A pan-cancer clinical platform to predict immunotherapy outcomes and prioritize immuno-oncology combinations in early-phase trials

Immunooncology; Predictive biomarkers; Tumor microenvironmentInmunooncología; Biomarcadores predictivos; Microambiente tumoralImmunooncologia; Biomarcadors predictius; Microambient tumoralBackground Immunotherapy is effective, but current biomarkers for patient selection have proven modest sensitivity. Here, we developed VIGex, an optimized gene signature based on the expression level of 12 genes involved in immune response with RNA sequencing. Methods We implemented VIGex using the nCounter platform (Nanostring) on a large clinical cohort encompassing 909 tumor samples across 45 tumor types. VIGex was developed as a continuous variable, with cutoffs selected to detect three main categories (hot, intermediate-cold and cold) based on the different inflammatory status of the tumor microenvironment. Findings Hot tumors had the highest VIGex scores and exhibited an increased abundance of tumor-infiltrating lymphocytes as compared with the intermediate-cold and cold. VIGex scores varied depending on tumor origin and anatomic site of metastases, with liver metastases showing an immunosuppressive tumor microenvironment. The predictive power of VIGex-Hot was observed in a cohort of 98 refractory solid tumor from patients treated in early-phase immunotherapy trials and its clinical performance was confirmed through an extensive metanalysis across 13 clinically annotated gene expression datasets from 877 patients treated with immunotherapy agents. Last, we generated a pan-cancer biomarker platform that integrates VIGex categories with the expression levels of immunotherapy targets under development in early-phase clinical trials. Conclusions Our results support the clinical utility of VIGex as a tool to aid clinicians for patient selection and personalized immunotherapy interventions.A.H.C. would like to acknowledge fellowship funding from the Spanish Society of Medical Oncology (SEOM), CRIS Contra el Cancer and Hold'em For Life Oncology Fellowship. This research has been funded by the Comprehensive Program of Cancer Immunotherapy & Immunology II (CAIMI-II) supported by the BBVA Foundation (grant 53/2021) and the 2020–2021 Division of Medical Oncology and Hematology (DMOH) Fellowship award at Princess Margaret Cancer Centre. VHIO would like to acknowledge the Cellex Foundation for providing research facilities and equipment and the CERCA Programme from the Generalitat de Catalunya for their support of this research. Authors from VHIO acknowledge the State Agency for Research (Agencia Estatal de Investigación) for providing financial support as a Center of Excellence Severo Ochoa (CEX2020-001024-S/AEI/10.13039/501100011033). A.V. was the recipient of a project award from the FAECC (AVP/18/AECC/3219) and received funding from the Advanced Molecular Diagnostic (DIAMAV) program from the FERO Foundation. Graphical abstract was created with BioRender.com. Diagram in Figure 3B was created with SankeyMATIC (sankeymatic.com)

Genetics of male infertility: molecular study of non-syndromic cryptorchidism and spermatogenic impairment

La presente tesis es una aportación al conocimiento de las bases genéticas de la criptorquidia no sindrómica y de las formas idiopáticas de espermatogénesis anómala. RXFP2 y ESR1 son dos genes candidatos en la etiología de la criptorquidia no sindrómica debido al rol del receptor RXFP2 en el control hormonal del descenso testicular y al posible papel del receptor ESR1 como mediador de los efectos de substancias capaces de interferir con el desarrollo del tracto urogenital masculino. La primera parte de la tesis está enfocada en el estudio del papel de dos variantes de estos genes en la etiología de la criptorquidia: la variante de secuencia p.T222P del gen RXFP2 (previamente descrita como una mutación patogénica causante de criptorquidia) y el microsatélite (TA)n del promotor del gen ESR1 estudiado por primera vez en relación con la criptorquidia. Para cada una de las variantes hemos analizado 550 sujetos de origen italiana y 570 de origen española (entre pacientes y controles). En la población española la variante p.T222P se ha encontrado en una proporción parecida de casos (1.6%) y controles (1.8%). Estos resultados indican que