Systematic genomic analysis of SARS-CoV-2 co-infections throughout the pandemic and segregation of the strains involved.

BACKGROUND: SARS-CoV-2 recombinants involving the divergent Delta and Omicron lineages have been described, and one of them, "Kraken" (XBB.1.5), has recently been a matter of concern. Recombination requires the coexistence of two SARS-CoV-2 strains in the same individual. Only a limited number of studies have focused on the identification of co-infections and are restricted to co-infections involving the Delta/Omicron lineages. METHODS: We performed a systematic identification of SARS-CoV-2 co-infections throughout the pandemic (7609 different patients sequenced), not biassed towards the involvement of highly divergent lineages. Through a comprehensive set of validations based on the distribution of allelic frequencies, phylogenetic consistency, re-sequencing, host genetic analysis and contextual epidemiological analysis, these co-infections were robustly assigned. RESULTS: Fourteen (0.18%) co-infections with ≥ 8 heterozygous calls (8-85 HZs) were identified. Co-infections were identified throughout the pandemic and involved an equal proportion of strains from different lineages/sublineages (including pre-Alpha variants, Delta and Omicron) or strains from the same lineage. Co-infected cases were mainly unvaccinated, with mild or asymptomatic clinical presentation, and most were at risk of overexposure associated with the healthcare environment. Strain segregation enabled integration of sequences to clarify nosocomial outbreaks where analysis had been impaired due to co-infection. CONCLUSIONS: Co-infection cases were identified throughout the pandemic, not just in the time periods when highly divergent lineages were co-circulating. Co-infections involving different lineages or strains from the same lineage were occurring in the same proportion. Most cases were mild, did not require medical assistance and were not vaccinated, and a large proportion were associated with the hospital environment.

Novel Candidate loci and Pathogenic Germline Variants Involved in Familial Hematological Malignancies Revealed by Whole-Exome Sequencing.

The familial occurrence of hematological malignancies has been underappreciated. Recent studies suggest that up to 15% of adults with myeloid neoplasms carry germline pathogenic variants in cancer-predisposing genes. This study aimed to identify the underlying germline predisposition variant in patients with a strong family or personal onco-hematological history using whole exome sequencing on sixteen uncharacterized individuals. It was carried out in two groups of patients, one with samples available from two affected relatives (Cohort A) and one with available samples from the index case (Cohort B). In Cohort A, six families were characterized. Two families shared variants in genes associated with DNA damage response and involved in cancer development (CHEK2 and RAD54L). Pathogenic or likely pathogenic germline variants were also found in novel candidate genes (NFATC2 and TC2N). In two families, any relevant pathogenic or likely pathogenic genomic variants were identified. In Cohort B, four additional index cases were analyzed. Three of them harbor clinically relevant variants in genes with a probable role in the development of inherited forms of hematological malignancies (GATA1, MSH4 and PRF1). Overall, whole exome sequencing is a useful approach to achieve a further characterization of these patients and their mutational spectra. Moreover, further investigations may help improve optimization for disease management of affected patients and their families.

First confirmation of importation and transmission in Spain of the newly identified SARS-CoV-2 B.1.1.7 variant.

INTRODUCTION: A newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the United Kingdom. The rapid spread in the UK of this new variant has caused other countries to be vigilant. MATERIAL AND METHODS: We based our initial screening of B.1.1.7 on the dropout of the S gene signal in the TaqPath assay, caused by the 69/70 deletion. Subsequently, we confirmed the B.1.1.7 candidates by whole genome sequencing. RESULTS: We describe the first three imported cases of this variant from London to Madrid, subsequent post-arrival household transmission to three relatives, and the two first cases without epidemiological links to UK. One case required hospitalization. In all cases, drop-out of gene S was correctly associated to the B.1.1.7 variant, as all the corresponding sequences carried the 17 lineage-marker mutations. CONCLUSION: The first identifications of the SARS-CoV-2 B.1.1.7 variant in Spain indicate the role of independent introductions from the UK coexisting with post-arrival transmission in the community, since the early steps of this new variant in our country.

