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- Actionable secondary findings following exome sequencing of 836 non-obstructive azoospermia cases and their value in patient managementPublication . Kasak, Laura; Lillepea, Kristiina; Nagirnaja, Liina; Aston, Kenneth I.; Schlegel, Peter N.; Gonçalves, João; Carvalho, Filipa; Moreno-Mendoza, Daniel; Almstrup, Kristian; Eisenberg, Michael L.; Jarvi, Keith A.; O’Bryan, Moira K.; Lopes, Alexandra M.; Conrad, Donald F.; Nagirnaja, Liina; Aston, Kenneth I.; Carrell, Douglas T.; Hotaling, James M.; Jenkins, Timothy G.; McLachlan, Rob; O’Bryan, Moira K.; Schlegel, Peter N.; Eisenberg, Michael L.; Sandlow, Jay I.; Jungheim, Emily S.; Omurtag, Kenan R.; Lopes, Alexandra M.; Seixas, Susana; Carvalho, Filipa; Fernandes, Susana; Barros, Alberto; Laan, Maris; Punab, Margus; Rajpert-De Meyts, Ewa; Jørgensen, Niels; Almstrup, Kristian; Krausz, Csilla G.; Jarvi, Keith A.; Punab, Margus; Laan, MarisStudy question: What is the load, distribution and added clinical value of secondary findings (SFs) identified in exome sequencing (ES) of patients with non-obstructive azoospermia (NOA)? Summary answer: One in 28 NOA cases carried an identifiable, medically actionable SF. What is known already: In addition to molecular diagnostics, ES allows assessment of clinically actionable disease-related gene variants that are not connected to the patient's primary diagnosis, but the knowledge of which may allow the prevention, delay or amelioration of late-onset monogenic conditions. Data on SFs in specific clinical patient groups, including reproductive failure, are currently limited. Study design, size, duration: The study group was a retrospective cohort of patients with NOA recruited in 10 clinics across six countries and formed in the framework of the international GEMINI (The GEnetics of Male INfertility Initiative) study. Participants/materials, setting, methods: ES data of 836 patients with NOA were exploited to analyze SFs in 85 genes recommended by the American College of Medical Genetics and Genomics (ACMG), Geisinger's MyCode, and Clinical Genome Resource. The identified 6374 exonic variants were annotated with ANNOVAR and filtered for allele frequency, retaining 1381 rare or novel missense and loss-of-function variants. After automatic assessment of pathogenicity with ClinVar and InterVar, 87 variants were manually curated. The final list of confident disease-causing SFs was communicated to the corresponding GEMINI centers. When patient consent had been given, available family health history and non-andrological medical data were retrospectively assessed. Main results and the role of chance: We found a 3.6% total frequency of SFs, 3.3% from the 59 ACMG SF v2.0 genes. One in 70 patients carried SFs in genes linked to familial cancer syndromes, whereas 1 in 60 cases was predisposed to congenital heart disease or other cardiovascular conditions. Retrospective assessment confirmed clinico-molecular diagnoses in several cases. Notably, 37% (11/30) of patients with SFs carried variants in genes linked to male infertility in mice, suggesting that some SFs may have a co-contributing role in spermatogenic impairment. Further studies are needed to determine whether these observations represent chance findings or the profile of SFs in NOA patients is indeed different from the general population. Limitations, reasons for caution: One limitation of our cohort was the low proportion of non-Caucasian ethnicities (9%). Additionally, as comprehensive clinical data were not available retrospectively for all men with SFs, we were not able to confirm a clinico-molecular diagnosis and assess the penetrance of the specific variants. Wider implications of the findings: For the first time, this study analyzed medically actionable SFs in men with spermatogenic failure. With the evolving process to incorporate ES into routine andrology practice for molecular diagnostic purposes, additional assessment of SFs can inform about future significant health concerns for infertility patients. Timely detection of SFs and respective genetic counseling will broaden options for disease prevention and early treatment, as well as inform choices and opportunities regarding family planning. A notable fraction of SFs was detected in genes implicated in maintaining genome integrity, essential in both mitosis and meiosis. Thus, potential genetic pleiotropy may exist between certain adult-onset monogenic diseases and NOA.
