Emiliano Giardina

Mapping a Dominant Form of Multinodular Goiter to Chromosome Xp22

Multinodular goiter (MNG) is a common disorder characterized by a nodular enlargement of the thyroid gland and occurring with a female&rcolon;male ratio of 5&rcolon;1. This article reports the analysis of an Italian three-generation pedigree MNG, including 10 affected females and 2 affected males. After linkage to candidate regions previously implicated in various forms of goiter was excluded, a novel MNG locus was searched. Because no male-to-male transmission was present in the study pedigree, an X-linked autosomal dominant pattern of inheritance was hypothesized. Therefore, 18 markers spaced at 10-cM intervals on the X chromosome were examined. A significant LOD score was observed in the Xp22 region, where marker DXS1226 generated a maximum LOD score of 4.73 at a recombination fraction of 0. Analysis of six flanking microsatellites confirmed these data, and haplotype inspection delimited a 9.6-cM interval lying between DXS1052 and DXS8039.

Full Sequencing of the FLG Gene in Italian Patients with Atopic Eczema: Evidence of New Mutations, but Lack of an Association

TO THE EDITOR Atopic eczema (AE) (OMIM %603165) is the most common chronic inflammatory skin disease, characterized by xerosis, pruritus, and erythematous lesions with increased transepidermal water loss. In recent years, it has been suggested that the epidermal skin barrier has a significant role in AE disease susceptibility and severity (Smith et al., 2006; Cork et al., 2009). Sandilands et al. (2006) demonstrated that null mutations within the filaggrin gene (FLG) strongly predispose individuals to AE. Two FLG null alleles (R501X and 2282del4) have been shown to be significantly associated with AE in several European populations (Palmer et al., 2006; Weidinger et al., 2007). Recently, a meta-analysis of the most common FLG variants in European populations, involving 5,791 eczema cases and 26,454 controls (Rodriguez et al., 2009), revealed that there is a high risk conferred by R501X and 2282del4 across the studies, with an overall odds ratio of 3.14 and 2.78, respectively. Indeed, large differences in carrier frequencies exist across Europe, ranging from 1.4% in an Italian population (Giardina et al., 2008) to 63% in an Irish population (Palmer et al., 2006). Recently, we observed that in Italian patients the frequencies of R501X and 2282del4 are strongly reduced with respect to those described in other patients of European origin, and the frequencies are similar between cases and controls (0.6 vs. 0.0% and 0.9 vs. 0.5%, respectively). In order to determine whether other mutations located elsewhere in FLG confer risk to AE, we performed a full sequencing of FLG in Italian patients. We performed a sequencing of the full FLG gene in a cohort of 220 Italian AE patients (recruited by IDI-Istituto Dermopatico dell’Immacolata and Fatebenefratelli Hospital). We then determined the frequency of variations and mutations in a cohort of 201 healthy subjects. The diagnosis of AE in our case cohort was made by experienced dermatologists or by a pediatric allergologist. The cohort consisted of 85% of cases with the intrinsic subtype and 15% with the extrinsic form of AE. These subtypes and severity of AE have been established based on the total IgE level (extrinsic subtype 4500 ng l ) and using the scoring atopic dermatitis. Further clinical details of Italian patients are available in Abbreviations: AE, atopic eczema; FLG, filaggrin gene; LD, linkage disequilibrium

Deletion of Late Cornified Envelope 3B and 3C Genes Is Not Associated with Atopic Dermatitis

Atopic dermatitis (AD) and psoriasis are common skin diseases characterized by cutaneous inflammation and disturbed epidermal differentiation. Genome-wide analyses have shown overlapping susceptibility loci, such as the epidermal differentiation complex on chromosome 1q21. Recently, a deletion on 1q21 (LCE3C_LCE3B-del), comprising LCE3B and LCE3C, two members of the late cornified envelope (LCE) gene cluster, was found to be associated with psoriasis. Although the mechanistic role of LCE proteins in psoriasis has not been identified, these proteins are putatively involved in skin barrier formation and repair. Considering the potential genetic overlap between the two diseases and the recent finding that mutations in the skin barrier protein filaggrin are associated with AD, we investigated a possible association between LCE3C_LCE3B-del and AD. Evaluation of four different cohorts of European ancestry, containing a total of 1075 AD patients and 1658 controls, did not provide evidence for such an association. Subgroup analysis did not reveal an association with concomitant asthma. Our data suggest that the potential roles of skin barrier defects in the pathogenesis of AD and psoriasis are based on distinct genetic causes.