First confirmation of importation and transmission in Spain of the newly identified SARS-CoV-2 B.1.1.7 variant / Primera confirmación de importación y transmisión en España de la variante B.1.1.7 del SARS-CoV-2 recientemente identificada

Introduction: A newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the United Kingdom. The rapid spread in the UK of this new variant has caused other countries to be vigilant. Material and methods: We based our initial screening of B.1.1.7 on the dropout of the S gene signal in the TaqPath assay, caused by the 69/70 deletion. Subsequently, we confirmed the B.1.1.7 candidates by whole genome sequencing. Results: We describe the first three imported cases of this variant from London to Madrid, subsequent post-arrival household transmission to three relatives, and the two first cases without epidemiological links to UK. One case required hospitalization. In all cases, drop-out of gene S was correctly associated to the B.1.1.7 variant, as all the corresponding sequences carried the 17 lineage-marker mutations. Conclusion: The first identifications of the SARS-CoV-2 B.1.1.7 variant in Spain indicate the role of independent introductions from the UK coexisting with post-arrival transmission in the community, since the early steps of this new variant in our country.(AU)

Introducción: Recientemente, ha surgido en Reino Unido una nueva variante de SARS-CoV-2, VOC202012/01, que origina el linaje B.1.1.7. Su rápida distribución en Reino Unido ha alertado a otros países a vigilar su presencia. Material y métodos: El rastreo inicial de la variante B.1.1.7 se basó en la ausencia de amplificación del gen S en el ensayo TaqPath, causado por la deleción 69/70. Todos los casos candidatos de corresponder a la variante B.1.1.7 con este criterio fueron posteriormente confirmados por secuenciación de genoma completo. Resultados: Describimos los primeros 3 casos importados de esta variante, desde Londres hasta Madrid, con la posterior transmisión domiciliaria de uno de estos casos a 3 familiares y, adicionalmente, los 2 primeros casos con la variante sin vínculo epidemiológico con Reino Unido. Uno de los casos requirió hospitalización. En todos los casos el criterio de no amplificación del gen S identificó con precisión la variante B.1.1.7, como demostró posteriormente la presencia de las 17 mutaciones marcadoras de este linaje. Conclusión: Las primeras identificaciones de la variante B.1.1.7 de SARS-CoV-2 indican un papel solapante de las introducciones independientes desde Reino Unido, con eventos de transmisión comunitaria, incluso desde los primeros momentos de la presencia de esta variante en nuestro país.(AU)

Association between gene polymorphisms in the cyclophosphamide metabolism pathway with complications after haploidentical hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for patients with hematologic malignances. Haploidentical HSCT (Haplo-HSCT) is an alternative option for patients who do not have an HLA-matched donor. The use of post-transplantation high dose cyclophosphamide (PT-Cy) is commonly employed for graft-versus-host disease (GVHD) prophylaxis in haplo-HSCT. Cyclophosphamide (Cy) is an alkylating agent with antineoplastic and immunosuppressive activity, whose bioactivation requires the activity of polymorphic enzymes in the liver to produce phosphoramide mustard, which is a DNA alkylating agent. To identify polymorphisms in the genes of Cy metabolism and correlate them with post-HSCT complications [GVHD, sinusoidal obstruction syndrome (SOS), hemorrhagic cystitis (HC) and transplant-related mortality (TRM)], we designed a custom next-generation sequencing panel with Cy metabolism enzymes. We analyzed 182 patients treated with haplo-HSCT with PT-Cy from 2007 to 2019, detecting 40 variants in 11 Cy metabolism genes. Polymorphisms in CYP2B6, a major enzyme involved in Cy activation, were associated with decreased activity of this enzyme and a higher risk of Graf-versus-host disease (GVHD). Variants in other activation enzymes (CYP2A6, CYP2C8, CYP2C9, CYP2C19) lead to decreased enzyme activity and were associated with GVHD. Polymorphisms in detoxification genes such as glutathione S-transferases decreased the ability to detoxify cyclophosphamide metabolites due to lower enzyme activity, which leads to increased amounts of toxic metabolites and the development of III-IV acute GVHD. GSMT1*0 a single nucleotide polymorphism previously recognized as a risk factor for SOS was associated with a higher risk of SOS. We conclude that polymorphisms of genes involved in the metabolism of cyclophosphamide in our series are associated with severe grades of GVHD and toxicities (SOS and TRM) after haplo-HSCT and could be used to improve the clinical management of transplanted patients.

