediterranean populations with 25 single-nucleotide polymorphisms (SNPs) located on the X-chromosome.ResultsA high overall homogeneity was found among the Mediterranean populations except for the population from Morocco, which seemed to differ genetically from the rest of the populations in the Mediterranean area. A very low genetic distance was found between populations in the Middle East and most of the western part of the Mediterranean Sea.A higher migration rate in females versus males was observed by comparing data from X-chromosome, mt-DNA and Y-chromosome SNPs both in the Mediterranean and a wider geographic area.Multilocus association was observed among the 25 SNPs on the X-chromosome in the populations from Ibiza and Cosenza.ConclusionOur results support both the hypothesis of (1) a reduced impact of the Neolithic Wave and more recent migration movements in NW-Africa, and (2) the importance of the Strait of Gibraltar as a geographic barrier. In contrast, the high genetic homogeneity observed in the Mediterranean area could be interpreted as the result of the Neolithic wave caused by a large demic diffusion and/or more recent migration events. A differentiated contribution of males and females to the genetic landscape of the Mediterranean area was observed with a higher migration rate in females than in males. A certain level of background linkage disequilibrium in populations in Ibiza and Cosenza could be attributed to their demographic background.

Forensic analysis of dog (Canis lupus familiaris) mitochondrial DNA sequences: an inter-laboratory study of the GEP-ISFG working group.

A voluntary collaborative exercise aiming at the mitochondrial analysis of canine biological samples was carried out in 2006-2008 by the Non-Human Forensic Genetics Commission of the Spanish and Portuguese Working Group (GEP) of the International Society for Forensic Genetics (ISFG). The participating laboratories were asked to sequence two dog samples (one bloodstain and one hair sample) for the mitochondrial D-loop region comprised between positions 15,372 and 16,083 using suggested primers and PCR conditions, and to compare their results against a reference sequence. Twenty-one participating laboratories reported a total of 67.5% concordant results, 15% non-concordant results, and 17.5% no results. The hair sample analysis presented more difficulty to the participants than the bloodstain analysis, with a high percentage (29%) failing to obtain a result. The high level of participation showed the interest of the community in the analysis of dog forensic samples but the results reveal that crucial methodological issues need to be addressed and further training is required in order to respond proficiently to the demands of forensic casework.

Estrogen modulation of adrenoceptor responsiveness in the female rat pineal gland: differential expression of intracellular estrogen receptors.

Abstract:  The effect of different doses of 17β‐estradiol (E2) on the pineal response to β‐adrenoceptor stimulation in female rats was examined. Pinealocytes from 21‐day‐old ovariectomized rats were exposed to different estrogen doses and treated with β‐adrenergic agonists. Estrogen treatment produced a dose‐dependent, biphasic response to β‐adrenoceptor‐induced accumulation of cAMP. This effect was inhibitory at estrogen doses up to 0.1 nm and fitted to a negative exponential curve, while at doses from 0.1 to 100 nm the effect was stimulatory and fitted to a standard positive hyperbola. For in vivo studies, ovariectomized rats were treated with equivalent estrogen concentrations plus a single dose of progesterone (250 μg per rat), and their pineals exposed in vitro to β‐adrenergic agonists. Low doses of E2 (0.1–100 ng per rat) reduced both pineal cAMP accumulation and N‐acetyltransferase activity after β‐adrenoceptor stimulation, while a high dose (10 μg per rat) induced the opposite response. Apparently, the final estrogen target was the pineal β‐adrenergic receptor, as a low dose of E2 (which had diminished cAMP accumulation after β‐adrenoceptor stimulation) also reduced its maximal binding capacity (Bmax) and its dissociation constant (Kd). We also found that the female rat pineal gland contains two different ER subtypes, α and β, which respond to estrogen exposure with nucleocytoplasmic shuttling. These results indicate that, in the female rat, estrogen directly modulates pineal sensitivity to adrenergic stimulation in a complex, dose‐dependent manner that may be related to differential expression and activity of two estrogen receptor subtypes within pineal cells.

Tyrosine hydroxylase activity in peripherally denervated rat pineal gland.

The presence of tyrosine hydroxylase (TH) in the rat pineal gland was studied using a combination of immunochemical and biochemical methods. In superior cervical ganglionectomized (SCGx) animals and in isolated pineals incubated for 72 h, both TH immunoreactive (TH-IR) fibers and TH biochemical activity were still present but reduced. Conversely, in dispersed pinealocytes incubated for only 24 h we were unable to detect either TH activity or TH-positive cells. Since in the pineal gland of intact rats total 3-methoxy-4-hydroxy phenylglycol (MHPG) was undetectable, and only traces of norepinephrine (NE) were present in the pineal of ganglionectomized animals, the results suggest a central pinealopetal catecholaminergic pathway which could use dopamine as a neurotransmitter.