Identification of multipotent cytotrophoblast cells from human first trimester chorionic villi.

In this article we used immunohistochemistry and FACS analyses to show that cells expressing markers typical of human stem cells such as SSEA4, OCT-4, ALP, and CD117 are present within the cytotrophoblastic tissue of human fetal chorionic villus samples (CVSs). After immunoselection of CV cells for SSEA4, FACS analyses showed an increased number of cells positive for OCT-4 and ALP and a small percentage (around 4%) of side population (SP) cells. In the same cell population, RT-PCR indicated the presence of OCT-4, NANOG, and SOX2 transcripts, also typical of stem cells. Depending on the in vitro conditions, a subset of SSEA4+ cells formed colonies resembling hESCs, with limited self renewal ability. At the same time, these cells were able to differentiate in vitro into derivatives of all three germ layers. When inoculated into immunocompromised mice, SSEA4+ cells did not form teratomas but were able to populate depleted hematopoietic tissues. Moreover, after injection into mouse blastocysts, they were incorporated into the inner cell mass and could be traced into several tissues of the adult chimeric mice. Finally, we show that SSEA4+ cells isolated from fetuses affected by Spinal Muscular Atrophy (SMA) can be genetically corrected with high efficiency in culture by Small Fragment Homologous Recombination (SFHR), a gene targeting approach. Taken together, our results indicate that SSEA4+ cells obtained from human CVSs contain a subpopulation of multipotent cells that we propose to name Human Cytotrophoblastic-derived Multipotent Cells (hCTMCs). These cells may be a safe and convenient source of cells for cell-based therapy, as well as an ideal target for in utero fetal gene therapy.

Whole genome amplification and real-time PCR in forensic casework

BackgroundWGA (Whole Genome Amplification) in forensic genetics can eliminate the technical limitations arising from low amounts of genomic DNA (gDNA). However, it has not been used to date because any amplification bias generated may complicate the interpretation of results. Our aim in this paper was to assess the applicability of MDA to forensic SNP genotyping by performing a comparative analysis of genomic and amplified DNA samples. A 26-SNPs TaqMan panel specifically designed for low copy number (LCN) and/or severely degraded genomic DNA was typed on 100 genomic as well as amplified DNA samples.ResultsAliquots containing 1, 0.1 and 0.01 ng each of 100 DNA samples were typed for a 26-SNPs panel. Similar aliquots of the same DNA samples underwent multiple displacement amplification (MDA) before being typed for the same panel. Genomic DNA samples showed 0% PCR failure rate for all three dilutions, whilst the PCR failure rate of the amplified DNA samples was 0% for the 1 ng and 0.1 ng dilutions and 0.077% for the 0.01 ng dilution. The genotyping results of both the amplified and genomic DNA samples were also compared with reference genotypes of the same samples obtained by direct sequencing. The genomic DNA samples showed genotype concordance rates of 100% for all three dilutions while the concordance rates of the amplified DNA samples were 100% for the 1 ng and 0.1 ng dilutions and 99.923% for the 0.01 ng dilution. Moreover, ten artificially-degraded DNA samples, which gave no results when analyzed by current forensic methods, were also amplified by MDA and genotyped with 100% concordance.ConclusionWe investigated the suitability of MDA material for forensic SNP typing. Comparative analysis of amplified and genomic DNA samples showed that a large number of SNPs could be accurately typed starting from just 0.01 ng of template. We found that the MDA genotyping call and accuracy rates were only slightly lower than those for genomic DNA. Indeed, when 10 pg of input DNA was used in MDA, we obtained 99.923% concordance, indicating a genotyping error rate of 1/1299 (7.7 × 10-4). This is quite similar to the genotyping error rate of STRs used in current forensic analysis. Such efficiency and accuracy of SNP typing of amplified DNA suggest that MDA can also generate large amounts of genome-equivalent DNA from a minimal amount of input DNA. These results show for the first time that MDA material is suitable for SNP-based forensic protocols and in general when samples fail to give interpretable STR results.