dicha variante genética es un polimorfismo común sin significado clínico en esta población. En cambio en la población italiana la frecuencia de la p.T222P ha resultado ser significativamente más alta en pacientes que en controles, lo que sugiere un posible papel para dicha variante como factor de riesgo para la criptorquidia. No hemos observado diferencias significativas en la distribución de los alelos (TA)n entre casos y controles. La frecuencia del genotipo A (supuestamente asociado a mayor actividad del promotor de ESR1) ha resultado ser parecida en casos y controles en ambas poblaciones de estudio. Estos resultados indican que el polimorfismo (TA)n no está asociado con la criptorquidia no sindrómica ni en población española ni en población italiana. La segunda parte de la tesis investiga el papel de variantes de número de copias (CNVs) en los cromosomas Y y X en la etiología de la espermatogenésis anómala. El estudio de las CNVs del cromosoma Y incluye: i) el análisis de más de 800 pacientes infértiles (mayoritariamente españoles) para las microdeleciones del cromosoma Y; ii) el estudio de deleciones y duplicaciones parciales de la región AZFc (estas últimas estudiadas por primera vez en población española) en 330 infértiles idiopáticos y 385 controles normozoospermicos de origen española. Nuestros datos basados en 27 portadores de microdeleciones clasicas del Y (3.3%) junto con una amplia revisión de la literatura indican que el diagnóstico genético de rutina para las microdeleciones del Y se podría limitar a varones con concentración espermática ≤2x106 /ml; ii) la técnica de recuperación espermática testicular más adecuada para los pacientes azoospérmicos portadores de deleciones AZFc es la microTESE; iii) el background del Y podría explicar en parte que las microedeleciones del Y si encuentren en diferente frecuencia en distintas poblaciones. Con respecto al estudio de los reordenamientos parciales en AZFc, hemos corroborado que las deleciones gr/gr (deleciones parciales AZFc) representan un factor de riesgo para la espermatogénesis alterada en población caucásica y hemos descartado que las duplicaciones AZFc contribuyan a esta anomalía. Para estudiar el papel de las CNVs del cromosoma X en la etiología de la espermatogenésis anómala, hemos analizado 199 sujetos con diferente concentración espermática utilizando un array de CGH de alta resolución específico para el cromosoma X. Hemos identificado 73 CNVs entre las cuales 29 perdidas (mayoritariamente raras) y 44 ganancias. El número de deleciones por sujeto y la cantidad media de DNA delecionado han resultado ser significativamente mayores en pacientes que en controles, lo que sugiere que un aumento de la “carga de deleción” podría estar implicado en la etiología de la espermatogenésis anómala. Tres deleciones recurrentes localizadas en Xq27.3 (CNV64) y en Xq28 (CNV67 y CNV69) han sido consideradas de mayor interés por su presencia exclusiva (CNV67) o prevalente (CNV64, CNV69) en el grupo de los pacientes. Dichas deleciones han sido objeto de un estudio profundizado que incluye: i) el screening de 627 infértiles idiopáticos y 628 controles normozoospérmicos de dos poblaciones mediterráneas (española e italiana); ii) la caracterización molecular de las deleciones; iii) la identificación de elementos funcionales afectados por las deleciones. Las CNV64 y CNV69 se han encontrado más frecuentemente en pacientes que en controles. La CNV69 presenta al menos dos patrones de deleción (tipo A y tipo B) de los cuales solo el tipo B ha resultado ser significativamente más representado en pacientes, indicando que este tipo de deleción podría ser responsable del efecto deletéreo de la CNV69 en la espermatogenésis. Las CNV64 y CNV69 no contienen genes en su interior pero podrían afectar una serie de elementos reguladores localizados <0.5 Mb. La CNV67, identificada únicamente en un 1.1% de los pacientes (p<0.01), podría directamente afectar el gen MAGEA9 y/o sus elementos reguladores. El análisis del pedigrí de dos portadores de CNV67 ha evidenciado que esta deleción es trasmitida por la madre. La condición de normozoospermico del hermano no portador de uno de los pacientes portador de CNV67 suporta el posible efecto patogénico de esta deleción en la espermatogenésis. Los resultados presentados en esta tesis suportan la visión