M.L. Giovannucci Uzielli

Premature ovarian failure (POF) and fragile X premutation females : From POF to fragile X carrier identification, from fragile X carrier diagnosis to POF association data

Early menopause in the fragile X carriers has been well documented in several reports. All surveys demonstrated that 13-25% of fragile X carriers experienced premature ovarian failure (POF), defined as menopause before the age of 40 years. In 1995 we started screening two groups of subjects as a part of a Fragile X Research Program: 1) women previously diagnosed as fragile X carriers from the register of our center and 2) women with POF and without a family history of fragile X or other forms of mental retardation. In this study we report the preliminary data collected from 75 fragile X families; in 30 of them, POF was present in one or several subjects, all of whom had a fragile X premutation. None of the women with a full mutation experienced POF in our series of patients. We also identified 89 families without a family history of fragile X or mental retardation, and there were 108 subjects who experienced POF, of which 6.5% had a fragile X premutation. This is 70-fold higher than the background prevalence of fragile X premutation in the Italian population and suggests an association with POF. These data confirm the results of other surveys.

Isochromosome not translocation in trisomy 21q21q

SummaryAfter primary trisomy, “de novo” 21q21q trisomy is the most frequent chromosomal aberration responsible for Down syndrome. This rearrangement is more commonly referred to as a Robertsonian translocation or centric fusion product than as an isochromosome, e.g., t(21q;21q) instead of i(21q); however, in practice, it has not so far proved possible to distinguish between these alternatives. The aim of this work was to establish which of the two alternatives is acceptable.

Spectrum and distribution of MECP2 mutations in 64 Italian Rett syndrome girls: tentative genotype/phenotype correlation

We report a direct DNA sequencing analysis of the MECP2 gene undertaken on a further 64 Italian patients with Rett syndrome by using a LICOR 4200 Automated Sequencer. All of the girls entering the study had a consistent clinical diagnosis for this disorder. All coding regions and the flanking intronic splice site sequences were amplified as three non-overlapping fragments by using both forward and reverse primers. The results were then compared to the MECP2 reference sequences published in GenBank. Mutations of the MECP2 gene were identified in 64 of 75 (85.33%) unrelated sporadic Rett syndrome girls. Genotype/phenotype correlation studies, in particular in groups of patients with the same mutation, did not offer definitive and interesting data.

Modified primers for D12S391 and a modified silver staining technique

Abstract In this paper we describe a new primer pair for the short tandem repeat (STR) D12S391 which makes it possible to obtain considerably shorter amplification fragments (125–173 bp), compared to the previously published primers (205–253 bp). The primers were tested on 70 samples with known genotypes, and no differing results were found. In sensitivity studies, forensic casework samples and DNA quality studies, we proved that the new primers can improve the efficiency of the amplification. Moreover, the resolution of this locus on denaturing PAGE followed by silver staining was dramatically improved. This improvement was found to be most valuable for typing the rare .3 variants known for this locus. We also present and propose a new method for silver staining denaturing acrylamide gels.

Y-chromosomal STR haplotype in Toscany (central Italy)

Here we show the Y-haplotype database consisting in the loci DYS19, DYS388, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385, YCAII and DXYS156Y of 107 males living in Toscany (central Italy).

Study on the STR TPOX in an Italian and an Austrian population using two different primer pairs and three different electrophoretic methods.

The short tandem repeat TPOX was studied using two different pairs of primers and three different electrophoretic methods with the aim of optimizing and standardizing the typing conditions for this locus. A genetic population study was subsequently conducted on two population samples from Central Italy (151 individuals) and from Austria (153 individuals) and compared using an R × C contingency table. With the aim of using this system for forensic samples, differences in sensitivity between the methods utilized were studied and several parameters of forensic interest for the two populations (PD, MEC, MEP, pM, PIC) were calculated. A new multiplex system for the loci CSF1PO, TPOX and CD4 is also presented.

