Inés Quintela

Distribution of choline acetyltransferase immunoreactivity in the brain of an elasmobranch, the lesser spotted dogfish (Scyliorhinus canicula).

Although the distribution of cholinergic cells is remarkably similar across the vertebrate species, no data are available on more primitive species, such as cartilaginous fishes. To extend the evolutionary analysis of the cholinergic systems, we studied the distribution of cholinergic neurons in the brain and rostral spinal cord of Scyliorhinus canicula by immunocytochemistry using an antibody against the enzyme choline acetyltransferase (ChAT). Western blot analysis of brain extracts of dogfish, sturgeon, trout, and rat showed that this antibody recognized similar bands in the four species. Putative cholinergic neurons were observed in most brain regions, including the telencephalon, diencephalon, cerebellum, and brainstem. In the retrobulbar region and superficial dorsal pallium of the telencephalon, numerous small pallial cells were ChAT‐like immunoreactive. In addition, tufted cells of the olfactory bulb and some cells in the lateral pallium showed faint immunoreactivity. In the preoptic‐hypothalamic region, ChAT‐immunoreactive (ChAT‐ir) cells were found in the preoptic nucleus, the vascular organ of the terminal lamina, and a small population in the caudal tuber. In the epithalamus, the pineal photoreceptors were intensely positive. Many cells of the habenula were faintly ChAT‐ir, but the neuropil of the interpeduncular nucleus showed intense ChAT immunoreactivity. In the pretectal region, ChAT‐ir cells were observed only in the superficial pretectal nucleus. In the brainstem, the somatomotor and branchiomotor nuclei, the octavolateral efferent nucleus, and a cell group just rostral to the Edinger‐Westphal (EW) nucleus contained ChAT‐ir neurons. In addition, the trigeminal mesencephalic nucleus, the nucleus G of the isthmus, some locus coeruleus cells, and some cell populations of the vestibular nuclei and of the electroreceptive nucleus of the octavolateral region exhibited ChAT immunoreactivity. In the reticular areas of the brainstem, the nucleus of the medial longitudinal fascicle, many reticular neurons of the rhombencephalon, and cells of the nucleus of the lateral funiculus were immunoreactive to this antibody. In the cerebellum, Golgi cells of the granule cell layer and some cells of the cerebellar nucleus were also ChAT‐ir. In the rostral spinal cord, ChAT immunoreactivity was observed in cells of the motor column, the dorsal horn, the marginal nucleus (a putative stretch‐receptor organ), and in interstitial cells of the ventral funiculus. These results demonstrate for the first time that cholinergic neurons are distributed widely in the central nervous system of elasmobranchs and that their cholinergic systems have evolved several characteristics that are unique to this group. J. Comp. Neurol. 420:139–170, 2000.

Human spermatogenic failure purges deleterious mutation load from the autosomes and both sex chromosomes, including the gene DMRT1.

Gonadal failure, along with early pregnancy loss and perinatal death, may be an important filter that limits the propagation of harmful mutations in the human population. We hypothesized that men with spermatogenic impairment, a disease with unknown genetic architecture and a common cause of male infertility, are enriched for rare deleterious mutations compared to men with normal spermatogenesis. After assaying genomewide SNPs and CNVs in 323 Caucasian men with idiopathic spermatogenic impairment and more than 1,100 controls, we estimate that each rare autosomal deletion detected in our study multiplicatively changes a mans risk of disease by 10% (OR 1.10 [1.04–1.16], p<2×10−3), rare X-linked CNVs by 29%, (OR 1.29 [1.11–1.50], p<1×10−3), and rare Y-linked duplications by 88% (OR 1.88 [1.13–3.13], p<0.03). By contrasting the properties of our case-specific CNVs with those of CNV callsets from cases of autism, schizophrenia, bipolar disorder, and intellectual disability, we propose that the CNV burden in spermatogenic impairment is distinct from the burden of large, dominant mutations described for neurodevelopmental disorders. We identified two patients with deletions of DMRT1, a gene on chromosome 9p24.3 orthologous to the putative sex determination locus of the avian ZW chromosome system. In an independent sample of Han Chinese men, we identified 3 more DMRT1 deletions in 979 cases of idiopathic azoospermia and none in 1,734 controls, and found none in an additional 4,519 controls from public databases. The combined results indicate that DMRT1 loss-of-function mutations are a risk factor and potential genetic cause of human spermatogenic failure (frequency of 0.38% in 1306 cases and 0% in 7,754 controls, p = 6.2×10−5). Our study identifies other recurrent CNVs as potential causes of idiopathic azoospermia and generates hypotheses for directing future studies on the genetic basis of male infertility and IVF outcomes.