- Bi-allelic Mutations in M1AP Are a Frequent Cause of Meiotic Arrest and Severely Impaired Spermatogenesis Leading to Male InfertilityPublication . Wyrwoll, Margot J.; Temel, Şehime G.; Nagirnaja, Liina; Oud, Manon S.; Lopes, Alexandra M.; van der Heijden, Godfried W.; Heald, James S.; Rotte, Nadja; Wistuba, Joachim; Wöste, Marius; Ledig, Susanne; Krenz, Henrike; Smits, Roos M.; Carvalho, Filipa; Gonçalves, João; Fietz, Daniela; Türkgenç, Burcu; Ergören, Mahmut C.; Çetinkaya, Murat; Başar, Murad; Kahraman, Semra; McEleny, Kevin; Xavier, Miguel J.; Turner, Helen; Pilatz, Adrian; Röpke, Albrecht; Dugas, Martin; Kliesch, Sabine; Neuhaus, Nina; Aston, Kenneth I.; Conrad, Donald F.; Veltman, Joris A.; Friedrich, Corinna; Tüttelmann, FrankMale infertility affects ∼7% of men, but its causes remain poorly understood. The most severe form is non-obstructive azoospermia (NOA), which is, in part, caused by an arrest at meiosis. So far, only a few validated disease-associated genes have been reported. To address this gap, we performed whole-exome sequencing in 58 men with unexplained meiotic arrest and identified the same homozygous frameshift variant c.676dup (p.Trp226LeufsTer4) in M1AP, encoding meiosis 1 associated protein, in three unrelated men. This variant most likely results in a truncated protein as shown in vitro by heterologous expression of mutant M1AP. Next, we screened four large cohorts of infertile men and identified three additional individuals carrying homozygous c.676dup and three carrying combinations of this and other likely causal variants in M1AP. Moreover, a homozygous missense variant, c.1166C>T (p.Pro389Leu), segregated with infertility in five men from a consanguineous Turkish family. The common phenotype between all affected men was NOA, but occasionally spermatids and rarely a few spermatozoa in the semen were observed. A similar phenotype has been described for mice with disruption of M1ap. Collectively, these findings demonstrate that mutations in M1AP are a relatively frequent cause of autosomal recessive severe spermatogenic failure and male infertility with strong clinical validity.
- Disruption of human meiotic telomere complex genes TERB1, TERB2 and MAJIN in men with non-obstructive azoospermiaPublication . Salas-Huetos, Albert; Tüttelmann, Frank; Wyrwoll, Margot J.; Kliesch, Sabine; Lopes, Alexandra M.; Gonçalves, João; Boyden, Steven E.; Wöste, Marius; Hotaling, James M.; GEMINI Consortium; Nagirnaja, Liina; Conrad, Donald F.; Carrell, Douglas T.; Aston, Kenneth I.Non-obstructive azoospermia (NOA), the lack of spermatozoa in semen due to impaired spermatogenesis affects nearly 1% of men. In about half of cases, an underlying cause for NOA cannot be identified. This study aimed to identify novel variants associated with idiopathic NOA. We identified a nonconsanguineous family in which multiple sons displayed the NOA phenotype. We performed whole-exome sequencing in three affected brothers with NOA, their two unaffected brothers and their father, and identified compound heterozygous frameshift variants (one novel and one extremely rare) in Telomere Repeat Binding Bouquet Formation Protein 2 (TERB2) that segregated perfectly with NOA. TERB2 interacts with TERB1 and Membrane Anchored Junction Protein (MAJIN) to form the tripartite meiotic telomere complex (MTC), which has been shown in mouse models to be necessary for the completion of meiosis and both male and female fertility. Given our novel findings of TERB2 variants in NOA men, along with the integral role of the three MTC proteins in spermatogenesis, we subsequently explored exome sequence data from 1495 NOA men to investigate the role of MTC gene variants in spermatogenic impairment. Remarkably, we identified two NOA patients with likely damaging rare homozygous stop and missense variants in TERB1 and one NOA patient with a rare homozygous missense variant in MAJIN. Available testis histology data from three of the NOA patients indicate germ cell maturation arrest, consistent with mouse phenotypes. These findings suggest that variants in MTC genes may be an important cause of NOA in both consanguineous and outbred populations.
