Y-chromosome evidence of southern origin of the East Asian - Specific haplogroup O3-M122

The prehistoric peopling of East Asia by modern humans remains controversial with respect to early population migrations. Here, we present a systematic sampling and genetic screening of an East Asian-specific Y-chromosome haplogroup (O3-M122) in 2,332 individuals from diverse East Asian populations. Our results indicate that the O3-M122 lineage is dominant in East Asian populations, with an average frequency of 44.3%. The microsatellite data show that the O3-M122 haplotypes in southern East Asia are more diverse than those in northern East Asia, suggesting a southern origin of the O3-M122 mutation. It was estimated that the early northward migration of the O3-M122 lineages in East Asia occurred similar to 25,000-30,000 years ago, consistent with the fossil records of modern humans in East Asia.

A simplified protocol for routine total DNA isolation from salmonid fishes

An efficient total DNA isolation protocol, suitable for routine population genetic screening purposes is described. This phenol based extraction can utilize fresh, frozen or ethanol preserved tissues.

Misfolding of galactose 1-phosphate uridylyltransferase can result in type I galactosemia

Type I galactosemia is a genetic disorder that is caused by the impairment of galactose-1-phosphate uridylyltransferase (GALT; EC 2.7.7.12). Although a large number of mutations have been detected through genetic screening of the human GALT (hGALT) locus, for many it is not known how they cause their effects. The majority of these mutations are missense, with predicted substitutions scattered throughout the enzyme structure and thus causing impairment by other means rather than direct alterations to the active site. To clarify the fundamental, molecular basis of hGALT impairment we studied five disease-associated variants p.D28Y, p.L74P, p.F171S, p.F194L and p.R333G using both a yeast model and purified, recombinant proteins. In a yeast expression system there was a correlation between lysate activity and the ability to rescue growth in the presence of galactose, except for p.R333G. Kinetic analysis of the purified proteins quantified each variant's level of enzymatic impairment and demonstrated that this was largely due to altered substrate binding. Increased surface hydrophobicity, altered thermal stability and changes in proteolytic sensitivity were also detected. Our results demonstrate that hGALT requires a level of flexibility to function optimally and that altered folding is the underlying reason of impairment in all the variants tested here. This indicates that misfolding is a common, molecular basis of hGALT deficiency and suggests the potential of pharmacological chaperones and proteostasis regulators as novel therapeutic approaches for type I galactosemia.

Next generation sequencing-based molecular diagnosis of retinitis pigmentosa: identification of a novel genotype-phenotype correlation and clinical refinements

Retinitis pigmentosa (RP) is a devastating form of retinal degeneration, with significant social and professional consequences. Molecular genetic information is invaluable for an accurate clinical diagnosis of RP due to its high genetic and clinical heterogeneity. Using a gene capture panel that covers 163 of the currently known retinal disease genes, including 48 RP genes, we performed a comprehensive molecular screening in a collection of 123 RP unsettled probands from a wide variety of ethnic backgrounds, including 113 unrelated simplex and 10 autosomal recessive RP (arRP) cases. As a result, 61 mutations were identified in 45 probands, including 38 novel pathogenic alleles. Interestingly, we observed that phenotype and genotype were not in full agreement in 21 probands. Among them, eight probands were clinically reassessed, resulting in refinement of clinical diagnoses for six of these patients. Finally, recessive mutations in CLN3 were identified in five retinal degeneration patients, including four RP probands and one cone-rod dystrophy patient, suggesting that CLN3 is a novel non-syndromic retinal disease gene. Collectively, our results underscore that, due to the high molecular and clinical heterogeneity of RP, comprehensive screening of all retinal disease genes is effective in identifying novel pathogenic mutations and provides an opportunity to discover new genotype-phenotype correlations. Information gained from this genetic screening will directly aid in patient diagnosis, prognosis, and treatment, as well as allowing appropriate family planning and counseling.

Deciphering the molecular role of DJ-1 in the etiology of Parkinson’s and Huntingtons’s disease

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2014

Postpacing abnormal repolarization in catecholaminergic polymorphic ventricular tachycardia associated with a mutation in the cardiac ryanodine receptor gene.

BACKGROUND Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disease for which electrophysiological studies (EPS) have shown to be of limited value.OBJECTIVE This study presents a CPVT family in which marked postpacing repolarization abnormalities during EPS were the only consistent phenotypic manifestation of ryanodine receptor (RyR2) mutation carriers.METHODS The study was prompted by the observation of transient marked QT prolongation preceding initiation of ventricular fibrillation during atrial fibrillation in a boy with a family history of sudden cardiac death (SCD). Family members underwent exercise and pharmacologic electrocardiographic testing with epinephrine, adenosine, and flecainide. Noninvasive clinical test results were normal in 10 patients evaluated, except for both epinephrine- and exercise-induced ventricular arrhythmias in 1. EPS included bursts of ventricular pacing and programmed ventricular extrastimulation reproducing short-long sequences. Genetic screening involved direct sequencing of genes involved in long QT syndrome as well as RyR2.RESULTS Six patients demonstrated a marked increase in QT interval only in the first beat after cessation of ventricular pacing and/or extrastimulation. All 6 patients were found to have a heterozygous missense mutation (M4109R) in RyR2. Two of them, presenting with aborted SCD, also had a second missense mutation (I406T- RyR2). Four family members without RyR2 mutations did not display prominent postpacing QT changes.CONCLUSION M4109R- RyR2 is associated with a high incidence of SCD. The contribution of I406T to the clinical phenotype is unclear. In contrast to exercise testing, marked postpacing repolarization changes in a single beat accurately predicted carriers of M4109R- RyR2 in this family.

