Variantes estruturais raras na disfunção severa da espermatogénese

Mestrado em Biomedicina MolecularA azoospermia afeta aproximadamente 15% de todos os homens inférteis e é frequentemente causada por anomalias cromossómicas e microdeleções do cromossoma Y. No entanto, em aproximadamente 70% dos casos de azoospermia não-obstrutiva (NOA) as causas permanecem por identificar. Nos últimos anos, a descoberta de variantes genómicas de número de cópia (CNVs), como as causadas por deleções, revelou uma fonte de variação genómica que afecta a dosagem génica e que poderá resultar em haploinsuficiência. De facto, observa-se uma sobre-representação de CNVs raros (<1% na população), sobretudo de grandes deleções de novo, em pacientes com diferentes distúrbios do desenvolvimento, comparados com controlos saudáveis. Porém, uma possível contribuição, para a infertilidade masculina, de variantes estruturais ligados ao cromossoma X e aos autossomas foi ainda pouco explorada. Este estudo foca-se na validação de deleções encontradas apenas em pacientes inférteis, no cromossoma X e em 11p13, que contêm genes candidatos a participar na espermatogénese. Estas deleções, previamente identificadas por arrays de oligonucleótidos, de elevada densidade (Affymetrix 6.0 SNP Array), numa coorte de 171 pacientes Portugueses com disfunção severa da espermatogénese (NOA e oligozoospermia severa), foram agora confirmadas por técnicas convencionais de genética molecular. Adicionalmente, a caraterização dos locais de quebra nestas deleções foi realizada por aCGH. Ainda que não se tenham validado as deleções menos extensas (em Xq21.1, Xq25, Xp11.4, Xq22.1 e Xq26.3), confirmou-se a nulizigotia em Xq28 nestes indivíduos, que abrange genes candidatos com uma função sugestiva na espermatogénese: MAGE-A8, expresso em testículo e em alguns cancros e o microRNA hsa-miR-4330, envolvido na regulação pós-transcricional de vários genes com expressão na linha germinal. Foi ainda validada, por MLPA, uma deleção extensa num paciente infértil não-sindrómico da nossa coorte. Estes resultados apontam a haploinsuficiência de WT1 como a causa mais provável de azoospermia neste paciente, já que não foram detetadas mutações germinais no alelo restante. Mutações no gene WT1, que codifica um factor de transcrição muito conservado, crucial para o desenvolvimento e manutenção gonadal em mamíferos, geralmente interferem com a ligação desta proteína ao DNA e estão principalmente associadas a síndromes que envolvem anomalias reprodutivas. Motivados pela nossa descoberta de uma deleção de WT1 num homem infértil embora saudável, decidimos abordar a contribuição de mutações exónicas no gene WT1 para a azoospermia isolada. Testámos a hipótese de que mutações localizadas em domínios que não aqueles essenciais à ligação ao DNA pudessem resultar na disfunção não-sindrómica da espermatogénese. Assim, analisámos a sequência codificante de WT1 num subgrupo de 40 pacientes azoospérmicos. Como resultado, descrevemos uma nova variação missense c.185C>T (P130L; ENST00000332351) no primeiro exão de WT1, inserida no domínio proteico de auto-associação. A nova variante descrita deverá ter um impacto menos drástico na função da proteína WT1, comparativamente com as mutações descritas no mesmo exão até à data, as quais resultam em proteínas truncadas e fenótipos severos de disfunção gonadal, incluindo a formação de tumores renais. Estes resultados revelam novos genes candidatos a um papel na espermatogénese e sugerem que a haploinsuficiência de proteínas importantes para o desenvolvimento do sistema reprodutor masculino podem resultar em azoospermia. Estudos futuros poderão clarificar a utilidade dos nossos genes candidatos como biomarcadores da infertilidade masculina. A implementação de novos biomarcadores beneficiaria os doentes azoospérmicos através da melhoria do diagnóstico, aconselhamento genético e acompanhamento destes pacientes, podendo vir a limitar a necessidade de procedimentos invasivos.Azoospermia affects approximately 15% of all infertile males and it is frequently caused by chromosomal abnormalities and Yq microdeletions. However, despite considerable research efforts in the last decades, in approximately 70% of the cases of non-obstructive azoospermia (NOA) the causes are yet to be identified. In the last years, the discovery of genomic copy number variants, such as those caused by deletions, revealed a source of genomic variation which impacts gene dosage and may result in haploinsufficiency. In fact, rare CNVs (<1% population), mainly large de novo deletions, are over-represented in patients with different developmental disorders, compared to healthy controls. However, a possible contribution of X-linked and autosomal structural variants to male infertility is still largely unexplored. This study focused on the validation of rare patient-specific deletions found on the X chromosome and at 11p13 of infertile patients, which harbor candidate spermatogenesis genes. These deletions had been previously identified by high density oligonucleotide arrays (Affymetrix 6.0 SNP Array), in a cohort of 171 Portuguese patients with severe spermatogenic impairment (non-obstructive azoospermia and severe oligozoospermia) and were now confirmed by conventional molecular genetics techniques. Additionally, breakpoint characterization was carried out by aCGH. In fact, even though the smaller deletions (at Xq21.1, Xq25, Xp11.4, Xq22.1 and Xq26.3) were not validated, we confirmed nullizygosity at Xq28 in two patients, spanning either MAGE-A8, a known cancer-testis antigen, or hsa-miR-4330, a microRNA involved in post-transcription regulation, both with a suggestive role in spermatogenesis pathways. We have also validated by MLPA a large deletion at 11p13, in a non-syndromic infertile patient from our cohort. These results support WT1 haploinsufficiency as the likely cause of azoospermia in this patient, as no other germline mutations were detected in the remaining WT1 copy. Mutations in WT1, an evolutionarily conserved transcription factor crucial for gonadal development and maintenance in mammals, typically interfere with the DNA-binding properties of the protein and are mainly associated with syndromes involving reproductive abnormalities. Motivated by our finding of a WT1 deletion in an infertile but otherwise healthy man we addressed the contribution of WT1 exonic mutations to isolated azoospermia. We reasoned that mutations located in domains not essential for DNA binding could result in non-syndromic spermatogenic impairment. Thus, we analyzed the WT1 coding sequence in a subgroup of 40 azoospermic patients. As a result of the exon screening, we report a novel c.185C>T (P130L; ENST00000332351) WT1 missense variant on exon 1, within the protein self-association domain. While all exon 1 mutations as yet reported result in truncated proteins and severe phenotypes, including the formation of renal tumors, this novel variant is expected to have a milder impact on WT1 function. These results reveal new candidate genes for a role in spermatogenesis and suggest that haploinsufficiency of proteins important for the development of the male reproductive system can lead to azoospermia. Further studies will clarify the utility of our candidate genes as biomarkers of male infertility. The implementation of new biomarkers would benefit azoospermic men by improving diagnosis, genetic counseling and patient care, eventually limiting the need for invasive procedures