- ABC system used as an add-on to clarify germline variants previously classified as VUS according to ACMG guidelinesPublication . Rodrigues, Pedro; Theisen, Patrícia; Silva, Catarina; Mendonça, Joana; Carpinteiro, Dina; Vieira, Luís; Gonçalves, JoãoThe increasing number of patients screened by NGS to identify germline variants associated with hereditary breast/ovarian cancer (HBOC) syndromes, is leading to a growing number of variants classified as Variants of Uncertain Significance (VUS) according to ACMG guidelines1. Since the ACMG system merges functional and clinical data into a one-dimensional system, it is not always clear how the classification was obtained. The ABC system (ABCs) of variant classification2 splits functional and clinical grading and aims to give a better guide to variant significance. The main goals of this work were i) to apply the ABCs to a group of previously classified ACMG-VUS and ii) to evaluate the potential clinical impact of this review/classification. Germline variants (36 - 29 missense, 1 synonymous and 6 intronic) detected in 5 genes (BRCA1, BRCA2, ATM, CHEK2, PALB2) previously classified as ACMG-VUS, were selected from our database of patients with HBOC, to be reclassified with the ABCs. Variant assessment included: query of clinical and population databases, literature and in silico tools (VEP, HSF, Alamut, Varsome). Eleven variants were classified as Class 0 (functional - fVUS); 17 as class E (functional - HFE (Hypothetical Function Effect), and 8 as Class D (functional - LFE (Likely Functional Effect). fVUS are not clinically graded. Considering that ACMG-VUS are not actionable, it is still an ongoing debate if they should be reported or not. Since the ACMG merges functional and clinical data, it might be difficult for clinicians to understand how VUS classification is achieved. The ABCs allowed us to distinguish between VUS classified due to lack of data from those that might have a functional impact. Class 0 variants (11) should not be reported and class E (17) reporting is optional. The use of ABCs highlighted 8 variants (class D) which might be a susceptibility factor with functional impact and should be reported. Functional and segregation studies are of major importance to clarify the clinical significance of these variants. 1-PMID: 25741868. 2-PMID: 33981013. Support: FCT/MCTES, ToxOmics and Human Health (UIDB/00009/2020). GenomePT(POCI-01-0145-FEDER-022184).
- Reporting of secondary findings in clinical genomic sequencing: national guidelines are requiredPublication . Theisen, Patrícia; Rodrigues, Pedro; Gonçalves, JoãoIntroduction: The rapid and growing integration of exome and genome sequencing into clinical genetic diagnosis raises awareness regarding the identification of variants of potential clinical value unrelated to the primary reason for testing (secondary findings, SF). SF pose major challenges, as multiple issues (medical, legal, ethical, economic) and different contexts (e.g. paediatric and prenatal diagnosis, patient and family management, research in rare diseases) must be considered, highlighting the importance to promote standardized reporting of SF. We aim to bring to the consideration of the Portuguese Society of Human Genetics (SPGH) the urgent need for issuing national guidelines for reporting SF from clinical sequencing. Methodology: Consultation and review of guidelines for reporting SF in clinical exome and genome sequencing from different organizations, focusing on the ones issued by the American College of Medical Genetics and Genomics (ACMG)1, the European Society of Human Genetics (ESHG)2 and the French Society of Predictive and Personalized Medicine (SFMPP)3. Results: The ACMG recently published SF v3.0 list4 includes 73 clinically actionable genes mainly related to cancer and cardiovascular phenotypes, for which causal SF should be reported unless patients opted out. The ESHG recommends a more cautious approach, stating that genomic analysis should be as targeted as possible for the time being because a broader analysis raises complex issues in clinical practice. The SFMPP restricts its guidelines for reporting pathogenic SF to a list of 36 actionable cancer genes, requiring a double consent from the patient. Discussion: Considering the diversity of approaches and the complexity involved in reporting SF, we propose that the SPGH should promote the creation of a multidisciplinary workgroup involving all the stakeholders to put forth official national guidelines for reporting SF in clinical sequencing. References 1. Miller DT et al. Genet Med. 2021;23:1381. 2. de Wert G et al. Eur J Hum Genet. 2021;29:365. 3. Pujol P et al. Eur J Hum Genet. 2018;26:1732. 4. Miller DT et al. Genet Med. 2021; 23:1391.