Differential expression of thymosins β4 and β10 during rat cerebellum postnatal development

The β-thymosins are a family of actin monomer-sequestering proteins widely distributed among vertebrate classes. The most abundant β-thymosins in mammalian species are thymosin β4 (Tβ4) and thymosin β10 (Tβ10), two small peptides (43 amino acids) sharing a high degree of sequence homology. In the present work, we have analyzed the distribution of Tβ4 and Tβ10 in the developing and adult rat cerebellum using in situ hybridization and immunohistochemistry techniques. Our results show that the temporal and cellular patterns of expression of both β-thymosins are different. In the young (7 and 18 postnatal days) and adult (1 and 4 months old) rat cerebellum, Tβ4 was mainly expressed in the glia (microglia, Golgi epithelial cells and oligodendrocytes), neurons (granule cells and Purkinje cells), and in the capillaries. In 14-month-old rats, the Tβ4 immunoreactivity was only detected in some microglia cells. In young and adult animals, most of the Tβ10 immunoreactivity was localized in several types of neuronal cells including granule cells, Golgi neurons and Purkinje cells. In old animals, a faint Tβ10 signal could be detected in a few Purkinje cells. Our results suggest that each β-thymosin could play a different function in the control of actin dynamics.

New technologies in the genetic approach to sudden cardiac death in the young

Sudden cardiac death (SCD) is a major health problem and constitutes one of the most important unsolved challenges in the practice of forensic pathology due to the failure to determine the cause of death. Particularly, an important number of previously healthy young people who have died suddenly and unexpectedly are consequence of genetic heart disorders, either structural cardiomyopathies or arrhythmogenic abnormalities. The technological approach to analyze this type of genetically heterogeneous disorders is far from easy but nowadays the variety of chemistries and methodologies improves choice. This review offers to the reader a state of the art of the available technologies for the study of genetics of sudden cardiac death, including mutation screening approaches, genome wide association studies, and the recently developed next-generation sequencing.

Enhanced localization of genetic samples through linkage-disequilibrium correction.

Characterizing the spatial patterns of genetic diversity in human populations has a wide range of applications, from detecting genetic mutations associated with disease to inferring human history. Current approaches, including the widely used principal-component analysis, are not suited for the analysis of linked markers, and local and long-range linkage disequilibrium (LD) can dramatically reduce the accuracy of spatial localization when unaccounted for. To overcome this, we have introduced an approach that performs spatial localization of individuals on the basis of their genetic data and explicitly models LD among markers by using a multivariate normal distribution. By leveraging external reference panels, we derive closed-form solutions to the optimization procedure to achieve a computationally efficient method that can handle large data sets. We validate the method on empirical data from a large sample of European individuals from the POPRES data set, as well as on a large sample of individuals of Spanish ancestry. First, we show that by modeling LD, we achieve accuracy superior to that of existing methods. Importantly, whereas other methods show decreased performance when dense marker panels are used in the inference, our approach improves in accuracy as more markers become available. Second, we show that accurate localization of genetic data can be achieved with only a part of the genome, and this could potentially enable the spatial localization of admixed samples that have a fraction of their genome originating from a given continent. Finally, we demonstrate that our approach is resistant to distortions resulting from long-range LD regions; such distortions can dramatically bias the results when unaccounted for.

Analysis of a claimed distant relationship in a deficient pedigree using high density SNP data

DNA markers are routinely used to reveal both simple and complex family relationships. Likelihood based approaches have been traditionally used to estimate relationships using relatively few unlinked markers. However it is widely recognized that when using such limited numbers of loci distant relationships between two individuals cannot be distinguished from the average level of allele sharing found in random pairwise comparisons in the same population. As a real example, we demonstrate the usefulness of genome-wide SNP genotyping to analyze a claimed second cousin relationship that could not be resolved using standard forensic markers, confirming theoretical expectations for very distant relationships. Genome profiles derived from Affymetrix 6.0 SNP arrays obtained from the claimed second cousins were compared to profiles obtained from unrelated individuals and simulated data. Significance of the high estimated probabilities in favor of the second cousin relationship hypothesis was proved from the results obtained with both real and simulated unrelated pairs. As a final cautionary note, it is important to consider that successful identification of the claimed distant relationship reported here is largely due to a well-founded hypothesis being compared to the alternative hypothesis of the claimants being unrelated, but where there are several possible alternative hypotheses, the approach we outline here can yield false indications of unfounded alternative relationships.

Thyroid cancer GWAS identifies 10q26.12 and 6q14.1 as novel susceptibility loci and reveals genetic heterogeneity among populations.