Physical, Transcriptional and Comparative Mapping on the Human X Chromosome

Progress in the study of the human genome has resulted in the production of a complete clone map and associated 'working draft' sequence. This will underpin the completion of the sequence itself, the annotation of genes and other features, and application of this new found knowledge. This thesis focuses on the evolving methods to determine the map, and to use the emerging sequence for the study of genes, incorporating new studies of other genomes to enhance progress in understanding and interpretation to biology and medicine. The success of the endeavours is necessarily accompanied by the development and evolution of new technologies and by critical assessment of the progress in acquiring knowledge of the genomic information. Evolution of mapping technologies included the development of the larger insert bacterial cloning systems (PACs) and (BACs), and an increase in available landmarks both from Y AC maps and RH maps. The work described in chapter 3, followed this evolution and was applied to construct a 6 Mb sequence-ready bacterial clone contig map in Xq22. A minimum set of clones was chosen for genomic sequencing. The resulting sequence map was compared to previously published maps and analysed both for common repeats, and previously unidentified low copy repeats. The availability of the emerging sequence of the human genome provided a resource for identification of the features encoded within. In chapter 4, the sequence of a 7 Mb region in Xq23-24 was analysed for the presence of genes using a combination of sequence similarity searches against both protein and DNA databases, and ab initio gene prediction. Predicted genes were confirmed where possible, by generating novel cDNA sequence. The region contained 33 confirmed genes (of which 14 were confirmed during this study), 11 predicted genes and 20 pseudogenes. Comparative genome sequence analysis is a powerful method both for aiding human gene identification and identifying other features encoded within the human genome such as regulatory elements. Comparing the genomes of two or more species also provides insights into the evolution of the species since the divergence from a common ancestor. Sequence from a 1 Mb region in human Xq24 was compared in two other species, mouse and zebrafish. In chapter 5, bacterial clone contigs for sequencing were constructed in the mouse by designing mouse-specific STSs orthologous to human sequence for clone isolation. In chapter 6, bacterial clone contigs for sequencing were constructed in zebrafish using STS from exons of human genes to identify zebrafish BAC clones by reduced stringency hybridisation. Comparative analysis of the region showed that humans and mice are more highly conserved than humans and zebrafish, in terms of gene content and organisation. A combination of comparative sequence analysis tools identified 14 novel potential conserved sequences between human and mouse, one of which was also conserved in zebrafish