The psoriasis genetics as a model of complex disease.

Psoriasis [OMIM*177900] is a common, chronic and papulosquamous inflammatory skin disease affecting approximately 2% of Caucasian. However, this disorder is rare among Japanese, Eskimos, West Africans and North American blacks and very uncommon in North American and South American natives. The causes for these variations are likely to be both genetic and environmental. Population-based studies and twin studies indicate that psoriasis is a heritable disease with a polygenic mode of inheritance with variable penetrance. Independent genome-wide scans have suggested the involvement of a large number of chromosomal regions (loci), and many candidate genes have been proposed. We discuss genetic approaches to the disease, results and interpretations of relevant studies, as well as future perspectives. Understanding the genetic basis of psoriasis will represent a major advance in our understanding of the disease and will reveal novel disease-specific biologic pathways.

Transmission ratio distortion in the spinal muscular atrophy locus Data from 314 prenatal tests

Background: Spinal muscular atrophy (SMA) is a recessive neurodegenerative disorder characterized by the loss of α-motor neurons in the spinal cord and subsequent death of motor neuron cells. SMA occurs with a frequency of 1 in 6,000 live births, with a carrier frequency of 1 in 40, and is a leading genetic cause of infant mortality. SMA is caused by loss or mutation of the telomeric survival motor neuron gene (SMN1), which is deleted in almost 94% of SMA patients Objective: To analyze the transmission ratio at the SMA locus, examining the segregation of the SMN1-deleted alleles in 314 fetuses from carrier parents who requested prenatal testing for the disease. Methods: Prenatal diagnosis of SMA in families at 25% risk of the disease has been performed on chorionic villous sampling specimens, through direct detection of the SMN1 gene mutation and linkage analysis using microsatellite markers from the 5q13 region. Analysis of the genotypic/allelic frequencies of the SMN1 gene was performed using the χ2 test, assuming a recessive mendelian inheritance. Results: Of 314 fetuses analyzed, 95 were homozygous for the wild-type allele (30.3%), 154 were carriers (49.0%), and the remaining 65 were homozygous for the mutated allele (20.7%). Statistical analysis demonstrated that proportion of fetuses predicted with SMA is lower than 25% expected for a recessive disorder, resulting in a transmission rate of the SMN1-deleted allele deviant from the 50% expected in a random the segregation of a mendelian tract (p = 0.016) Conclusions: This is the first study to evaluate the genotypic frequencies at the spinal muscular atrophy (SMA) locus based on data derived from prenatal analysis, which are not subject to ascertainment bias. The analysis showed a transmission ratio distortion at the SMA locus in favor of the SMN1 wild-type alleles.

Generation of Human Induced Pluripotent Stem Cells from Extraembryonic Tissues of Fetuses Affected by Monogenic Diseases

The generation of human induced pluripotent stem cells (hiPSCs) derived from an autologous extraembryonic fetal source is an innovative personalized regenerative technology that can transform own-self cells into embryonic stem-like ones. These cells are regarded as a promising candidate for cell-based therapy, as well as an ideal target for disease modeling and drug discovery. Thus, hiPSCs enable researchers to undertake studies for treating diseases or for future applications of in utero therapy. We used a polycistronic lentiviral vector (hSTEMCCA-loxP) encoding OCT4, SOX2, KLF4, and cMYC genes and containing loxP sites, excisible by Cre recombinase, to reprogram patient-specific fetal cells derived from prenatal diagnosis for several genetic disorders, such as myotonic dystrophy type 1 (DM1), β-thalassemia (β-Thal), lymphedema-distichiasis syndrome (LDS), spinal muscular atrophy (SMA), cystic fibrosis (CF), as well as from wild-type (WT) fetal cells. Because cell types tested to create hiPSCs influence both the reprogramming process efficiency and the kinetics, we used chorionic villus (CV) and amniotic fluid (AF) cells, demonstrating how they represent an ideal cell resource for a more efficient generation of hiPSCs. The successful reprogramming of both CV and AF cells into hiPSCs was confirmed by specific morphological, molecular, and immunocytochemical markers and also by their teratogenic potential when inoculated in vivo. We further demonstrated the stability of reprogrammed cells over 10 and more passages and their capability to differentiate into the three embryonic germ layers, as well as into neural cells. These data suggest that hiPSCs-CV/AF can be considered a valid cellular model to accomplish pathogenesis studies and therapeutic applications.