First importations of SARS-CoV-2 P.1 and P.2 variants from Brazil to Spain and early community transmission.

INTRODUCTION: SARS-CoV-2variants of concern (VOC) have been described in the UK (B.1.1.7), South Africa (B.1.351) and Brazil (P.1). Among them, the most scarce information has been obtained from the P.1 variant and more data on its global presence and about its spreading dynamics are needed. METHODS: Whole genome sequencing was performed prospectively on travellers arriving from Brazil and on a random selection of SARS-CoV-2 positive cases from our population. RESULTS: In this study we report the first SARS-CoV-2 P.1 and P.2 variants exported from Brazil to Spain. The case infected with the P.1 variant, who had only stayed in Rio de Janeiro, required hospitalisation. The two P.2 cases remained asymptomatic. A wider distribution for P.1 variant beyond the Brazilian Amazonia should be considered. The exportation of the P.2 variant, carrying the E484K mutation, deserves attention. One month after the first description of P.1 and P.2 importations from Brazil to Madrid, these variants were identified circulating in the community, in cases without a travel history, and involved in household transmissions CONCLUSION: Whole genome sequencing of SARS-CoV-2 positive travellers arriving from Brazil allowed us to identify the first importations of P.1 and P.2 variants to Spain and their early community transmission.

First importations of SARS-CoV-2 P.1 and P.2 variants from Brazil to Spain and early community transmission / Primeras importaciones de las variantes SARS-CoV-2 P.1 y P.2 de Brasil a Madrid y transmisión comunitaria

Introduction: SARS-CoV-2variants of concern (VOC) have been described in the UK (B.1.1.7), South Africa (B.1.351) and Brazil (P.1). Among them, the most scarce information has been obtained from the P.1 variant and more data on its global presence and about its spreading dynamics are needed.Methods: Whole genome sequencing was performed prospectively on travellers arriving from Brazil and on a random selection of SARS-CoV-2 positive cases from our population.Results: In this study we report the first SARS-CoV-2 P.1 and P.2 variants exported from Brazil to Spain. The case infected with the P.1 variant, who had only stayed in Rio de Janeiro, required hospitalisation. The two P.2 cases remained asymptomatic. A wider distribution for P.1 variant beyond the Brazilian Amazonia should be considered. The exportation of the P.2 variant, carrying the E484K mutation, deserves attention. One month after the first description of P.1 and P.2 importations from Brazil to Madrid, these variants were identified circulating in the community, in cases without a travel history, and involved in household transmissionsConclusion: Whole genome sequencing of SARS-CoV-2 positive travellers arriving from Brazil allowed us to identify the first importations of P.1 and P.2 variants to Spain and their early community transmission.

Introducción: Se han descrito «variantes de preocupación» (VOC) de SARS-CoV-2 en el Reino Unido (B.1.1.7), Sudáfrica (B.1.351) y Brasil (P.1). Entre ellas, se dispone de información más escasa para la variante P.1 y se necesitan más datos sobre su presencia global y sobre su dinámica de expansión.Métodos: Se realizó secuenciación del genoma completo de forma prospectiva de SARS-CoV-2 en viajeros procedentes de Brasil y en una selección aleatoria de casos positivos de SARS-CoV-2 de nuestra población.Resultados: En este estudio reportamos las primeras variantes de SARS-CoV-2 P.1 y P.2 exportadas desde Brasil a España. El caso infectado por la variante P.1, que solo había permanecido en Río de Janeiro, requirió hospitalización. Los 2 casos de la variante P.2 permanecieron asintomáticos. Se debe considerar una distribución más amplia para la variante P.1 más allá de la Amazonía brasileña. La exportación de la variante P.2, que porta la mutación E484K, merece asimismo atención adicional. Un mes después de la primera descripción de las importaciones de P.1 y P.2 de Brasil a Madrid, se identificaron estas variantes circulando en la comunidad, en casos sin antecedentes de viaje, e implicadas en transmisiones domiciliarias.Conclusión: La secuenciación de genoma completo de viajeros positivos para SARS-CoV-2 procedentes de Brasil nos permitió identificar las primeras importaciones de variantes P.1 y P.2 a España y su transmisión comunitaria precoz.