Targeted resequencing as diagnostic tool in patients with epilepsy

Epilepsy is one of the most common neurological disorder, affecting 5–8/1.000 individuals worldwide. Approximately 20–30 % of epilepsy cases are caused by acquired conditions such as stroke, tumor or head injury, but the remaining 70–80 % of cases are believed to be due to one or more genetic factors. In the last decade, advances in genomic technologies have led to a rapid increase in understanding of epilepsy genetics and to date, to the best of our knowledge, about 1000 genes have been associated with epilepsy. The aim of this study is to determine the contribution of some currently known disease-causing genes in a cohort of Italian patients affected by syndromic or non-syndromic forms of epilepsy. We designed a genes panel for Targeted Resequencing (TRS) containing 85 relevant epilepsy genes responsible for the most common epilepsy phenotypes known so far. A cohort of 49 patients (23 male and 26 female) with a clinical diagnosis of epilepsy, including both sporadic and familial cases, has been enrolled for the study and analyzed by TRS. This approach allowed us to identify variants in 25/49 (51%) patients analyzed. In detail, disease-causing mutations (classified as pathogenic or likely pathogenic following the American College of Medical genetics guidelines), has been identified in 10/25 (40%) affecting the genes ARX, GAMT, KCNQ2, MECP2, SCN1A, POLG, SPTAN1, STXBP1 and TCF4, while variants of uncertain clinical significance (VUS) has been identified in the remaining 15/25 patients (60%) affecting the genes ATP1A2, CACNB4, CLN3, CLN6, CNTN4, CACNA1H, CNTNAP2, GRIN2A, GRIN2B, KCNMA1, LIAS, POLG, PNKP, PRICKLE2, SCN1A, SCN2A, SPTAN1, SCN9A, TSC1. Next Generation Sequencing technologies have revolutionized our approach to genetic epilepsies both from research than clinical perspective. The identification of novel mutations in known epilepsy associated genes is useful to increase our knowledge about the molecular mechanisms of the disease. More importantly, our study highlight once again the utility of next generation sequencing in establishing an etiological basis in clinically and genetically heterogeneous conditions such as epilepsy. Knowing the genetic basis of the disease can be valuable not only for diagnosis but also for guiding treatment and, above all, estimating recurrence risk

Functional and pharmacological evaluation of novel GLA variants in Fabry disease identifies six (two de novo) causative mutations and two amenable variants to the chaperone DGJ

Abstract Background Allelic heterogeneity is an important feature of the GLA gene for which almost 900 known genetic variants have been discovered so far. Pathogenetic GLA variants cause alpha-galactosidase A (α-Gal A) enzyme deficiency leading to the X-linked lysosomal storage disorder Fabry disease (FD). Benign GLA intronic and exonic variants ( e.g. pseudodeficient p.Asp313Tyr) have also been described. Some GLA missense variants, previously deemed to be pathogenetic ( e.g. p.Glu66Gln and p.Arg118Cys), they have been reclassified as benign after re-evaluation by functional and population studies. Hence, the functional role of novel GLA variants should be investigated to assess their clinical relevance. Results We identified six GLA variants in 4 males and 2 females who exhibited symptoms of FD: c.159C>G p.(Asn53Lys), c.400T>C p.(Tyr134His), c.680G>C (p.Arg227Pro), c.815A>T p.(Asn272Ile), c.907A>T p.(Ile303Phe) and c.1163_1165delTCC (p.Leu388del). We evaluated their impact on the α-Gal A protein by bioinformatic analysis and homology modelling, by analysis of the GLA mRNA, and by site-directed mutagenesis and in vitro expression studies. We also measured their responsiveness to the pharmacological chaperone DGJ. Conclusions The six detected GLA variants cause deficient α-Gal A activity and impairment or loss of the protein wild-type structure. We found p.Asn53Lys and p.Ile303Phe variants to be susceptible to DGJ

