Recomendaciones de calidad para el diagnóstico prenatal molecular / Quality recommenations in molecular prenatal diagnosis

El diagnóstico prenatal molecular consiste en la aplicación de técnicas de genética molecular sobre una muestra fetal obtenida generalmente mediante una técnica invasiva con el objetivo de diagnosticar una determinada enfermedad hereditaria ante un riesgo clínico evidente. Desde que en 1975 se realizara el primer diagnóstico prenatal molecular hasta la fecha, el panorama ha cambiado considerablemente. El descubrimiento y aplicación de nuevas técnicas moleculares, la mejora de las técnicas invasivas con la consiguiente reducción del riesgo de pérdida fetal y, en particular, la gran experiencia adquirida en las unidades multidisciplinaresde genética han convertido al diagnóstico prenatalmolecular en una opción diagnóstica rutinaria con una importante demanda social. Además, el número de enfermedades hereditarias que pueden ser diagnosticadas y el número de mutaciones identificadas en pacientes afectados por enfermedades raras es cada vez mayor, lo que posibilita cada vez más la aplicación de esta opción diagnóstica. Ante esta situación de crecimiento, tanto en cantidad de diagnósticos como en su diversidad y complejidad, y teniendo en cuenta las importantes implicaciones, tanto hereditarias como éticas y sociales, resulta necesario disponer de unos criteriosmínimos de calidad que garanticen un adecuado diagnósticoprenatal y de este modo posibilitar la posterior toma dedecisiones. Estas recomendaciones deben tener en cuentano sólo los aspectos técnicos que rodean el análisis de la muestra de ADN fetal, sino también la planificación previa y la viabilidad de un determinado tipo de diagnóstico, así como la correcta interpretación y comunicación a la pareja de las implicaciones de los resultados obtenidos

The molecular prenatal diagnosis consists in theapplication of molecular genetics techniques on a foetalsample obtained, generally, using an invasive techniqueto diagnose a particular inherited disease when an evident clinical risk is present. Since in 1975 the first molecular prenatal diagnosis was performed up to now, the picture has changed considerably. The discovery and implementation of new molecular techniques, the improving of the invasive techniques with a consequent reduction of the risk for foetal loss and in particular the great experience acquired in the multidisciplinary genetics units have become the molecular prenatal diagnosis in a routine diagnostic option with an important social demand. In addition, the number of inherited diseases that can be diagnosed and the number of mutations identified in patients affected by rare diseases is increasing, enabling more and more the application of this diagnosis option. In this situation of growth, both in quantity of diagnoses as in its diversity and complexity, and taking into account the important implications, not only hereditary but also ethical and social, is necessary to have minimum of quality standards in order to guarantee an adequate prenatal diagnosis and thus enabling the subsequent decision making process. These recommendationsshould take into account, not just the technicalaspects surrounding the analysis of the sample offoetal DNA, but also the planning and the feasibility of a particular type of diagnosis, as well as the correct interpretation and communication of all the results implications to the couple (AU)

Genotype and phenotype analysis of Friedreich's ataxia compound heterozygous patients.

Friedreich's ataxia is caused by mutations in the FRDA gene that encodes frataxin, a nuclear-encoded mitochondrial protein. Most patients are homozygous for the expansion of a GAA triplet repeat within the FRDA gene, but a few patients show compound heterozygosity for a point mutation and the GAA-repeat expansion. We analyzed DNA samples from a cohort of 241 patients with autosomal recessive or isolated spinocerebellar ataxia for the GAA triplet expansion. Patients heterozygous for the GAA expansion were screened for point mutations within the FRDA coding region. Molecular analyses included the single-strand conformation polymorphism analysis, direct sequencing, and linkage analysis with FRDA locus flanking markers. Seven compound heterozygous patients were identified. In four patients, a point mutation that predicts a truncated frataxin was detected. Three of them associated classic early-onset Friedreich's ataxia with an expanded GAA allele greater than 800 repeats. The other patient associated late-onset disease at the age of 29 years with a 350-GAA repeat expansion. In two patients manifesting the classical phenotype, no changes were observed by single-strand conformation polymorphism (SSCP) analysis. Linkage analysis in a family with two children affected by an ataxic syndrome, one of them showing heterozygosity for the GAA expansion, confirmed no linkage to the FRDA locus. Most point mutations in compound heterozygous Friedreich's ataxia patients are null mutations. In the present patients, clinical phenotype seems to be related to the GAA repeat number in the expanded allele. Complete molecular definition in these patients is required for clinical diagnosis and genetic counseling.