- NGS Panels applied to Hereditary Cancer SyndromesPublication . Rodrigues, Pedro; Theisen, Patrícia; Silva, Catarina; Vieira, Luís; Gonçalves, JoãoCancer is among the leading causes of morbidity and mortality worldwide (Okur et al, 2017). Germline pathogenic variants for monogenic, highly penetrant cancer susceptibility genes are observed in 5%–10% of all cancers (Lu et al, 2014). Hereditary cancers due to monogenic causes are characterized by earlier age of onset, other associated cancers, and often a family history of specific cancers. From the clinical perspective, it is important to recognize the affected individuals to provide them the best clinical management (Hennessy et al, 2010; Ledermann et al, 2014; Pennington et al, 2014) and to identify at-risk family members who will benefit from predictive genetic testing and enhanced surveillance, including early detection and/or risk reduction measures (Kurian et al, 2010; Okur et al, 2017). Germline variants identified in major cancer susceptibility genes associated with hereditary breast or ovarian cancer (HBOC) or hereditary colorectal cancer (HCRC), also account for 5-10% of the patients with these cancers. In the last years, new susceptibility genes, with different penetrance degrees, have been identified. Variants in any of those genes are rare and classical methodologies (e.g. Sanger sequencing - SS) are time consuming and expensive. Next-generation sequencing (NGS) has several advantages compared to SS, including the simultaneous analysis of many samples and sequencing of a large set of genes, higher sensitivity (down to 1% vs 15-20% in SS), lower cost and faster turnaround time, reasons that make NGS the best approach for molecular diagnosis. It is possible nowadays to choose between whole-genome sequencing (WGS), whole-exome sequencing (WES) and NGS limited to a set of genes (NGS-Panel). In cases where a suspected genetic disease or condition has been identified, targeted sequencing of specific genes or genomic regions is preferred (Grada et al, 2013). For that reason, we use NGS-Panel approach using TruSight Cancer (Illumina) to sequence DNA extracted from blood samples of patients with personal and/or familiar history of cancer. This hereditary cancer gene panel sequences 94 genes associated with both common (e.g., breast, colorectal) and rare hereditary cancers and allows the creation of virtual gene panels according to each phenotype or disease under study. NGS workflow analysis (Figure 1) includes five steps: quality assessment of raw data, read alignment to a reference genome, variant identification/calling, variant annotation and data visualization (Pabinger et al, 2013). The establishment of the most appropriate bioinformatics pipeline is crucial in order to achieve the best results. NGS data allows the identification of several types of variants like single nucleotide variants (SNVs), small insertions/deletions, inversions and also copy number variants (CNVs).