Thyroid cancer is the most heritable cancer of all those not displaying typical Mendelian inheritance. However, most of the genetic factors that would explain the high heritability remain unknown. Our aim was to identify additional common genetic variants associated with susceptibility to this disease. In order to do so, we performed a genome‐wide association study in a series of 398 cases and 502 controls from Spain, followed by a replication in four well‐defined Southern European case‐control collections contributing a total of 1,422 cases and 1,908 controls. The association between the variation at the 9q22 locus near FOXE1 and thyroid cancer risk was consistent across all series, with several SNPs identified (rs7028661: OR = 1.64, p = 1.0 × 10−22, rs7037324: OR = 1.54, p = 1.2 × 10−17). Moreover, the rare alleles of three SNPs (rs2997312, rs10788123 and rs1254167) at 10q26.12 showed suggestive evidence of association with higher risk of the disease (OR = 1.35, p = 1.2 × 10−04, OR = 1.26, p = 5.2 × 10−04 and OR = 1.38, p = 5.9 × 10−05, respectively). Finally, the rare allele of rs4075570 at 6q14.1 conferred protection in the series studied (OR = 0.82, p = 2.0 × 10−04). This study suggests that heterogeneity in genetic susceptibility between populations is a key feature to take into account when exploring genetic risk factors related to this disease.

A new approach to long QT syndrome mutation detection by Sequenom MassARRAY® system

Congenital long QT syndrome is an inherited cardiac disorder characterized by a prolonged QT interval and polymorphic ventricular arrhythmias that could result in recurrent syncope, seizures or sudden death as the most dramatic event. Until now QT interval mutations have been described in 12 genes, where the majority of mutations reside in three genes KCNQ1, KCNH2, and SCN5A. Diagnosis and prognosis are directly related with the gene and mutation involved. We have developed a diagnostic approach for long QT syndrome and Brugada syndrome based on published mutations and Sequenom MassArray® system. Three diagnostic tests have been developed, oriented to each of the three most prevalent genes in the long QT syndrome. A total of 433 mutations are analyzed in 38 multiplex reactions, allowing their detection in about 48 h. Tests were validated on 502 samples from individuals with different clinical conditions and family history. The average call rates obtained for each of the tests were 93, 83, and 73% in KCNQ1, KCNH2, and SCNA, respectively. Sequenom MassARRAY mutation detection is a reliable, highly flexible, and cost‐efficient alternative to conventional methods for genetic testing in long QT syndrome and Brugada syndrome, facilitating flexible upgrades of the version of the test presented here with the inclusion of new mutations.

Genome-wide association analysis implicates dysregulation of immunity genes in chronic lymphocytic leukaemia

Several chronic lymphocytic leukaemia (CLL) susceptibility loci have been reported; however, much of the heritable risk remains unidentified. Here we perform a meta-analysis of six genome-wide association studies, imputed using a merged reference panel of 1,000 Genomes and UK10K data, totalling 6,200 cases and 17,598 controls after replication. We identify nine risk loci at 1p36.11 (rs34676223, P=5.04 × 10−13), 1q42.13 (rs41271473, P=1.06 × 10−10), 4q24 (rs71597109, P=1.37 × 10−10), 4q35.1 (rs57214277, P=3.69 × 10−8), 6p21.31 (rs3800461, P=1.97 × 10−8), 11q23.2 (rs61904987, P=2.64 × 10−11), 18q21.1 (rs1036935, P=3.27 × 10−8), 19p13.3 (rs7254272, P=4.67 × 10−8) and 22q13.33 (rs140522, P=2.70 × 10−9). These new and established risk loci map to areas of active chromatin and show an over-representation of transcription factor binding for the key determinants of B-cell development and immune response.

Exon-focused genome-wide association study of obsessive-compulsive disorder and shared polygenic risk with schizophrenia.

Common single-nucleotide polymorphisms (SNPs) account for a large proportion of the heritability of obsessive-compulsive disorder (OCD). Co-ocurrence of OCD and schizophrenia is commoner than expected based on their respective prevalences, complicating the clinical management of patients. This study addresses two main objectives: to identify particular genes associated with OCD by SNP-based and gene-based tests; and to test the existence of a polygenic risk shared with schizophrenia. The primary analysis was an exon-focused genome-wide association study of 370 OCD cases and 443 controls from Spain. A polygenic risk model based on the Psychiatric Genetics Consortium schizophrenia data set (PGC-SCZ2) was tested in our OCD data. A polygenic risk model based on our OCD data was tested on previous data of schizophrenia from our group. The most significant association at the gene-based test was found at DNM3 (P=7.9 × 10−5), a gene involved in synaptic vesicle endocytosis. The polygenic risk model from PGC-SCZ2 data was strongly associated with disease status in our OCD sample, reaching its most significant value after removal of the major histocompatibility complex region (lowest P=2.3 × 10−6, explaining 3.7% of the variance). The shared polygenic risk was confirmed in our schizophrenia data. In conclusion, DNM3 may be involved in risk to OCD. The shared polygenic risk between schizophrenia and OCD may be partially responsible for the frequent comorbidity of both disorders, explaining epidemiological data on cross-disorder risk. This common etiology may have clinical implications.