Spanish family with Machado-Joseph disease: neurophysiological features and neuropathy study.

OBJECTIVES: We have carried out electrophysiological studies and sural nerve biopsy evaluation in a Spanish family with genetically proven Machado-Joseph disease (SCA3/MJD) phenotype III. PATIENTS AND METHODS: Two symptomatic and other two asymptomatic members of the family were clinically examined. Electrophysiological evaluation included multimodal evoked potentials, quantitative electromyography and nerve conduction studies, and central motor conduction time. We also report neuropathological findings in the sural nerve biopsy in the proband. RESULTS: Analysis of the SCA3/MJD CAG trinucleotide repeat at the ataxin 3 gene in the DNA of the proband and one of his daughters demonstrated an expanded allele of 63 CAG repeat units. Ataxic pursuit was primary disturbed in MJD, followed by gaze evoked nystagmus, hypermetric saccades and glissades. Limitation of vertical and horizontal gaze, impaired sinusoidal vestibulo-ocular reflex and vestibulo-ocular reflex-fixation-suppression, and active and passive optokinetic nistagmus loss appeared at later stages. Evoked potential studies showed multimodal abnormalities. Electrophysiological and sural nerve biopsy findings correspond well to a pattern of both anterior horn and root ganglion cell distal dominant degeneration. Central motor conduction time was normal in our patients up to advanced stages of the disease. CONCLUSIONS: Electrophysiological and neuropathological studies suggested widespread peripheral and central affection in MJD. Repeated application of electrophysiological techniques may prove useful for monitoring disease progress.

Enfermedad de machado-joseph en una familia española: datos neurofisiológicos y estudio de la neuropatía / Spanish family with Machado-Joseph disease: neurophysiological features and neuropathy study

Objetivos: Se han realizado estudios neurofisiológicos y biopsia del nervio sural en una familia española con diagnóstico genético de enfermedad de Machado-Joseph (SCA3/MJD) y fenotipo clínico III. Pacientes y métodos: Se examinaron dos miembros de la familia clínicamente afectados y dos asintomáticos. El estudio neurofisiológico incluyó los siguientes estudios: potenciales evocados multimodales, electromiografía y conducción de nervio, tiempo de conducción motora central y otoneurología. Se realizó biopsia del nervio sural en el caso índice. Resultados: El análisis de la expansión del triplete CAG en el gen de la ataxina 3 de la SCA3/MJD demostró en el ADN del caso índice y de una de sus hijas una expansión alélica de 63 repeticiones. La presencia de un seguimiento ocular atáxico parece ser el primer signo oculomotor de la SCA3/MJD, seguido del nistagmo espontáneo, las sacadas hipermétricas y glissades. La limitación de los movimientos oculares conjugados verticales y horizontales, y las alteraciones del reflejo vestíbulo-ocular en la prueba pendular, de la supresión visual del reflejo vestíbulo-ocular y del nistagmo optocinético (activo y pasivo) se presentan en los estadios posteriores. Los estudios electromiográficos y de conducciones de nervios y los resultados de la biopsia del nervio sural concuerdan con un patrón de afectación degenerativa de las neuronas del asta anterior y del ganglio raquídeo. El tiempo de conducción motora central se mantuvo normal hasta estadios avanzados de la enfermedad. Conclusiones: El resultado de los estudios neurofisiológicos y los hallazgos de la biopsia del nervio sural sugieren una participación extensa del sistema nervioso central y del periférico en la SCA3/MJD. La valoración periódica mediante técnicas neurofisiológicas resulta útil para la monitorización del progreso de esta enfermedad. (AU)

Dnop56, a Drosophila gene homologous to the yeast nucleolar NOP56 gene.