The development of two new STR multiplex systems

Abstract Here we show two new amplification systems, which were realised for the amplification of 80 highly polymorphic loci, in combination with an Infrared Automated DNA Sequencer LI-COR 4200 (LI-COR, Nebraska, USA). The two amplification systems were indicated as MU5 and MU6. The amplification system MU5 contained D12S391, F13A01, SE33 and Penta D loci, the amplification system MU6 the loci D2S1338, D19S433, F13B and Penta E. Both systems were performed using forward IRDye™800 labelled and reverse unlabelled primers, in combination with the QIAGEN Multiplex PCR kit (Qiagen, Germany). The extension of the set of STRs in our laboratory is useful to further improve our ability to resolve complex kinship cases (e.g. deficiency cases, incest, paternity tests with mutations). Moreover, these new multiplex systems can also be used for manual typing, because the STRs do not overlap in size.

De novo mutations at D3S1358, D8S1179 and D18S51 loci emerged during paternity testing: confirmation of biological paternal lineage by using a panel of Y-chromosome STRs

Abstract In three paternity tests performed in our laboratories with a battery of autosomal STRs, we discovered three separate incompatibilities for the loci D3S1358, D8S1179 and D18S51. Since the other employed markers confirmed the paternity of the alleged father, the observed incompatibility for these markers was probably due to mutation events. However, taking into account the mutation value for the markers, the paternity index (PI) and posterior probability ( W ) results were very low. In these cases, we normally analyse an extra panel of polymorphic markers to increase the PI value. However, in these three cases, since the disputed children were males and the Y-chromosome is transmitted exclusively to the male lineage, we studied a battery of 10 Y-STR in the children and in the alleged fathers. Evaluation of Y-STR haplotype frequencies was deduced from a European available database.

Fingernail, as an Alternative Source of DNA for Forensic Investigations and Medical Diagnostics A new technique to obtain template DNA in critical situations

The use of PCR (polymerase chain reaction) is having a profound impact on forensic science and medical diagnostics. Limited amounts of biological material and/or degraded low molecular weight DNA can be anticipated for forensic identification and often also for diagnostic investigations in fetuses, stillbirths and children with birth defects. In vitro amplification of DNA, via PCR, represents an important tool for overcoming some of the limitations. Nevertheless, the problem of availability of biological samples as DNA source is often critic. In order to obtain a useful and rapid procedure of DNA analysis in such difficult situations, we have recently developed a simple DNA extraction method using Chelex 100 and a PCR based amplification technique, and using fingernail as an alternative source of DNA. Chelex 100 procedures result in denaturated DNA samples, not suitable for RFLPS analysis. Nevertheless we demonstrated that no differences are detectable between the genotypes obtained by PCR amplification using the conventional phenol-chlorophorm or saline extraction and the Chelex based procedure. A DNA sample of 3-5 micrograms weight was easily obtained from fingernail clippings, and enabled us to perform several tests by using DNA typing methodologies, both for personal identification (in various forensic, medical and social situations) and in disputed parentage. Our laboratory uses as polymorphic markers a set of several VNTRs (variable number of tandem repeats) and, more recently, a series of unlinked STRs (short tandem repeats). The use of DNA typing with STR loci provides an accurate, highly sensitive and rapid assay for parentage testing, forensic identification and medical applications.

Validation of STR system FXIIIB for forensic investigation in a population of Central Italy

Abstract The principle purpose of this study was to investigate the frequency distribution of the short tandem repeat system FXIIIB in a sample of individuals from Tuscany (Central Italy). Samples (204), taken from donors residing in the area of Florence, Prato and Pistoia, were analysed by PCR and vertical denaturing polyacrylamide gel electrophoresis. A sensitivity study was also undertaken and the method was optimised for the study of forensic samples. The research resulted in the identification of 5 alleles and 11 genotypes. The system was tested for Hardy–Weinberg equilibrium and the Central Italian population was compared with populations from Northern Italy and Austria. Moreover, we introduced the use of this microsatellite in an automatic platform, in combination with other STRs in a new multiplex amplification system. An infrared automatic DNA sequencer was used to analyse this marker. The introduction of this marker in our protocol could be useful to resolve particular intriguing cases of forensic interest, as for example incest or deficitary paternity/maternity tests, in which additional loci must be investigated.