- Molecular characterization of a new CYP21A2 allele and classification of its pathogenicityPublication . Gomes, Susana; Saraiva, Jorge; Gonçalves, JoãoBackground: The CYP21A2 gene, coding for 21-Hydroxylase (21-OH), is located on 6p21.3 within the major histocompatibility complex, and integrated in a cluster of genes (RP1, C4A, C4B, TNXB) and pseudogenes (RP2, CYP21A1P, TNXA). This genomic region is variable in size and gene copy number. Due to the high homology between genes and their pseudogenes, recombination is common, deletions, insertions and duplications are frequent. The great diversity of this cluster and rare alleles contributes to additional difficulties on molecular analysis and pathogenicity classification. Methods: The CYP21A cluster was characterized using genomic DNA obtained from four healthy brothers (parents not available). Two long-PCR products, specific for each CYP21A2 copy of a trimodular allele (with two CYP21A2 copies), and for a normal/bimodular allele present in this family, were characterized by Sanger cycling sequencing and MLPA (MRC-Holland, P050-C1 kit). Results: The molecular studies revealed that one sister, who asked for genetic counselling, has a very rare trimodular allele, with two CYP21A2 genes. One of these genes has a deletion covering exons 4 to 7 and an insertion of exons 4 to 7 of the pseudogene (CYP21A1P) which has the pathogenic variants c.518T>A, c.710T>A, c.713T>A, c.719T>A, c.844G>T and c.923dupT, all in phase. This alteration can be described as: CYP21A2ex4_7delinsCYP21A1Pex4_7. Conclusion: The developed molecular approach, which was specifically designed for this family and included segregation analysis of all brothers, allowed the characterization of a new CYP21A2 trimodular allele that, even containing six pathogenic variants, is non-pathogenic as it also has (in phase) a normal CYP21A2 copy.
- Importância do estudo multigénico no diagnóstico molecular de doenças raras por sequenciação de nova geraçãoPublication . Gonçalves, João; Rodrigues, Pedro; Oliveira, Beatriz; Pereira Caetano, Iris; Theisen, PatríciaIntrodução: A sequenciação de nova geração (NGS) revolucionou o diagnóstico molecular das doenças raras (DR) proporcionando a análise de um maior número de genes, resultados mais rápidos e custos reduzidos. A NGS usando diferentes abordagens, possibilita o estudo do genoma (WGS), do exoma (WES), de genes individuais ou de painéis multigénicos (NGS-PMG). Objetivos: Aplicação de NGS-PMG no estudo de doenças raras específicas. Métodos: Preparação de bibliotecas de sequências-alvo a partir de DNA genómico, utilizando a captura com sondas (Trusight Cancer-Rapid Capture, Illumina) ou amplicões (painéis customizados - AmpliSeq, Illumina). Sequenciação no MiSeq (Illumina) e análise bioinformática usando software da Illumina e programas disponíveis on line. Validação de alterações pontuais por sequenciação de Sanger. Resultados: A utilização da metodologia Trusight-Cancer, que inclui a sequenciação de 94 genes de suscetibilidade para cancro hereditário (CH), permite-nos a análise seletiva de PMG, aplicados ao estudo de por ex., síndrome de Lynch, Polipose Adenomatose Familiar, cancro Hereditário da Mama e Ovário). A aplicação de PMG por AmpliSeq a um painel de genes desenhado in silico e específico de patologias do desenvolvimento sexual (PDS), permite-nos a análise molecular de 40 genes associados a hipogonadismo, Síndrome de Kallmann, insensibilidade aos androgénios, androgenização precoce, ciliopatias, falância ovárica prematura. Os resultados obtidos (sem falsos negativos), permitiram detetar diferentes tipos de alterações moleculares em doentes com diferentes DR, proporcionando em muitos um diagnóstico definitivo. Destaca-se que a estratégia de NGS implementada para o CH, integra também a avaliação de variações do número de cópias (deleções e duplicações) e a validação destes resultados por metodologia diferente - MLPA. Conclusões: A utilização de NGS-PMG possibilitando a identificação de novas variantes em novos genes, permite confirmar o diagnóstico clínico e contribuir para um melhor acompanhamento dos doentes e prevenção da doença genética hereditária. No caso de doenças cujo fenótipo não permite inferir um PMG a analisar, o alargamento ao WES ou ao WGS, para além de poder contribuir para o diagnóstico, gerará novo conhecimento e eventualmente o desenvolvimento de terapias inovadores.