Nosocomial Transmission of SARS-CoV-2 Involving Vaccinated Health Care Workers.

COVID-19 vaccination has proven to be effective at preventing symptomatic disease but there are scarce data to fully understand whether vaccinated individuals can still behave as SARS-CoV-2 transmission vectors. Based on viral genome sequencing and detailed epidemiological interviews, we report a nosocomial transmission event involving two vaccinated health care-workers (HCWs) and four patients, one of them with fatal outcome. Strict transmission control measures, as during the prevaccination period, must be kept between HCWs and HCWs-patients in nosocomial settings. IMPORTANCE COVID-19 vaccination has proven to be effective at preventing symptomatic disease. Although some transmission events involving vaccinated cases have also been reported, scarce information is still available to fully understand whether vaccinated individuals may still behave as vectors in SARS-CoV-2 transmission events. Here, we report a SARS-CoV-2 nosocomial transmission event, supported on whole genome sequencing, in early March 2021 involving two vaccinated HCWs and four patients in our institution. Strict transmission control measures between HCWs and HCWs - patients in nosocomial settings must not be relaxed, and should be kept as strictly as during the prevaccination period.

First importations of SARS-CoV-2 P.1 and P.2 variants from Brazil to Spain and early community transmission.

Introduction: SARS-CoV-2variants of concern (VOC) have been described in the UK (B.1.1.7), South Africa (B.1.351) and Brazil (P.1). Among them, the most scarce information has been obtained from the P.1 variant and more data on its global presence and about its spreading dynamics are needed. Methods: Whole genome sequencing was performed prospectively on travellers arriving from Brazil and on a random selection of SARS-CoV-2 positive cases from our population. Results: In this study we report the first SARS-CoV-2 P.1 and P.2 variants exported from Brazil to Spain. The case infected with the P.1 variant, who had only stayed in Rio de Janeiro, required hospitalisation. The two P.2 cases remained asymptomatic. A wider distribution for P.1 variant beyond the Brazilian Amazonia should be considered. The exportation of the P.2 variant, carrying the E484K mutation, deserves attention. One month after the first description of P.1 and P.2 importations from Brazil to Madrid, these variants were identified circulating in the community, in cases without a travel history, and involved in household transmissions. Conclusion: Whole genome sequencing of SARS-CoV-2 positive travellers arriving from Brazil allowed us to identify the first importations of P.1 and P.2 variants to Spain and their early community transmission.

Introducción: Se han descrito «variantes de preocupación¼ (VOC) de SARS-CoV-2 en el Reino Unido (B.1.1.7), Sudáfrica (B.1.351) y Brasil (P.1). Entre ellas, se dispone de información más escasa para la variante P.1 y se necesitan más datos sobre su presencia global y sobre su dinámica de expansión. Métodos: Se realizó secuenciación del genoma completo de forma prospectiva de SARS-CoV-2 en viajeros procedentes de Brasil y en una selección aleatoria de casos positivos de SARS-CoV-2 de nuestra población. Resultados: En este estudio reportamos las primeras variantes de SARS-CoV-2 P.1 y P.2 exportadas desde Brasil a España. El caso infectado por la variante P.1, que solo había permanecido en Río de Janeiro, requirió hospitalización. Los 2 casos de la variante P.2 permanecieron asintomáticos. Se debe considerar una distribución más amplia para la variante P.1 más allá de la Amazonía brasileña. La exportación de la variante P.2, que porta la mutación E484K, merece asimismo atención adicional. Un mes después de la primera descripción de las importaciones de P.1 y P.2 de Brasil a Madrid, se identificaron estas variantes circulando en la comunidad, en casos sin antecedentes de viaje, e implicadas en transmisiones domiciliarias. Conclusión: La secuenciación de genoma completo de viajeros positivos para SARS-CoV-2 procedentes de Brasil nos permitió identificar las primeras importaciones de variantes P.1 y P.2 a España y su transmisión comunitaria precoz.

Systematic Genomic and Clinical Analysis of Severe Acute Respiratory Syndrome Coronavirus 2 Reinfections and Recurrences Involving the Same Strain.

