A GHEP-ISFG collaborative study on the genetic variation of 38 autosomal indels for human identification in different continental populations.

A collaborative effort was carried out by the Spanish and Portuguese Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) to promote knowledge exchange between associate laboratories interested in the implementation of indel-based methodologies and build allele frequency databases of 38 indels for forensic applications. These databases include populations from different countries that are relevant for identification and kinship investigations undertaken by the participating laboratories. Before compiling population data, participants were asked to type the 38 indels in blind samples from annual GHEP-ISFG proficiency tests, using an amplification protocol previously described. Only laboratories that reported correct results contributed with population data to this study. A total of 5839 samples were genotyped from 45 different populations from Africa, America, East Asia, Europe and Middle East. Population differentiation analysis showed significant differences between most populations studied from Africa and America, as well as between two Asian populations from China and East Timor. Low FST values were detected among most European populations. Overall diversities and parameters of forensic efficiency were high in populations from all continents.

Autosomal SNPs study of a population sample from north of Portugal and a sample of immigrants from the Eastern Europe living in Portugal.

The use of autosomal single nucleotide polymorphisms (SNPs) for forensic research has been widely discussed in recent years, mainly because SNPs have important advantages compared to short tandem repeats (STRs). In this study a total of 131 non related individuals from the North of Portugal and 85 immigrant individuals from the Eastern Europe, mainly Ukrainians, equally non related and residing in Portugal, were typed for 52 loci included in the in the SNP for ID 52plex with the SNaPshot™ assay.

The GHEP-EMPOP collaboration on mtDNA population data--A new resource for forensic casework.

Mitochondrial DNA (mtDNA) population data for forensic purposes are still scarce for some populations, which may limit the evaluation of forensic evidence especially when the rarity of a haplotype needs to be determined in a database search. In order to improve the collection of mtDNA lineages from the Iberian and South American subcontinents, we here report the results of a collaborative study involving nine laboratories from the Spanish and Portuguese Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) and EMPOP. The individual laboratories contributed population data that were generated throughout the past 10 years, but in the majority of cases have not been made available to the scientific community. A total of 1019 haplotypes from Iberia (Basque Country, 2 general Spanish populations, 2 North and 1 Central Portugal populations), and Latin America (3 populations from São Paulo) were collected, reviewed and harmonized according to defined EMPOP criteria. The majority of data ambiguities that were found during the reviewing process (41 in total) were transcription errors confirming that the documentation process is still the most error-prone stage in reporting mtDNA population data, especially when performed manually. This GHEP-EMPOP collaboration has significantly improved the quality of the individual mtDNA datasets and adds mtDNA population data as valuable resource to the EMPOP database (www.empop.org).

2006 GEP-ISFG collaborative exercise on mtDNA: reflections about interpretation, artefacts, and DNA mixtures.

We report the results of the seventh edition of the GEP-ISFG mitochondrial DNA (mtDNA) collaborative exercise. The samples submitted to the participant laboratories were blood stains from a maternity case and simulated forensic samples, including a case of mixture. The success rate for the blood stains was moderate ( approximately 77%); even though four inexperienced laboratories concentrated about one-third of the total errors. A similar success was obtained for the analysis of mixed samples (78.8% for a hair-saliva mixture and 69.2% for a saliva-saliva mixture). Two laboratories also dissected the haplotypes contributing to the saliva-saliva mixture. Most of the errors were due to reading problems and misinterpretation of electropherograms, demonstrating once more that the lack of a solid devised experimental approach is the main cause of error in mtDNA testing.

Association of insulin resistance and nocturnal fall of blood pressure in GH-deficient adults during GH replacement.

The GH deficiency syndrome in adults is characterized by changes in body composition, metabolic, cardiovascular and psychological profile. Such alterations fit the metabolic syndrome. Changes of blood pressure (BP) levels related to the presence of insulin resistance (IR) may be present in the GH-deficient adult prior to or after therapy with recombinant GH (hGH). The purpose of the study was to assess the relationship between BP and IR in GH-deficient adults after 24 months of replacement with hGH. Thirteen GH-deficient adults were studied [7 men and 6 women, with an average age of 38.6+/-14.14 yr body mass index (BMI) 25.83+/-2.26 kg/m2]. The BP was assessed by means of ambulatory monitoring of BP (AMBP), prior to the treatment and 12 and 24 months after replacement with hGH. Glucose metabolism was assessed by the homeostatic model assessment (HOMA), during the same periods. The average dosage of hGH utilized was 0.67+/-0.15 mg/day. In the analysis of BP levels, we observed a decrease of the diurnal systolic BP (SB P) (p=0.043) and a reduction of the diurnal systolic (p=0.002) and diastolic pressure loads (p=0.038). During the night there were no changes in BP levels. We observed an increase in the percentage of patients with a non-physiological nocturnal fall (non dippers) after replacement with hGH (61.53%). The mean HOMA, insulin and glucose in the fasting state did not present any statistically significant changes. Although the patients within the nondipper group had higher HOMA and insulin levels throughout the study, there were no changes in any of these parameters after GH replacement. All patients with HOMA >2.5 were within the non-dipper group, whereas all dippers had HOMA <2.5. In conclusion, 24 months of therapy with hGH do not seem to have affected glucose homeostasis, and since there is no relationship with the increase of the percentage of non-physiological nocturnal fall, we will need a longer observation time to discover the effects of this finding.