In silico and in vitro comparative analysis to select, validate and test SNPs for human identification

BackgroundThe recent advances in human genetics have recently provided new insights into phenotypic variation and genome variability. Current forensic DNA techniques involve the search for genetic similarities and differences between biological samples. Consequently the selection of ideal genomic biomarkers for human identification is crucial in order to ensure the highest stability and reproducibility of results.ResultsIn the present study, we selected and validated 24 SNPs which are useful in human identification in 1,040 unrelated samples originating from three different populations (Italian, Benin Gulf and Mongolian). A Rigorous in silico selection of these markers provided a list of SNPs with very constant frequencies across the populations tested as demonstrated by the Fst values. Furthermore, these SNPs also showed a high specificity for the human genome (only 5 SNPs gave positive results when amplified in non-human DNA).ConclusionComparison between in silico and in vitro analysis showed that current SNPs databases can efficiently improve and facilitate the selection of markers because most of the analyses performed (Fst, r2, heterozigosity) in more than 1,000 samples confirmed available population data.

Gonadal mosaicism in hereditary angioedema.

To the Editor: Hereditary angioneurotic edema (HAE; OMIM #106100) is a rare autosomal dominant disorder resulting from the congenital deficiency of functional C1 esterase inhibitor protein (C1INH). Patients affected with HAE clinically are characterized by recurrent episodes of swelling of the extremities, face, trunk, airways or abdominal viscera, occurring either spontaneously or following stress and/or trauma. Laryngeal manifestations are often life threatening (1–3). Affected individuals carry a mutation in the C1-INH gene (C1-INH, SERPING1; OMIM #606860) (4, 5). C1-INH encodes for an esterase, which is the only inhibitor of the first component of the complement system (6). The C1-INH gene maps onto chromosome 11q12-q13.1, and it is organized into eight exons and seven introns (7). A large spectrum of mutations have been described in the C1-INH gene, leading to a failure in secretion or production of C1-INH protein (8, 9). Approximately 25% of these mutations occur de novo (10). We describe here the molecular genetic analysis of a family in which only the sons but not the parents show either clinical or laboratory findings typical of HAE and provide evidence for the first time of gonadic mosaicism in this disease. In 2003, two brothers, aged respectively, 4 and 1 years, were referred to the Department of Rheumatology/Allergology and Clinical Immunology of the University of Rome, Tor Vergata, for the continued presentation of repeated bouts of edema. The first child had his first attack at the age of 2 (in 2001) localized in periorbital region. The attack spontaneously resolved in few hours. In 2002, he had a new severe episode of edema localized at the left foot, which required hospitalization. He was screened for allergies and infectious diseases with no positive result, and neither treatment with steroids and antihistaminics brought any improvement. At the same time, the younger brother manifested flares in periorbital region. We decided to study both children for angioedema assaying C1-INH and C4. C1-INH plasma concentrations were less than 0.048 g/dl in both sibs (normal value 0.15– 0.35 g/dl); functional C1-INH was 17% and 30%, respectively (n.v. 70–130%). C4 levels were 7 and 3 mg/dl, respectively (n.v. 10–40 mg/dl). A critical laryngeal edema episode that occurred in one of the children was successfully treated by administration of C1-INH concentrate, confirming HAE diagnosis. Surprisingly, no clinical and laboratory findings were detected in parents and relatives. Genomic DNA was isolated from peripheral blood lymphocytes, buccal cells, hair roots, and urinary cells. PCR amplification of C1-INH gene was carried out on 200 ng of DNA using primers reported in Table 1 (11). Each amplicon was analyzed using Denaturing High Performance Liquid Chromatography (DHPLC) in a WAVE DNA Fragment Analysis System (Transgenomic Inc., Crewe, UK). Amplicons exhibiting a heterozygous pattern were purified and directly sequenced using an ABI Prism 3100 Genetic