que la etiología de la criptorquidia es probablemente poligénica y en la mayoría de los casos multifactorial. Es plausible que la aplicación de nuevas tecnologías de secuenciación masiva, garantizando un análisis más a gran escala del background genético del individuo, contribuyan a desenredar la complejidad de la etiología de esta enfermedad. Futuros estudios serán necesarios para corroborar el significado clínico de la CNV67 y confirmar, en otras poblaciones, la significativa asociación entre CNV64, CNV69 tipo B y espermatogenésis anómala.The present thesis explores the role of specific genetic variants in the etiology of two forms of disturbed male reproductive fitness: non-syndromic cryptorchidism and idiopathic spermatogenic failure. The etiology of non-syndromic cryptorchidism, still largely unknown, is likely to be multifactorial reflecting the involvement of environmental and genetic factors. RXFP2 and ESR1 are interesting candidate genes due to the involvement of RXFP2 receptor in testis descent and the potential role of ESR1 receptor as mediator of substances able to interfere with the development of the male urogenital tract. The first part of the thesis presents the study of two genetic variants of RXFP2 and ESR1, aimed to explore their contribution to non syndromic cryptorchidism. These are the missense substitution T222P in exon 8 of RXFP2, previously proposed as a pathogenic mutation for cryptorchidism, and the VNTR polymorphism (TA)n within the ESR1 promoter, previously reported as a potential functional polymorphism in relation to bone mineralization and spermatogenesis and never studied so far in relation to cryptorchidism. Complessively 550 subjects from Italy and 570 from Spain (with and without history of cryptorchidism) were screened for each variant. The T222P was found at a similar frequency in both patients (1.6%) and controls (1.8%) in the Spanish population thus indicating that in this population it is a common polymorphism with no pathogenic effect. In the Italian study population the frequency of T222P was significantly higher in patients (p = 0.031) supporting a role for this variant as mild risk factor for cryptorchidism (OR= 3.17, 95% CI: 1.07–9.34). Nevertheless, the screening for this variant for diagnostic purposes is not advised because of the relatively high frequency of control carriers (1.4%). Two (TA)n genotypes (A and B) were defined based on the possible allelic combinations of high, medium or low number of repeats and their frequency was compared between cases and controls from the two study populations. Allelic distribution of (TA)n did not show significant differences between cases and controls. The frequency of genotype A, considered the functionally most active one, was also similar in cases and controls both in the Italian and Spanish study populations. These results indicate that the (TA)n within the ESR1 promoter is not associated with non syndromic cryptorchidism neither in the Spanish nor in the Italian population. The second part of the thesis explores the role of Y and X-linked Copy Number Variations (CNVs) in the etiology of spermatogenic impairment. The study of Y-linked CNVs includes: i) the retrospective analysis of 806 mainly Spanish infertile patients screened for Y microdeletions and ii) the study of partial AZFc deletions and duplications (the latter studied here for the first time in the Spanish population) including 330 idiopathic infertile patients and 385 controls from Spain. A total of 27/806 (3.3%) patients carried complete AZF deletions. All were azoo/cryptozoospermic, except for one whose sperm concentration was 1- 2x106/ml. This finding integrated with the literature suggests that routine AZF microdeletion testing could eventually include only men with ≤2x106/ml. In AZFc deleted men a lower sperm recovery rate was observed upon conventional TESE (9.1%) compared to the literature (60%-80% with microTESE) indicating that microTESE ensuring better outcomes, should be regarded as the best option. Haplogroup (hgr) E was the most represented among non- Spanish whereas hgr P among Spanish AZF deletion carriers supporting a potential contribution of Y background to the inter-population variability in deletion frequency. The gr/gr deletion was significantly associated with spermatogenic impairment, further