Analysis of body fluid mixtures by mtDNA sequencing: An inter-laboratory study of the GEP-ISFG working group.

The mitochondrial DNA (mtDNA) working group of the GEP-ISFG (Spanish and Portuguese Group of the International Society for Forensic Genetics) carried out an inter-laboratory exercise consisting of the analysis of mtDNA sequencing patterns in mixed stains (saliva/semen and blood/semen). Mixtures were prepared with saliva or blood from a female donor and three different semen dilutions (pure, 1:10 and 1:20) in order to simulate forensic casework. All labs extracted the DNA by preferential lysis and amplified and sequenced the first mtDNA hypervariable region (HVS-I). Autosomal and Y-STR markers were also analysed in order to compare nuclear and mitochondrial results from the same DNA extracts. A mixed stain prepared using semen from a vasectomized individual was also analysed. The results were reasonably consistent among labs for the first fractions but not for the second ones, for which some laboratories reported contamination problems. In the first fractions, both the female and male haplotypes were generally detected in those samples prepared with undiluted semen. In contrast, most of the mixtures prepared with diluted semen only yielded the female haplotype, suggesting that the mtDNA copy number per cell is smaller in semen than in saliva or blood. Although the detection level of the male component decreased in accordance with the degree of semen dilution, it was found that the loss of signal was not consistently uniform throughout each electropherogram. Moreover, differences between mixtures prepared from different donors and different body fluids were also observed. We conclude that the particular characteristics of each mixed stain can deeply influence the interpretation of the mtDNA evidence in forensic mixtures (leading in some cases to false exclusions). In this sense, the implementation of preliminary tests with the aim of identifying the fluids involved in the mixture is an essential tool. In addition, in order to prevent incorrect conclusions in the interpretation of electropherograms we strongly recommend: (i) the use of additional sequencing primers to confirm the sequencing results and (ii) interpreting the results to the light of the phylogenetic perspective.

Allele frequencies and population data for 17 Y-STR loci (AmpFlSTR Y-filer) in a Northern Portuguese population sample.

Allele frequencies and population data for 17 Y-STR loci included in a new commercial kit that has recently been available, the AmpFlSTR Y-filer PCR amplification kit (Applied Biosystems), that permits the simultaneous amplification of all the markers included in the actually used European "extended haplotype", DYS19, DYS189I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385I/II, DYS438, DYS439 and also DYS437, DYS448, DYS456, DYS458, DYS635 and Y GATA H4, were obtained from a sample of 175 healthy unrelated males and 45 father-son pairs from the North of Portugal. A total of 171 haplotypes were identified, of which 167 were unique and 4 were found in 2 individuals. The haplotype diversity (99.97%) and discrimination capacity (95.43%) were calculated. We report some non-standard situations, such as allele duplications and mutations. We also report a case of disputed paternity in which duplicated alleles plus an inconsistency of the transmitted alleles appeared.

Mutation rates at Y chromosome specific microsatellites.

A collaborative work was carried out by the Spanish and Portuguese ISFG Working Group (GEP-ISFG) to estimate Y-STR mutation rates. Seventeen Y chromosome STR loci (DYS19, DYS385, DYS389I and II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS460, DYS461, DYS635 [GATA C4], GATA H4, and GATA A10) were analyzed in a sample of 3,026 father/son pairs. Among 27,029 allele transfers, 54 mutations were observed, with an overall mutation rate across the 17 loci of 1.998 x 10(-3) (95% CI, 1.501 x 10(-3) to 2.606 x 10(-3)). With just one exception, all of the mutations were single-step, and they were observed only once per gametogenesis. Repeat gains were more frequent than losses, longer alleles were found to be more mutable, and the mutation rate seemed to increase with the father's age. Hum Mutat 26(6), 520-528, 2005. (c) 2005 Wiley-Liss, Inc.