Enjeux éthiques et légaux des applications du diagnostic préimplantatoire au Canada

"Mémoire présenté à la Faculté des études supérieures en vue de l'obtention du grade de LLM en maîtrise option recherche axe Droit, Biotechnologies et Société"

Le passé eugénique canadien et ses leçons au regard des nouvelles technologies génétiques

L'eugénisme fascine. L'eugénisme fait peur. D'ailleurs, le spectre de cette idéologie, lié à un passé riche en histoire, revient en force et suscite des craintes de dérives quant à l'avancement des connaissances scientifiques et de leurs applications principalement dans le débat sur le diagnostic préimplantatoire. Alors que nous nous intéressons à son encadrement normatif, une approche historique permet de guider le législateur afin de ne pas répéter des erreurs passées et de mieux en comprendre les enjeux. Or, une loi eugénique a déjà existé au Canada en matière de stérilisation des personnes mentalement handicapées. À la lumière de celle-ci et de son histoire, l'auteur s'interroge sur les leçons que nous pouvons appliquer au diagnostic préimplantatoire, une technologie génétique permettant de sélectionner un enfant créé in vitro en fonction de son profil génétique.

Progress in the genetics of common obesity and type 2 diabetes

The prevalence of obesity and diabetes, which are heritable traits that arise from the interactions of multiple genes and lifestyle factors, continues to rise worldwide, causing serious health problems and imposing a substantial economic burden on societies. For the past 15 years, candidate gene and genome-wide linkage studies have been the main genetic epidemiological approaches to identify genetic loci for obesity and diabetes, yet progress has been slow and success limited. The genome-wide association approach, which has become available in recent years, has dramatically changed the pace of gene discoveries. Genome-wide association is a hypothesis-generating approach that aims to identify new loci associated with the disease or trait of interest. So far, three waves of large-scale genome-wide association studies have identified 19 loci for common obesity and 18 for common type 2 diabetes. Although the combined contribution of these loci to the variation in obesity and diabetes risk is small and their predictive value is typically low, these recently identified loci are set to substantially improve our insights into the pathophysiology of obesity and diabetes. This will require integration of genetic epidemiological methods with functional genomics and proteomics. However, the use of these novel insights for genetic screening and personalised treatment lies some way off in the future.

Comparative Cytogenetic Analysis of the Genus Symphysodon (Discus Fishes, Cichlidae): Chromosomal Characteristics of Retrotransposons and Minor Ribosomal DNA

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular and Cytogenetic Analyses on Brazilian Youths with Pervasive Developmental Disorders

The Pervasive Developmental Disorders (PDDs) constitute a group of behavioral and neurobiological impairment conditions whose main features are delayed communicative and cognitive development. Genetic factors are reportedly associated with PDDs and particular genetic abnormalities are frequently found in specific diagnostic subgroups such as the autism spectrum disorders. This study evaluated cytogenetic and molecular parameters in 30 youths with autism or other PDDs. The fragile X syndrome was the most common genetic abnormality detected, presented by 1 patient with autism and 1 patient with PPD not-otherwise specified (PPD-NOS). One girl with PDD-NOS was found to have tetrasomy for the 15q11-q13 region, and one patient with autism exhibited in 2/100 metaphases an inv(7)(p15q36), thus suggesting a mosaicism 46,XX/46,XX,inv(7)(p15q36) or representing a coincidental finding. The high frequency of chromosomopathies support the hypothesis that PDDs may develop as a consequence to chromosomal abnormalities and justify the cytogenetic and molecular assessment in all patients with PDDs for establishment of diagnosis.

Use of a synthetic lethal screen to identify genes related to TIF51A in Saccharomyces cerevisiae

The putative eukaryotic translation initiation factor 5A (eIF5A) is an essential protein for cell viability and the only cellular protein known to contain the unusual amino acid residue hypusine. eIF5A has been implicated in translation initiation, cell proliferation, nucleocytoplasmic transport, mRNA decay, and actin polarization, but the precise biological function of this protein is not clear. However, eIF5A was recently shown to be directly involved with the translational machinery. A screen for synthetic lethal mutations was carried out with one of the temperature-sensitive alleles of TIF51A (tif51A-3) to identify factors that functionally interact with eIF5A and revealed the essential gene YPT1. This gene encodes a small GTPase, a member of the rab family involved with secretion, acting in the vesicular trafficking between endoplasmatic reticulum and the Golgi. Thus, the synthetic lethality between TIF51A and YPT1 may reveal the connection between translation and the polarized distribution of membrane components, suggesting that these proteins work together in the cell to guarantee proper protein synthesis and secretion necessary for correct bud formation during G1/ S transition. Future studies will investigate the functional interaction between eIF5A and Ypt1 in order to clarify this involvement of eIF5A with vesicular trafficking. ©FUNPEC-RP.