Whole exome sequencing revealed a novel dystrophin-related protein-2 (DRP2) deletion in an Iranian family with symptoms of polyneuropathy

Objective(s): Charcot-Marie Tooth disease (CMT) is one of the main inherited causes of motor and sensory neuropathies with variable expressivity and age-of onset. Although more than 70 genes have been identified for CMT, more studies are needed to discover other genes involved in CMT. Introduction of whole exome sequencing (WES) to capture all the exons may help to find these genes.Materials and Methods: Here, we tried to find the genetic cause of the neuropathy in two Iranian brothers using WES. Blood sample was collected from probands and their family members to extract the genomic DNA. The extracted DNA from one of the affected case was subjected for WES. The variant calls were filtered  to reveal the pathogenic variant. Presence of the candidate mutation was confirmed using Sanger sequencing. The pathogenic potential of the variant was examined using in silico software. Using ClustalW multiple alignment, the presence of variant in conserved domain of protein was investigated. The parent and another affected boy were also checked for presence of the variant using PCR-sequencing. Results: The obtained data presented a novel TTC del mutation in CDS 738 of dystrophin related protein 2 (DRP2) gene, which was validated by sequencing. The variant was located in a conserved domain of DRP2 protein and predicted as pathogenic. Two affected boys were hemizygous for the mutation and received the mutation from mother. Conclusion: Here, we provided the evidence for the contribution of DRP2 in CMT. Also, the symptoms shed light on molecular aspect of this genetically heterogeneous disease

An Alu element-based model of human genome instability

The human genome is strewn with repetitive sequence. An early estimate derived from the draft human genome sequence placed this repetitive content at ~45%. More detailed recent analyses have advanced the idea that the human repetitive and repeat derived contribution to the genome may be closer to 66-69%. The most commonly repeated sequence in the human genome is the Alu element. Alus make up 10.6 percent of all human DNA and have expanded to over one million copies in the human genome reproducing through a copy and paste mechanism. New Alu germline insertions are estimated to occur at a rate of 1 in 20 human births. In addition to their insertional impact, Alus have also been associated with various forms of genomic sequence disruptions including inversions, rearrangements, translocations and deletions. Chimeric Alus are frequently located at the breakpoints of these various forms of structural variations. This observation has led to the putative conclusion that chimeric Alus primarily result from the non-allelic homologous recombination between Alu elements. However, little proof is available regarding the actual mechanism(s) that catalyze this activity. This dissertation reveals a newly recognized pattern among human Alu pairs that may provide additional insight into the mechanism(s) driving chimeric Alu formation. After adjusting for directional biases associated with clustering, Alu pairs in the same orientation (direct) outnumber Alu pairs in the opposite orientation (inverted pairs) by over two percent (p\u3c0.05). If this imbalance was generated by deletions resulting from interactions between inverted Alu elements, many chimeric Alus may have formed from the homologous repair of these deletions. This dissertation characterizes the human Alu pair imbalance and constructs an Alu-based model of human genome instability. This model was used to compare the relative instabilities of 50 human deletion-prone cancer genes and 50 randomly chosen genes. Taken as separate groups, the 50 deletion-prone cancer genes were estimated to be 58% more unstable than the 50 randomly chosen genes. This approach to estimating human gene instability may lay the foundation for comparing genetic risks unique to specific individuals, families and people groups