Mitochondrial DNA error prophylaxis: assessing the causes of errors in the GEP'02-03 proficiency testing trial.

We report the results of the Spanish and Portuguese working group (GEP) of the International Society for Forensic Genetics (ISFG) Collaborative Exercise 2002-2003 on mitochondrial DNA (mtDNA) analysis. Six different samples were submitted to the participating laboratories: four blood stains (M1-M2-M3-M4), one mixture blood sample (M5), and two hair shaft fragments (M6). Most of the labs reported consensus results for the blood stains, slightly improving the results of previous collaborative exercises. Although hair shaft analysis is still carried out by a small number of laboratories, this analysis yielded a high rate of success. On the contrary, the analysis of the mixture blood stain (M5) yielded a lower rate of success; in spite of this, the whole results on M5 typing demonstrated the suitability of mtDNA analysis in mixture samples. We have found that edition errors are among the most common mistakes reported by the different labs. In addition, we have detected contamination events as well as other minor problems, i.e. lack of standarization in nomenclature for punctual and length heteroplasmies, and indels. In the present edition of the GEP-ISFG exercise we have paid special attention to the visual phylogenetic inspection for detecting common sequencing errors.

Allele frequencies of sixteen STRs in the population of Northern Portugal.

Allele frequencies of sixteen autossomal short tandem repeats (STRs), D3S1358, VWA, D16S539, D8S1179, D21S11, D18S51, TH01, FGA, D5S818, D13S317, D7S820, TPOX, CSF1PO, Penta D, Penta E (included in the PowerPlex 16 kit), and the SE33 (PowerPlex ES Monoplex System SE33) were determined in a sample of 200 healthy unrelated individuals from the north of Portugal.

Allele distribution of 15 PCR-based loci in the Rwanda Tutsi population by multiplex amplification and capillary electrophoresis.

A genetic study of 15 autosomal STRs is carried out (D2S1338, D3S1358, D5S818, D7S820, D8S1 79, D13S317, D16S359, D18S51, D19S433, D21S11, CSF1PO, FGA, TPOX, THO1, VWA) in a sample of unrelated Tutsis. The molecular phenotypes were determined by means of multiplex strategies (AmpFlSTR Identifiler PCR Amplification Kit, Applied Biosystems) followed by capillary electrophoresis.

A review of the collaborative exercises on DNA typing of the Spanish and Portuguese ISFH Working Group. International Society for Forensic Haemogenetics.

Since 1992 the Spanish and Portuguese Working Group (GEP) of the International Society for Forensic Haemogenetics (ISFH) has been organizing collaborative exercises on DNA profiling with the aim of making progress on standardization and discussing technical and statistical problems in DNA analysis. A total of four exercises (GEP-92 to GEP-95) have been carried out until now. A consequence of these exercises was the creation of a quality control programme in Spain and Portugal in 1995 which was carried out simultaneously with the GEP-95 exercise. The number of participating laboratories increased from 10 in the first exercise (GEP-92) to 19 in the last exercise (GEP-95). Despite this increasing number of participating laboratories, results remained satisfactory. In the last exercises, all the laboratories used PCR-based DNA polymorphisms with an increasing number of markers obtaining good results. SLPs were used by only 30% of laboratories in the last two exercises but the results indicated a good level of expertise in most of these laboratories. The reasons for these successful results are the common use of the EDNAP protocol for SLP analysis and commercially available kits or common sequenced allelic ladders for PCR-based DNA polymorphisms.

Study of three AMPFLPs (D1S80, 3'ApoB and YNZ22) in the population of the north of Portugal.

Allele and genotype frequencies for D1S80, 3'ApoB and YNZ22 loci have been determined in a population sample of the North of Portugal using the polymerase chain reaction (PCR) amplification and nonradioactive detection. The distribution of genotypes in the three polymorphisms studied is in agreement with expected values according to the Hardy-Weinberg equilibrium. The combined chance of exclusion for the three systems is 0.96, and the combined power of discrimination is 0.99.