Y chromosome SNP haplogroups in Danes, Greenlanders and Somalis

We have developed a PCR-based assay with co-amplification of 25 DNA fragments and detection of 35 Y chromosome SNPs with the SNaPshot technique. The Y SNP package can define 34 Y chromosome haplogroups and it can identify the majority of the Y chromosome haplogroups of interest in the populations relevant to forensic genetics in Denmark. We typed 194 Danes, 215 Greenlanders, and 201 Somalis, all males. A total of 21 different haplogroups were identified. In Danes, 11 haplogroups with frequencies from 0.5% to 38% were identified and three of the haplogroups, I, R1b*(xR1b1, R1b6, R1b8) and R1a1*(xR1a1b), were found in f91% of the population. In Greenlanders, 10 haplogroups with frequencies from 0.5% to 50% were identified and the haplogroups P*(xQ3a, R1), R1b*(xR1b1, R1b6, R1b8) and I, were found in f86% of the population. In Somalis, 14 haplogroups with frequencies from 0.5% to 79% were identified and the haplogroups E3b1*(xE3b1b) and K*(xN3, O, P) were found in f88% of the population. The distribution of haplogroups was compared to the distribution found in 65 males from West Africa. D 2003 Elsevier B.V. All rights reserved.

A novel geometry for circular series connected multilevel inductors for CMOS RF integrated circuits

The scope of this brief is to introduce a novel geometry for circular series connected multilevel inductors. The idea is to improve the overlapping of the different metal layers that form the integrated inductor to maximize the magnetic flux shared by them and so the inductance. The performance of this new geometry has been compared with the conventional one, using Agilent HFSS field solver. After that, two multilevel inductors using this new geometry have been fabricated in a standard 0.6 /spl mu/m three-metal CMOS process and measured.

GHEP-ISFG collaborative simulated exercise for DVI/MPI: Lessons learned about large-scale profile database comparisons

The GHEP-ISFG Working Group has recognized the importance of assisting DNA laboratories to gain expertise in handling DVI or missing persons identification (MPI) projects which involve the need for large-scale genetic profile comparisons. Eleven laboratories participated in a DNA matching exercise to identify victims from a hypothetical conflict with 193 missing persons. The post mortem database was comprised of 87 skeletal remain profiles from a secondary mass grave displaying a minimal number of 58 individuals with evidence of commingling. The reference database was represented by 286 family reference profiles with diverse pedigrees. The goal of the exercise was to correctly discover re-associations and family matches. The results of direct matching for commingled remains re-associations were correct and fully concordant among all laboratories. However, the kinship analysis for missing persons identifications showed variable results among the participants. There was a group of laboratories with correct, concordant results but nearly half of the others showed discrepant results exhibiting likelihood ratio differences of several degrees of magnitude in some cases. Three main errors were detected: (a) some laboratories did not use the complete reference family genetic data to report the match with the remains, (b) the identity and/or non-identity hypotheses were sometimes wrongly expressed in the likelihood ratio calculations, and (c) many laboratories did not properly evaluate the prior odds for the event. The results suggest that large-scale profile comparisons for DVI or MPI is a challenge for forensic genetics laboratories and the statistical treatment of DNA matching and the Bayesian framework should be better standardized among laboratories.

Species identification in forensic samples using the SPInDel approach: A GHEP-ISFG inter-laboratory collaborative exercise

DNA is a powerful tool available for forensic investigations requiring identification of species. However, it is necessary to develop and validate methods able to produce results in degraded and or low quality DNA samples with the high standards obligatory in forensic research. Here, we describe a voluntary collaborative exercise to test the recently developed Species Identification by Insertions/Deletions (SPInDel) method. The SPInDel kit allows the identification of species by the generation of numeric profiles combining the lengths of six mitochondrial ribosomal RNA (rRNA) gene regions amplified in a single reaction followed by capillary electrophoresis. The exercise was organized during 2014 by a Working Commission of the Spanish and Portuguese-Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG), created in 2013. The 24 participating laboratories from 10 countries were asked to identify the species in 11 DNA samples from previous GHEP-ISFG proficiency tests using a SPInDel primer mix and control samples of the 10 target species. A computer software was also provided to the participants to assist the analyses of the results. All samples were correctly identified by 22 of the 24 laboratories, including samples with low amounts of DNA (hair shafts) and mixtures of saliva and blood. Correct species identifications were obtained in 238 of the 241 (98.8%) reported SPInDel profiles. Two laboratories were responsible for the three cases of misclassifications. The SPInDel was efficient in the identification of species in mixtures considering that only a single laboratory failed to detect a mixture in one sample. This result suggests that SPInDel is a valid method for mixture analyses without the need for DNA sequencing, with the advantage of identifying more than one species in a single reaction. The low frequency of wrong (5.0%) and missing (2.1%) alleles did not interfere with the correct species identification, which demonstrated the advantage of using a method based on the analysis of multiple loci. Overall, the SPInDel method was easily implemented by laboratories using different genotyping platforms, the interpretation of results was straightforward and the SPInDel software was used without any problems. The results of this collaborative exercise indicate that the SPInDel method can be applied successfully in forensic casework investigations.