supporting the inclusion of this genetic test in the work-up of infertile men, while partial AZFc duplications do not represent a risk for spermatogenic failure in the Spanish population. The present thesis addresses the topic of X-linked CNVs in spermatogenic defects by presenting the X-chromosome array-CGH (a-CGH) analysis of 199 men with different sperm count. A total of 73 CNVs were identified including 29 mostly rare losses and 44 gains. A significantly higher burden of deletions was found in patients compared to controls due to an excessive rate of deletions/person (0.57 versus 0.21, respectively; p =8.78x10-6) and to a higher mean sequence loss/person (11.79 Kb and 8.13 Kb, respectively; p = 3.43 x10-6). This finding suggests that an increased X chromosome deletion burden may be involved in the etiology of spermatogenic impairment. X-linked Cancer Testis Antigen (CTA) genes resulted to be frequently affected, indicating that their dosage variation may play a role in X-linked CNV-related spermatogenic failure. Three recurrent deletions, mapping to the Xq27.3 (CNV64) and Xq28 (CNV67 and CNV69), were considered of interest for their exclusive (CNV67) or prevalent (CNV64 and CNV69) presence in patients. These deletions were object of an in-depth analysis including: i) the screening of 627 idiopathic patients and 628 normozoospermic controls from two Mediterranean populations (Spanish and Italian); ii) the molecular characterization of deletions; iii) the exploration for functional elements. CNV64 and CNV69 were significantly more frequent in patients than controls (OR=1.9 and 2.2, for CNV64 and CNV69 respectively). CNV69 displayed at least two deletion patterns (type A and type B), of which type B, being significantly more represented in patients than controls, may account for the potential deleterious effect of CNV69 on sperm production. No genes have been identified inside CNV64 and CNV69, nevertheless a number of regulatory elements, have been found to be potentially affected. CNV67 deletion was exclusively found in patients at a frequency of 1,1% (p<0.01) thus resembling the AZF region of the Y chromosome. This deletion may involve the CTA gene MAGEA9 and/or of its regulatory elements. It may also affect regulatory elements of HSFX1/2 showing testis-specific expression. Pedigree analyses of two CNV67 carriers indicated that CNV67 deletion is maternally inherited. One of these families was especially informative since the pathological semen phenotype of the carrier versus his normozoospermic non-carrier brother was a strong indicator for a pathogenic effect of the deletion on spermatogenesis. The results of the present thesis are in line with the prevalent view that the etiology of nonsyndromic cryptorchidism is likely to be polygenic and in most of cases multifactorial. We are confident that high throughput technologies, especially next generation sequencing, will help to unravel the complexity of the etiology of this disease. We provide evidence for a significant association between recurrent X-linked deletions and impaired sperm production. Further studies in other ethnic-geographic groups are needed to confirm the association of CNV64 and CNV69 type B with spermatogenic failure. Due to the specific association of CNV67 with spermatogenic impairment a parallelism with AZF deletions of the Y chromosome is tempting. This CNV merits further investigation in order to provide a feasible substrate for fine molecular characterization, and to further evaluate its putative diagnostic value

Genetics of male infertility : molecular study of non-syndromic cryptorchidism and spermatogenic impairment

La presente tesis es una aportación al conocimiento de las bases genéticas de la criptorquidia no sindrómica y de las formas idiopáticas de espermatogénesis anómala. RXFP2 y ESR1 son dos genes candidatos en la etiología de la criptorquidia no sindrómica debido al rol del receptor RXFP2 en el control hormonal del descenso testicular y al posible papel del receptor ESR1 como mediador de los efectos de substancias capaces de interferir con el desarrollo del tracto urogenital masculino. La primera parte de la tesis está enfocada en el estudio del papel de dos variantes de estos genes en la etiología de la criptorquidia: la variante de secuencia p.T222P del gen RXFP2 (previamente descrita