Genetics of primary ovarian insufficiency: new developments and opportunities

BACKGROUND Primary ovarian insufficiency (POI) is characterized by marked heterogeneity, but with a significant genetic contribution. Identifying exact causative genes has been challenging, with many discoveries not replicated. It is timely to take stock of the field, outlining the progress made, framing the controversies and anticipating future directions in elucidating the genetics of POI. METHODS A search for original articles published up to May 2015 was performed using PubMed and Google Scholar, identifying studies on the genetic etiology of POI. Studies were included if chromosomal analysis, candidate gene screening and a genome-wide study were conducted. articles identified were restricted to English language full-text papers. RESULTS Chromosomal abnormalities have long been recognized as a frequent cause of POI, with a currently estimated prevalence of 10?13%. Using the traditional karyotype methodology, monosomy X, mosaicism, X chromosome deletions and rearrangements, X-autosome translocations, and isochromosomes have been detected. Based on candidate gene studies, single gene perturbations unequivocally having a deleterious effect in at least one population include Bone morphogenetic protein 15 (BMP15), Progesterone receptor membrane component 1 (PGRMC1), and Fragile X mental retardation 1 (FMR1) premutation on the X chromosome; Growth differentiation factor 9 (GDF9), Folliculogenesis specific bHLH transcription factor (FIGLA), Newborn ovary homeobox gene (NOBOX), Nuclear receptor subfamily 5, group A, member 1 (NR5A1) and Nanos homolog 3 (NANOS3) seem likely as well, but mostly being found in no more than 1?2% of a single population studied. Whole genome approaches have utilized genome-wide association studies (GWAS) to reveal loci not predicted on the basis of a candidate gene, but it remains difficult to locate causative genes and susceptible loci were not always replicated. Cytogenomic methods (array CGH) have identified other regions of interest but studies have not shown consistent results, the resolution of arrays has varied and replication is uncommon. Whole-exome sequencing in non-syndromic POI kindreds has only recently begun, revealing mutations in the Stromal antigen 3 (STAG3), Synaptonemal complex central element 1 (SYCE1), minichromosome maintenance complex component 8 and 9 (MCM8, MCM9) and ATP-dependent DNA helicase homolog (HFM1) genes. Given the slow progress in candidate-gene analysis and relatively small sample sizes available for GWAS, family-based whole exome and whole genome sequencing appear to be the most promising approaches for detecting potential genes responsible for POI. CONCLUSION Taken together, the cytogenetic, cytogenomic (array CGH) and exome sequencing approaches have revealed a genetic causation in ?20?25% of POI cases. Uncovering the remainder of the causative genes will be facilitated not only by whole genome approaches involving larger cohorts in multiple populations but also incorporating environmental exposures and exploring signaling pathways in intragenic and intergenic regions that point to perturbations in regulatory genes and networks

Integrated high-resolution physical and comparative gene maps in horses

High-resolution physically ordered gene maps for the horse (Equus caballus, ECA) are essential to the identification of genes associated with hereditary diseases and traits of interest like fertility, coat color, and disease resistance or susceptibility. Such maps also serve as foundations for genome comparisons across species and form the basis to study chromosome evolution. In this study seven equine chromosomes (ECA6, 7, 10, 15, 18, 21 and X) corresponding to human chromosomes (HSA) 2, 19 and X were selected for high-resolution mapping on the basis of their potential involvement in diseases and conditions of importance to horses. To accomplish this, gene- and sequence-specific markers were generated and genotyped on the TAMU 5000rad horse x hamster RH panel. Additionally, screening of a BAC library by overgoes and subsequent STS content mapping and fingerprinting approaches were used to assemble and verify a BAC contig along a ~5 Mb span on ECA21. Dense gene maps were generated for each of the seven equine chromosomes by adding 408 new markers (285 type I and 123 type II) to the current maps of these chromosomes, thereby greatly improving overall map resolution to one mapped marker every 960kb on average (range: 700 kb â 1.3 Mb). Moreover, the contig on ECA21 contained 47 markers (42 genes and 5 microsatellites) as well as 106 STS markers distributed along 207 BAC clones. Comparisons of these maps with other species revealed a remarkably high level of horse-human X chromosome conservation, as well as two evolutionary breakpoints unique to Perissodactyls or Equids for the equine homologues of HSA19 and HSA2, one of which has been more precisely localized by the ECA21 contig. Thus, high resolution maps developed for these chromosomes i) provide a basis to map traits of interest rapidly to specific chromosomal regions, ii) facilitate searches for candidate genes for these traits by fine comparisons of the equine regions with corresponding segments in other species, and iii) enable understanding the evolution of the chromosomes. Expansion of this work to the entire equine genome will be important for developing novel strategies for diagnosis, prevention, and treatment of equine diseases