Clinical and molecular phenotype of Aicardi-Goutières syndrome

Aicardi-Goutières syndrome (AGS) is a genetic encephalopathy whose clinical features mimic those of acquired in utero viral infection. AGS exhibits locus heterogeneity, with mutations identified in genes encoding the 3′→5′ exonuclease TREX1 and the three subunits of the RNASEH2 endonuclease complex. To define the molecular spectrum of AGS, we performed mutation screening in patients, from 127 pedigrees, with a clinical diagnosis of the disease. Biallelic mutations in TREX1, RNASEH2A, RNASEH2B, and RNASEH2C were observed in 31, 3, 47, and 18 families, respectively. In five families, we identified an RNASEH2A or RNASEH2B mutation on one allele only. In one child, the disease occurred because of a de novo heterozygous TREX1 mutation. In 22 families, no mutations were found. Null mutations were common in TREX1, although a specific missense mutation was observed frequently in patients from northern Europe. Almost all mutations in RNASEH2A, RNASEH2B, and RNASEH2C were missense. We identified an RNASEH2C founder mutation in 13 Pakistani families. We also collected clinical data from 123 mutation-positive patients. Two clinical presentations could be delineated: an early-onset neonatal form, highly reminiscent of congenital infection seen particularly with TREX1 mutations, and a later-onset presentation, sometimes occurring after several months of normal development and occasionally associated with remarkably preserved neurological function, most frequently due to RNASEH2B mutations. Mortality was correlated with genotype; 34.3% of patients with TREX1, RNASEH2A, and RNASEH2C mutations versus 8.0% RNASEH2B mutation-positive patients were known to have died (P = .001). Our analysis defines the phenotypic spectrum of AGS and suggests a coherent mutation-screening strategy in this heterogeneous disorder. Additionally, our data indicate that at least one further AGS-causing gene remains to be identified. © 2007 by The American Society of Human Genetics. All rights reserved.

Detection of classical 17p11.2 deletions, an atypical deletion and RAI1 alterations in patients with features suggestive of Smith-Magenis syndrome

Smith-Magenis syndrome (SMS) is a complex disorder whose clinical features include mild to severe intellectual disability with speech delay, growth failure, brachycephaly, flat midface, short broad hands, and behavioral problems. SMS is typically caused by a large deletion on 17p11.2 that encompasses multiple genes including the retinoic acid induced 1, RAI1, gene or a mutation in the RAI1 gene. Here we have evaluated 30 patients with suspected SMS and identified SMS-associated classical 17p11.2 deletions in six patients, an atypical deletion of ∼139 kb that partially deletes the RAI1 gene in one patient, and RAI1 gene nonsynonymous alterations of unknown significance in two unrelated patients. The RAI1 mutant proteins showed no significant alterations in molecular weight, subcellular localization and transcriptional activity. Clinical features of patients with or without 17p11.2 deletions and mutations involving the RAI1 gene were compared to identify phenotypes that may be useful in diagnosing patients with SMS. © 2012 Macmillan Publishers Limited All rights reserved.

An Integrative Genomic and Transcriptomic Analysis Reveals Potential Targets Associated with Cell Proliferation in Uterine Leiomyomas

Background: Uterine Leiomyomas (ULs) are the most common benign tumours affecting women of reproductive age. ULs represent a major problem in public health, as they are the main indication for hysterectomy. Approximately 40-50% of ULs have non-random cytogenetic abnormalities, and half of ULs may have copy number alterations (CNAs). Gene expression microarrays studies have demonstrated that cell proliferation genes act in response to growth factors and steroids. However, only a few genes mapping to CNAs regions were found to be associated with ULs. Methodology: We applied an integrative analysis using genomic and transcriptomic data to identify the pathways and molecular markers associated with ULs. Fifty-one fresh frozen specimens were evaluated by array CGH (JISTIC) and gene expression microarrays (SAM). The CONEXIC algorithm was applied to integrate the data. Principal Findings: The integrated analysis identified the top 30 significant genes (P<0.01), which comprised genes associated with cancer, whereas the protein-protein interaction analysis indicated a strong association between FANCA and BRCA1. Functional in silico analysis revealed target molecules for drugs involved in cell proliferation, including FGFR1 and IGFBP5. Transcriptional and protein analyses showed that FGFR1 (P = 0.006 and P<0.01, respectively) and IGFBP5 (P = 0.0002 and P = 0.006, respectively) were up-regulated in the tumours when compared with the adjacent normal myometrium. Conclusions: The integrative genomic and transcriptomic approach indicated that FGFR1 and IGFBP5 amplification, as well as the consequent up-regulation of the protein products, plays an important role in the aetiology of ULs and thus provides data for potential drug therapies development to target genes associated with cellular proliferation in ULs. © 2013 Cirilo et al.