Associaçäo entre auto-anticorpos anti-pro-insulina e anti-insulina com diabetes mellitus do tipo I / The association between insulin and proinsulin autoantibodies with tipe I Diabetes Mellitus

O presente trabalho compara a incidência de auto-anticorpos anti-insulina (IAA) e anti-pró-insulina (PAA) em diabéticos do tipo I de início recente e em seus parentes de primeiro grau. Foram estudados 33 indivíduos normais (grupo I), 16 diabéticos do tipo I de início recente (grupo II) e 141 parentes em primeiro grau de diabéticos do tipo I (grupo III). Os IAA e PAA foram determinados pelo método de radioensaio, sendo considerados anormais níveis de IAA acima de 0,584 pmol/L e de PAA acima de 0,441 pmol/L. Näo foram observadas diferenças significantes quanto a idade e sexo entre os 3 grupos. Nos indivíduos normais os níveis de PAA foram significantemente menores do que os de IAA. Entre os diabéticos de início recente foi encontrada uma incidência de IAA de 37,5 por cento e de PAA de 25,0 por cento, näo ocorrendo, entretanto, diferenças significantes entre os níveis destes dois anticorpos. Entre os parentes em primeiro grau a incidência de IAA foi de 3,5 por cento e de PAA de 7,8 por cento, näo ocorrendo também diferenças entre os dois testes. Houve uma correlaçäo significante entre os níveis dos IAA e dos PAA no grupo de diabéticos de início recente (r=0,64; p < 0,05). Os IAA e PAA parecem estar dirigidos contra o mesmo determinante antigênico e, portanto, têm o mesmo valor preditivo para o DM tipo I

Insulin autoantibodies in first degree relatives of type I diabetic patients.

1. Insulin autoantibodies (IAA) of first-degree relatives of type I diabetic patients and recent-onset type I diabetics were measured by radioimmunoassay. A cut-off of 60 nU/ml was established on the basis of the values of normal control individuals. The intra-assay coefficient of variation was 9.2% for a moderately positive serum (1908 +/- 176 nU/ml (mean +/- SD), N = 7; range, 1708 to 2158 nU/ml). The interassay coefficient of variation was 23.8% for a negative (normal control) serum (28.1 +/- 6.7 nU/ml, N = 6; range, 22 to 39 nU/ml) and 14.5% in a highly positive serum (6185 +/- 899 nU/ml, N = 7; range, 5053 to 7009 nU/ml). 2. Insulin autoantibody levels (mean +/- SEM) were 19.3 +/- 2.8 nU/ml (range, -19 to 40 nU/ml) in 25 controls, 24.8 +/- 3.4 nU/ml (range, -17 to 59 nU/ml) in 41 type II diabetic patients, 18.5 +/- 2.4 nU/ml (range, -58 to 268 nU/ml) in 171 first-degree relatives of type I diabetic patients and 208.9 +/- 87.0 nU/ml (range, 10 to 1101 nU/ml) in 16 recent-onset type I diabetic patients. IAA levels were significantly higher in the last group compared with the other groups (P < 0.01). 3. None of the controls or type II diabetics exceeded the upper limit of normality. In contrast, 9 of 171 (5.3%) first-degree relatives and 9 of 16 (56.0%) recent-onset type I diabetic patients had IAA levels above the 60 nU/ml cut-off point.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin autoantibodies in first degree relatives of type I diabetic patients

Insulin autoantibodies (IAA) of first-degree relatives of type diabetic patient and recent-onset type I diabetics were measured by radioimmunoassay. A cut-off of 60 nU/ml was established on the basis of the values of normal control individuals. The intra-assay coefficient of variation was 9.2% for a moderately positive serum (1908 ñ 176 nU/ml (mean ñ SD), N=7; range, 1708 to 2158 nU/ml). The interassay coefficient of variation was 23.8% for a negative (normal control) serum (28.1 ñ 6.7 nU/ml, N=6; range, 22 to 39 nU/ml) and 14.5% in a highly positive serum (6185 ñ 899 nU/ml, N=7; range, 5053 to 7009 nU/ml). Insulin autoantibody levels (mean ñ SEM) were 19.3 ñ 2.8 nU/ml (range, =-19 to 40 nU/ml) in 25 controls, 24.8 ñ 3.4 nU/ml (range, -17 to 59 nU/ml) in 41 type II diabetic patients, 18.5 ñ 2.4 nU/ml (range, -58 to 268 nU/ml) in 171 first-degree relatives of type I diabetic patients and 208.9 ñ 87.0 nU/ml (range, 10 to 1101 nU/ml) in 16 recent-onset type I diabetic patients. IAA levels were significantly higher in the last group compared with the other groups (P<0.01). None of the controls or type II diabetics exceeded the upper limit of normalyity. In contrast, 9 of 171 (5.3%) first-degree relatives and 9 of 16 (56.0%) recent-onset type I diabetic patients had IAA levels above the 60 nU/ml cut-off point. These data indicate that this method is effective for the detection of individuals who are at high risk to develop type I diabetes