- Reclassification of BRCA1/2 variants previously classified as VUS (ACMG-AMP guidelines) with gene-specific guidelines from ClinGen ENIGMA and CanVIG-UKPublication . Rodrigues, Pedro; Theisen, Patrícia; Gonçalves, JoãoIn recent years, the number of BRCA1/2 germline variants associated with hereditary breast/ovarian cancer syndrome (HBOC), classified as variants of uncertain significance (VUS) according to ACMG-AMP guidelines (ACMGg) has been increasing. Reclassification of VUS as (likely) benign or (likely) pathogenic is crucial for maximizing diagnostic yield and appropriately managing HBOC patients. Recently, specific guidelines to improve classification of BRCA1/2 variants were independently developed by ClinGen ENIGMA1 (CG-Eg) and CanVIG-UK2 (CV-UKg). Main goals: i) independently reclassify BRCA1/2 variants previously classified as VUS (ACMGg) with the new guidelines (CG-Eg and CV-UKg); ii) compare the results between the different guidelines iii) evaluate the potential clinical impact of this reclassification. BRCA1/2 germline variants identified in patients with suspected HBOC and previously classified as VUS (8 missense, 5 intronic) were independently reclassified according to CG-Eg and CV-UKg. Variant assessment included: query of clinical/population databases and use of VEP, Alamut, VarSome and Franklin-Genoox. VUS reclassification (using CG-Eg versus CV-UKg) was in agreement for 10 variants (2 VUS, 6 likely benign (LB) and 2 benign (B)). The remaining 3 VUS were reclassified as LB with CG-Eg and kept as VUS with CV-UK. Application of specific guidelines reduced the number of VUS from 10 to 2 (CG-Eg) or to 5 (CV-UKg). The main difference between CG-Eg and CV-UKg is related with the downgrading strength of PM2 and the upgrading strength of BP1 criteria (in CG-Eg) for missense variants present outside clinically important functional domains and without splicing impact. The difference in BP1 strength has a major impact, making CG-Eg more stringent and reducing the number of VUS. The use of different guidelines, even if gene-specific, can lead to dissimilar classifications, a general consensus leading to a unique international guideline will be useful.
- Molecular diagnosis of haemophilia A: four novel variants identified in five patientsPublication . Certã, Rita; Moreira, Isabel; Antunes, Ema; Cruz, Eugénia; Diniz, Maria João; Kjollerstrom, Paula; Morais, Sara; Gonçalves, JoãoAims/Context: Haemophilia A (HMA) is an X-linked bleeding disorder caused by reduced levels of the coagulation factor VIII (FVIII) due to alterations in the F8 gene. Decreased levels of FVIII activity leads to a loss of clotting activity and to bleeding (predominantly into joins, muscles and inner organs). The severity of HMA ranges from mild (5-30% activity) to moderate (2-5% activity) to severe (<1% activity). During the last five years, we have found four novel variants identified in five index patients with no family history of HMA. Three frameshift variants were detected in patients presenting severe HMA and one missense variant was identified in two unrelated patients with a mild phenotype. Methods: Analysis of the F8 gene was performed in five index patients using PCR followed by Sanger sequencing, after F8 IVS22 and IVS1 inversions being excluded in severe HMA cases. Bioinformatics analysis was performed with several pathogenicity prediction tools (Alamut Visual, VarSome, VEP and Human Splicing Finder). Results and Conclusions: In the three patients with severe HMA, three different novel F8 variants were identified: c.1060_1061delCT, p.(Leu354Thrfs*5), c.4804delC, p.(Gln602Lysfs*19) and c.3561dupT, p.(Pro1188Serfs*10). All these variants create a frameshift, leading to a premature termination codon and presumably resulting in non-functional truncated proteins, confirming the patient’s phenotypes. The novel F8 missense variant c.5836G>T, p.(Asp1946Tyr) was identified in two unrelated patients, both with mild HMA. The Asp1946 is a highly conserved amino acid in the FVIII protein. Additionally, physicochemical properties between Asp and Tyr are significantly different, and in silico analysis classified it as pathogenic due to the amino acid substitution. Normal mRNA splicing process can also be disturbed due to the creation of a new donor splice site. RNA studies and other functional assays are essential in order to establish this variant clinical significance. Identification of novel pathogenic F8 variants in HMA patients allows genotype-phenotype correlations, appropriate genetic counseling and new knowledge about the molecular bases of this pathology.
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