Small nucleolar RNAs (snoRNAs) are trans-acting factors involved in maturation of rRNA and have been classified into Box C/D and Box H/ACA families. Most of the snoRNAs occur as ribonucleoprotein complexes with snoRNA-associated proteins (snoRNPs). All Box C/D snoRNAs in yeast form complexes with Nop1p, Nop56p and Nop58p. Similarly, it has been reported that Box H/ACA-containing snoRNAs form complexes with yeast Gar1p. Nop56p and Nop58p homologs have been described in several species. Here we report the isolation and molecular characterization of the Dnop56 genes from D. melanogaster and D. subobscura which show a very similar structure. Drosophila Nop56p proteins contain lysine-rich regions at their carboxy-terminus, and show a high degree of similarity to other Nop56p proteins from different organisms. Phylogenetic relationships among these proteins and other snoRNPs have been established.

Genetics of the SCA6 gene in a large family segregating an autosomal dominant "pure" cerebellar ataxia.

Spinocerebellar ataxia type 6 (SCA6) is an autosomal dominant cerebellar degeneration caused by the expansion of a CAG trinucleotide repeat in the CACNA1A gene. Mutations in patients are characterised by expanded alleles of between 21 and 30 repeat units and by extreme gonadal stability when transmitted from parents to children. We have investigated the SCA6 mutation in a large Spanish kindred in which previously reported spinocerebellar SCA genes and loci had been excluded. We observed a 23 CAG repeat expanded allele in the 13 clinically affected subjects and in three out of 10 presymptomatic at risk subjects. Transmission of the mutant allele was stable in six parent to child pairs and in 29 meioses through the pedigree. Linkage analysis with the SCA6-CAG polymorphism and marker D19S221 confirmed the location of SCA6 on chromosome 19p13. The molecular findings in this large family confirm the expansion of the CAG repeat in the CACNA1A gene as the cause of SCA6 and the high meiotic stability of the repeat.

GEM, a cluster of repetitive sequences in the Drosophila subobscura genome.

GEM is a new family of repetitive sequences detected in the D. subobscura genome. Two of the four described GEM elements encompass a heterogeneous central module, with no detectable ORF, flanked by two long inverted repeats. These elements are composed of a set of repetitive modules, which are inverted repeat (IR), direct repeat (DR), palindromic sequence (PS), long sequence (LS) and short sequence (SS). These five modules can be found either clustered or dispersed as single modules in the D. subobscura genome, in euchromatic and heterochromatic regions. In addition to the 3' region of Adh retrosequences, single IR and LS blocks were found associated with the promoter region of different genes, in particular, LS-like blocks have also been found associated with functional genes in D. melanogaster and D. virilis. Conversely, the DR block is highly similar to satellite DNAs from some other species of the obscura group. In addition, GEM elements share some structural features with IS elements described in different Drosophila species. It is likely that both GEM and IS sequences would be vestiges of an ancestral transposable element.

Molecular evolution of P transposable elements in the Genus drosophila. II. The obscura species group.

A phylogenetic analysis of P transposable elements in the Drosophila obscura species group is described. Multiple P sequences from each of 10 species were obtained using PCR primers that flank a conserved region of exon 2 of the transposase gene. In general, the P element phylogeny is congruent with the species phylogeny, indicating that the dominant mode of transmission has been vertical, from generation to generation. One manifestation of this is the distinction of P elements from the Old World obscura and subobscura subgroups from those of the New World affinis subgroup. However, the overall distribution of elements within the obscura species group is not congruent with the phylogenetic relationships of the species themselves. There are at least four distinct subfamilies of P elements, which differ in sequence from each other by as much as 34%, and some individual species carry sequences belonging to different subfamilies. P sequences from D. bifasciata are particularly interesting. These sequences belong to two subfamilies and both are distinct from all other P elements identified in this survey. Several mechanisms are postulated to be involved in determining phylogenetic relationships among P elements in the obscura group. In addition to vertical transmission, these include retention of ancestral polymorphisms and horizontal transfer by an unknown mating-independent mechanism.