como una mutación patogénica causante de criptorquidia) y el microsatélite (TA)n del promotor del gen ESR1 estudiado por primera vez en relación con la criptorquidia. Para cada una de las variantes hemos analizado 550 sujetos de origen italiana y 570 de origen española (entre pacientes y controles). En la población española la variante p.T222P se ha encontrado en una proporción parecida de casos (1.6%) y controles (1.8%). Estos resultados indican que dicha variante genética es un polimorfismo común sin significado clínico en esta población. En cambio en la población italiana la frecuencia de la p.T222P ha resultado ser significativamente más alta en pacientes que en controles, lo que sugiere un posible papel para dicha variante como factor de riesgo para la criptorquidia. No hemos observado diferencias significativas en la distribución de los alelos (TA)n entre casos y controles. La frecuencia del genotipo A (supuestamente asociado a mayor actividad del promotor de ESR1) ha resultado ser parecida en casos y controles en ambas poblaciones de estudio. Estos resultados indican que el polimorfismo (TA)n no está asociado con la criptorquidia no sindrómica ni en población española ni en población italiana. La segunda parte de la tesis investiga el papel de variantes de número de copias (CNVs) en los cromosomas Y y X en la etiología de la espermatogenésis anómala. El estudio de las CNVs del cromosoma Y incluye: i) el análisis de más de 800 pacientes infértiles (mayoritariamente españoles) para las microdeleciones del cromosoma Y; ii) el estudio de deleciones y duplicaciones parciales de la región AZFc (estas últimas estudiadas por primera vez en población española) en 330 infértiles idiopáticos y 385 controles normozoospermicos de origen española. Nuestros datos basados en 27 portadores de microdeleciones clasicas del Y (3.3%) junto con una amplia revisión de la literatura indican que el diagnóstico genético de rutina para las microdeleciones del Y se podría limitar a varones con concentración espermática ≤2x106 /ml; ii) la técnica de recuperación espermática testicular más adecuada para los pacientes azoospérmicos portadores de deleciones AZFc es la microTESE; iii) el background del Y podría explicar en parte que las microedeleciones del Y si encuentren en diferente frecuencia en distintas poblaciones. Con respecto al estudio de los reordenamientos parciales en AZFc, hemos corroborado que las deleciones gr/gr (deleciones parciales AZFc) representan un factor de riesgo para la espermatogénesis alterada en población caucásica y hemos descartado que las duplicaciones AZFc contribuyan a esta anomalía. Para estudiar el papel de las CNVs del cromosoma X en la etiología de la espermatogenésis anómala, hemos analizado 199 sujetos con diferente concentración espermática utilizando un array de CGH de alta resolución específico para el cromosoma X. Hemos identificado 73 CNVs entre las cuales 29 perdidas (mayoritariamente raras) y 44 ganancias. El número de deleciones por sujeto y la cantidad media de DNA delecionado han resultado ser significativamente mayores en pacientes que en controles, lo que sugiere que un aumento de la "carga de deleción" podría estar implicado en la etiología de la espermatogenésis anómala. Tres deleciones recurrentes localizadas en Xq27.3 (CNV64) y en Xq28 (CNV67 y CNV69) han sido consideradas de mayor interés por su presencia exclusiva (CNV67) o prevalente (CNV64, CNV69) en el grupo de los pacientes. Dichas deleciones han sido objeto de un estudio profundizado que incluye: i) el screening de 627 infértiles idiopáticos y 628 controles normozoospérmicos de dos poblaciones mediterráneas (española e italiana); ii) la caracterización molecular de las deleciones; iii) la identificación de elementos funcionales afectados por las deleciones. Las CNV64 y CNV69 se han encontrado más frecuentemente en pacientes que en controles. La CNV69 presenta al menos dos patrones de deleción (tipo A y tipo B) de los cuales solo el tipo B ha resultado ser significativamente más representado en pacientes, indicando que este tipo de deleción podría ser responsable del efecto deletéreo de la CNV69 en la espermatogenésis. Las CNV64 y CNV69 no contienen genes en su interior pero podrían afectar una serie de elementos reguladores localizados 0.5 Mb. La CNV67, identificada únicamente en