Estimates of the burden of severe acute respiratory syndrome coronavirus 2 reinfections are limited by the scarcity of population-level studies incorporating genomic support. We conducted a systematic study of reinfections in Madrid, Spain, supported by genomic viral analysis and host genetic analysis, to cleanse laboratory errors and to discriminate between reinfections and recurrences involving the same strain. Among the 41,195 cases diagnosed (March 2020-March 2021), 93 (0.23%) had 2 positive reverse transcription PCR tests (55-346 days apart). After eliminating cases with specimens not stored, of suboptimal sequence quality, or belonging to different persons, we obtained valid data from 22 cases. Of those, 4 (0.01%) cases were recurrences involving the same strain; case-patients were 39-93 years of age, and 3 were immunosuppressed. Eighteen (0.04%) cases were reinfections; patients were 19-84 years of age, and most had no relevant clinical history. The second episode was more severe in 8 cases.

Incorporation of next-generation sequencing in clinical practice using solid and liquid biopsy for patients with non-Hodgkin's lymphoma.

Although next-generation sequencing (NGS) data on lymphomas require further validation before being implemented in daily practice, the clinical application of NGS can be considered right around the corner. The aim of our study was to validate an NGS lymphoid panel for tissue and liquid biopsy with the most common types of non-Hodgkin's lymphoma [follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL)]. In this series, 372 somatic alterations were detected in 93.6% (44/47) of the patients through tissue biopsy. In FL, we identified 93 somatic alterations, with a median of 7.4 mutations per sample. In DLBCL, we detected 279 somatic variants with a median of 8.6 mutations (range 0-35). In 92% (24/26) of the cases, we were able to detect some variant in the circulating tumor DNA. We detected a total of 386 variants; 63.7% were detected in both types of samples, 13.2% were detected only in the circulating tumor DNA, and 23% were detected only in the tissue biopsy. We found a correlation between the number of circulating tumor DNA mutations, advanced stage, and bulky disease. The genetic alterations detected in this panel were consistent with those previously described at diagnosis. The liquid biopsy sample is therefore a complementary tool that can provide new genetic information, even in cases where a solid biopsy cannot be performed or an insufficient sample was obtained. In summary, we describe and analyze in this study the findings and difficulties encountered when incorporating liquid biopsy into clinical practice in non-Hodgkin's lymphoma at diagnosis.

Complete Analysis of the Epidemiological Scenario around a SARS-CoV-2 Reinfection: Previous Infection Events and Subsequent Transmission.

The first descriptions of reinfection by SARS-CoV-2 have been recently reported. However, these studies focus exclusively on the reinfected case, without considering the epidemiological context of the event. Our objectives were to perform a complete analysis of the sequential infections and community transmission events around a SARS-CoV-2 reinfection, including the infection events preceding it, the exposure, and subsequent transmissions. Our analysis was supported by host genetics, viral whole-genome sequencing, phylogenomic viral population analysis, and refined epidemiological data obtained from interviews with the involved subjects. The reinfection involved a 53-year-old woman with asthma (Case A), with a first COVID-19 episode in April 2020 and a much more severe second episode 4-1/2 months later, with SARS-CoV-2 seroconversion in August, that required hospital admission. An extended genomic analysis allowed us to demonstrate that the strain involved in Case A's reinfection was circulating in the epidemiological context of Case A and was also transmitted subsequently from Case A to her family context. The reinfection was also supported by a phylogenetic analysis, including 348 strains from Madrid, which revealed that the strain involved in the reinfection was circulating by the time Case A suffered the second episode, August-September 2020, but absent at the time range corresponding to Case A's first episode. IMPORTANCE We present the first complete analysis of the epidemiological scenario around a reinfection by SARS-CoV-2, more severe than the first episode, including three cases preceding the reinfection, the reinfected case per se, and the subsequent transmission to another seven cases.

Next Generation Cytogenetics in Myeloid Hematological Neoplasms: Detection of CNVs and Translocations.