un 1.1% de los pacientes (p 0.01), podría directamente afectar el gen MAGEA9 y/o sus elementos reguladores. El análisis del pedigrí de dos portadores de CNV67 ha evidenciado que esta deleción es trasmitida por la madre. La condición de normozoospermico del hermano no portador de uno de los pacientes portador de CNV67 suporta el posible efecto patogénico de esta deleción en la espermatogenésis. Los resultados presentados en esta tesis suportan la visión que la etiología de la criptorquidia es probablemente poligénica y en la mayoría de los casos multifactorial. Es plausible que la aplicación de nuevas tecnologías de secuenciación masiva, garantizando un análisis más a gran escala del background genético del individuo, contribuyan a desenredar la complejidad de la etiología de esta enfermedad. Futuros estudios serán necesarios para corroborar el significado clínico de la CNV67 y confirmar, en otras poblaciones, la significativa asociación entre CNV64, CNV69 tipo B y espermatogenésis anómala.The present thesis explores the role of specific genetic variants in the etiology of two forms of disturbed male reproductive fitness: non-syndromic cryptorchidism and idiopathic spermatogenic failure. The etiology of non-syndromic cryptorchidism, still largely unknown, is likely to be multifactorial reflecting the involvement of environmental and genetic factors. RXFP2 and ESR1 are interesting candidate genes due to the involvement of RXFP2 receptor in testis descent and the potential role of ESR1 receptor as mediator of substances able to interfere with the development of the male urogenital tract. The first part of the thesis presents the study of two genetic variants of RXFP2 and ESR1, aimed to explore their contribution to non syndromic cryptorchidism. These are the missense substitution T222P in exon 8 of RXFP2, previously proposed as a pathogenic mutation for cryptorchidism, and the VNTR polymorphism (TA)n within the ESR1 promoter, previously reported as a potential functional polymorphism in relation to bone mineralization and spermatogenesis and never studied so far in relation to cryptorchidism. Complessively 550 subjects from Italy and 570 from Spain (with and without history of cryptorchidism) were screened for each variant. The T222P was found at a similar frequency in both patients (1.6%) and controls (1.8%) in the Spanish population thus indicating that in this population it is a common polymorphism with no pathogenic effect. In the Italian study population the frequency of T222P was significantly higher in patients (p = 0.031) supporting a role for this variant as mild risk factor for cryptorchidism (OR= 3.17, 95% CI: 1.07-9.34). Nevertheless, the screening for this variant for diagnostic purposes is not advised because of the relatively high frequency of control carriers (1.4%). Two (TA)n genotypes (A and B) were defined based on the possible allelic combinations of high, medium or low number of repeats and their frequency was compared between cases and controls from the two study populations. Allelic distribution of (TA)n did not show significant differences between cases and controls. The frequency of genotype A, considered the functionally most active one, was also similar in cases and controls both in the Italian and Spanish study populations. These results indicate that the (TA)n within the ESR1 promoter is not associated with non syndromic cryptorchidism neither in the Spanish nor in the Italian population. The second part of the thesis explores the role of Y and X-linked Copy Number Variations (CNVs) in the etiology of spermatogenic impairment. The study of Y-linked CNVs includes: i) the retrospective analysis of 806 mainly Spanish infertile patients screened for Y microdeletions and ii) the study of partial AZFc deletions and duplications (the latter studied here for the first time in the Spanish population) including 330 idiopathic infertile patients and 385 controls from Spain. A total of 27/806 (3.3%) patients carried complete AZF deletions. All were azoo/cryptozoospermic, except for one whose sperm concentration was 1- 2x106/ml. This finding integrated with the literature suggests that routine AZF microdeletion testing could eventually include only men with ≤2x106/ml. In AZFc deleted men a lower sperm recovery rate was observed upon conventional TESE (9.1%) compared to the literature (60%-80% with microTESE) indicating that microTESE ensuring better outcomes, should be regarded as the best option. Haplogroup (hgr) E was the most represented among non- Spanish whereas hgr P among Spanish AZF deletion carriers supporting a potential contribution of Y background to the inter-population variability in deletion frequency. The gr/gr deletion was significantly associated with spermatogenic impairment, further supporting the inclusion of this genetic test in the work-up of infertile men, while partial AZFc duplications do not represent a risk for spermatogenic failure in the Spanish population. The present thesis addresses the topic of X-linked CNVs in spermatogenic defects by presenting the X-chromosome array-CGH (a-CGH) analysis of 199 men with different sperm count. A total of 73 CNVs were identified including 29 mostly rare losses and 44 gains. A significantly higher burden of deletions was found in patients compared to controls due to an excessive rate of deletions/person (0.57 versus 0.21, respectively; p =8.78x10-6) and to a higher mean sequence loss/person (11.79 Kb and 8.13 Kb, respectively; p = 3.43 x10-6). This finding suggests that an increased X chromosome deletion burden may be involved in the etiology of spermatogenic impairment. X-linked Cancer Testis Antigen (CTA) genes resulted to be frequently affected, indicating that their dosage variation may play a role in X-linked CNV-related spermatogenic failure. Three recurrent deletions, mapping to the Xq27.3 (CNV64) and Xq28 (CNV67 and CNV69), were considered of interest for their exclusive (CNV67) or prevalent (CNV64 and CNV69) presence in patients. These deletions were object of an in-depth analysis including: i) the screening of 627 idiopathic patients and 628 normozoospermic controls from two Mediterranean populations (Spanish and Italian); ii) the molecular characterization of deletions; iii) the exploration for functional elements. CNV64 and CNV69 were significantly more frequent in patients than controls (OR=1.9 and 2.2, for CNV64 and CNV69 respectively). CNV69 displayed at least two deletion patterns (type A and type B), of which type B, being significantly more represented in patients than controls, may account for the potential deleterious effect of CNV69 on sperm production. No genes have been identified inside CNV64 and CNV69, nevertheless a number of regulatory elements, have been found to be potentially affected. CNV67 deletion was exclusively found in patients at a frequency of 1,1% (p 0.01) thus resembling the AZF region of the Y chromosome. This deletion may involve the CTA gene MAGEA9 and/or of its regulatory elements. It may also affect regulatory elements of HSFX1/2 showing testis-specific expression. Pedigree analyses of two CNV67 carriers indicated that CNV67 deletion is maternally inherited. One of these families was especially informative since the pathological semen phenotype of the carrier versus his normozoospermic non-carrier brother was a strong indicator for a pathogenic effect of the deletion on spermatogenesis. The results of the present thesis are in line with the prevalent view that the etiology of nonsyndromic cryptorchidism is likely to be polygenic and in most of cases multifactorial. We are confident that high throughput technologies, especially next generation sequencing, will help to unravel the complexity of the etiology of this disease. We provide evidence for a significant association between recurrent X-linked deletions and impaired sperm production. Further studies in other ethnic-geographic groups are needed to confirm the association of CNV64 and CNV69 type B with spermatogenic failure. Due to the specific association of CNV67 with spermatogenic impairment a parallelism with AZF deletions of the Y chromosome is tempting. This CNV merits further investigation in order to provide a feasible substrate for fine molecular characterization, and to further evaluate its putative diagnostic value

X Chromosome-Linked CNVs in Male Infertility: Discovery of Overall Duplication Load and Recurrent, Patient-Specific Gains with Potential Clinical Relevance