Conventional cytogenetics are the gold standard for the identification of chromosomal alterations recurrent in myeloid neoplasms. Some next-generation sequencing (NGS) panels are designed for the detection of copy number variations (CNV) or translocations; however, their use is far from being widespread. Here we report on the results of a commercial panel including frequent mutations, CNVs and translocations in myeloid neoplasms. Frequent chromosomal alterations were analyzed by NGS in 135 patients with myeloid neoplasms and three with acute lymphoblastic leukemia. NGS analysis was performed using the enrichment-capture Myeloid Neoplasm-GeneSGKit (Sistemas Genómicos, Spain) gene panel including 35 genes for mutational analysis and frequent CNVs and translocations. NGS results were validated with cytogenetics and/or MLPA when possible. A total of 66 frequent alterations included in NGS panel were detected, 48 of them detected by NGS and cytogenetics. Ten of them were observed only by cytogenetics (mainly trisomy 8), and another eight only by NGS (mainly deletion of 12p). Aside from this, 38 secondary CNVs were detected in any of the genes included mainly for mutational analysis. NGS represents a reliable complementary source of information for the analysis of CNVs and translocations. Moreover, NGS could be a useful tool for the detection of alterations not observed by conventional cytogenetics.

Proper Assignation of Reactivation in a COVID-19 Recurrence Initially Interpreted as a Reinfection.

A 77-year-old man (case R) with previous diagnosis of a mild COVID-19 episode was hospitalized 35 days later. On day 23 postadmission, he developed a second COVID-19 episode, now severe, and finally died. Initially, case R's COVID-19 recurrence was interpreted as a reinfection due to the exposure to a SARS-CoV-2 RT-PCR-positive roommate. However, whole-genome sequencing indicated that case R's recurrence corresponded to a reactivation of the strain involved in his first episode. Case R's reactivation had major consequences, leading to a more severe episode, and causing subsequent transmission to another 2 hospitalized patients, 1 of them with fatal outcome.

Clinical Utility of the Detection of the Loss of the Mismatched HLA in Relapsed Hematological Patients After Haploidentical Stem Cell Transplantation With High-Dose Cyclophosphamide.

Haploidentical hematopoietic stem cell transplantation (Haplo-HSCT) with high-dose cyclophosphamide (PTCy) has resulted in a low incidence of graft-vs.-host disease (GVHD), graft failure, and non-relapse mortality. However, post-transplantation relapse remains a common cause of treatment failure in high-risk patients. Unraveling the mechanisms of relapse is therefore crucial for designing effective relapse treatment strategies. One of these mechanisms is the loss of the mismatched HLA on the recipient's leukemic cells. To study the incidence and clinical relevance of this phenomenon, we analyzed 181 patients treated with Haplo-HSCT with PTCy (2007-2019), of which 37 relapsed patients after transplantation. According to the kit employed for HLA-loss analysis, among 22 relapsed patients, we identified HLA loss at relapse in 6 of the 22 patients (27%) studied. Based on the results obtained, the genomic loss of HLA was more common in females than males (66 vs. 33%) and HLA-loss relapses occurred later than classical relapses (345 vs. 166 days). Moreover, the patients with HLA-loss had a greater presence of active disease at the time of transplantation and had undergone a larger number of treatment lines than the group with classical relapses (66 vs. 43% and 66 vs. 18%, respectively). Four of these relapses were studied retrospectively, while two were studied prospectively, the results of which could be considered for patient management. Additionally, two relapsed patients analyzed retrospectively had myeloid neoplasms. One patient had not undergone any treatment, and three had undergone donor lymphocyte infusions (DLIs) and chemotherapy. All presented severe GVHD and disease progression. In contrast, the two patients studied prospectively had a lymphoid neoplasm and were not treated with DLIs. One of them was treated with chemotherapy but died from disease progression, and the other patient underwent a second Haplo-HSCT from a different donor and is still alive. We can conclude that the detection of HLA-loss at the onset of relapse after Haplo-HSCT with PTCy could help in clinical practice to select appropriate rescue treatment, thereby avoiding the use of DLIs or a second transplantation from the same donor.

First confirmation of importation and transmission in Spain of the newly identified SARS-CoV-2 B.1.1.7 variant.

INTRODUCTION: A newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the United Kingdom. The rapid spread in the UK of this new variant has caused other countries to be vigilant. MATERIAL AND METHODS: We based our initial screening of B.1.1.7 on the dropout of the S gene signal in the TaqPath assay, caused by the 69/70 deletion. Subsequently, we confirmed the B.1.1.7 candidates by whole genome sequencing. RESULTS: We describe the first three imported cases of this variant from London to Madrid, subsequent post-arrival household transmission to three relatives, and the two first cases without epidemiological links to UK. One case required hospitalization. In all cases, drop-out of gene S was correctly associated to the B.1.1.7 variant, as all the corresponding sequences carried the 17 lineage-marker mutations. CONCLUSION: The first identifications of the SARS-CoV-2 B.1.1.7 variant in Spain indicate the role of independent introductions from the UK coexisting with post-arrival transmission in the community, since the early steps of this new variant in our country.

Novel biallelic variant in BBS9 causative of Bardet-Biedl syndrome: expanding the spectrum of disease-causing genetic alterations.

BACKGROUND: Bardet-Biedl syndrome (BBS) is a rare autosomal recessive ciliopathy disorder. Many BBS disease-causing genetic variants have been identified due to the advancement of molecular diagnostic tools. We report on a novel pathogenic variant in a consanguineous Pakistani family with an affected child. CASE PRESENTATION: Clinical exome sequencing was used to search for BBS causing variants in the affected individual and identified a novel homozygous splice-site variant in the BBS9 gene (c.702 + 1del). Sanger sequencing was performed for variant validation and segregation studies. Expression analysis using mRNA levels to assess the functional impact of the novel variant demonstrated skipping of exon 7 in the affected alleles, suggesting a truncating effect. Three-dimensional structural modelling was used to predict pathogenicity of the variant residue and the alteration leads to a partial deletion of the PHTB1_N domain and a total deletion of the PHTB1_C domain. CONCLUSION: The study of this case expands the spectrum of biallelic variants in the BBS9 gene associated with BBS and increased the knowledge on the molecular consequences of splicing variation c.702 + 1del.

Clinical utility of targeted next-generation sequencing for the diagnosis of myeloid neoplasms with germline predisposition.

Myeloid neoplasms (MN) with germline predisposition (MNGP) are likely to be more common than currently appreciated. Many of the genes involved in MNGP are also recurrently mutated in sporadic MN. Therefore, routine analysis of gene panels by next-generation sequencing provides an effective approach to detect germline variants with clinical significance in patients with hematological malignancies. Gene panel sequencing was performed in 88 consecutive and five nonconsecutive patients with MN diagnosis. Disease-causing germline mutations in CEBPα, ASXL1, TP53, MPL, GATA2, DDX41, and ETV6 genes were identified in nine patients. Six out of the nine patients with germline variants had a strong family history. These patients presented great heterogeneity in the age of diagnosis and phenotypic characteristics. In our study, there were families in which all the affected members presented the same subtype of disease, whereas members of other families presented various disease phenotypes. This intrafamiliar heterogeneity suggests that the acquisition of particular somatic variants may drive the evolution of the disease. This approach enabled high-throughput detection of MNGP in patients with MN diagnosis, which is of great relevance for both the patients themselves and the asymptomatic mutation carriers within the family. It is crucial to make a proper diagnosis of these patients to provide them with the most suitable treatment, follow-up, and genetic counseling.

Genetic biomarkers identify a subgroup of high-risk patients within low-risk NPM1-mutated acute myeloid leukemia.

Although acute myeloid leukemia (AML) with NPM1mut/FLT3-ITDneg is a low-risk entity, its relapse rate remains high. Out of 333 AML patients, 27 were NPM1mut, and were analyzed in greater detail in order to find associations between clinical and molecular features and cumulative incidence of relapse. Next-generation sequencing (NGS) was performed on diagnosis and remission samples using two capture-based panels. The presence of the FLT3D835 variant at diagnosis and a qPCR value of NPM1mut ≥0.1% after induction chemotherapy were associated with an increased probability of relapse, especially if both conditions are present together. By contrast, patients in which the main clone found at diagnosis harbored NPM1 variant had a lower risk of relapse. Nineteen of the 85 variants found at diagnosis were detected by NGS in remission. AML Subgroup with NPM1mut/FLT3-ITDneg is a heterogeneous entity, which can be further risk-stratified based on molecular biomarkers.