<div><p>Introduction</p><p>Spermatogenesis is a highly complex process involving several thousand genes, only a minority of which have been studied in infertile men. In a previous study, we identified a number of Copy Number Variants (CNVs) by high-resolution array-Comparative Genomic Hybridization (a-CGH) analysis of the X chromosome, including 16 patient-specific X chromosome-linked gains. Of these, five gains (DUP1A, DUP5, DUP20, DUP26 and DUP40) were selected for further analysis to evaluate their clinical significance.</p><p>Materials and Methods</p><p>The copy number state of the five selected loci was analyzed by quantitative-PCR on a total of 276 idiopathic infertile patients and 327 controls in a conventional case-control setting (199 subjects belonged to the previous a-CGH study). For one interesting locus (intersecting DUP1A) additional 338 subjects were analyzed.</p><p>Results and Discussion</p><p>All gains were confirmed as patient-specific and the difference in duplication load between patients and controls is significant <i>(p = 1.65×10⁻⁴).</i> Two of the CNVs are private variants, whereas 3 are found recurrently in patients and none of the controls. These CNVs include, or are in close proximity to, genes with testis-specific expression. DUP1A, mapping to the PAR1, is found at the highest frequency (1.4%) that was significantly different from controls (0%) (<i>p = 0.047</i> after Bonferroni correction). Two mechanisms are proposed by which DUP1A may cause spermatogenic failure: i) by affecting the correct regulation of a gene with potential role in spermatogenesis; ii) by disturbing recombination between PAR1 regions during meiosis. This study allowed the identification of novel spermatogenesis candidate genes linked to the 5 CNVs and the discovery of the first recurrent, X-linked gain with potential clinical relevance.</p></div

High Resolution X Chromosome-Specific Array-CGH Detects New CNVs in Infertile Males

<div><h3>Context</h3><p>The role of CNVs in male infertility is poorly defined, and only those linked to the Y chromosome have been the object of extensive research. Although it has been predicted that the X chromosome is also enriched in spermatogenesis genes, no clinically relevant gene mutations have been identified so far.</p> <h3>Objectives</h3><p>In order to advance our understanding of the role of X-linked genetic factors in male infertility, we applied high resolution X chromosome specific array-CGH in 199 men with different sperm count followed by the analysis of selected, patient-specific deletions in large groups of cases and normozoospermic controls.</p> <h3>Results</h3><p>We identified 73 CNVs, among which 55 are novel, providing the largest collection of X-linked CNVs in relation to spermatogenesis. We found 12 patient-specific deletions with potential clinical implication. Cancer Testis Antigen gene family members were the most frequently affected genes, and represent new genetic targets in relationship with altered spermatogenesis. One of the most relevant findings of our study is the significantly higher global burden of deletions in patients compared to controls due to an excessive rate of deletions/person (0.57 versus 0.21, respectively; p = 8.785×10⁻⁶) and to a higher mean sequence loss/person (11.79 Kb and 8.13 Kb, respectively; p = 3.435×10⁻⁴).</p> <h3>Conclusions</h3><p>By the analysis of the X chromosome at the highest resolution available to date, in a large group of subjects with known sperm count we observed a deletion burden in relation to spermatogenic impairment and the lack of highly recurrent deletions on the X chromosome. We identified a number of potentially important patient-specific CNVs and candidate spermatogenesis genes, which represent novel targets for future investigations.</p> </div

Case-control study of selected losses preliminarily identified by array-CGH as patient-specific (not found in normozoospermic controls).

<p>Genes inside the CNV minimum size are depicted in bold;</p>*<p>genes inside the CNV maximum size;</p>§<p>the first proximal flanking gene;</p>†<p>the first distal flanking gene; the remaining genes are situated <500 Kb from the minimum size border. Azoosp = Azoospermia; Oligozoosp = Oligozoospermia; Cryptozoosp = Cryptozoospermia; SCOS = Sertoli Cell Only Syndrome; SGA = Spermatogenic Arrest.</p

List of the 31 patient-specific (not found in normozoospermic controls) CNVs detected by array-CGH and their description according to type, gene location (<i>NO</i> = no gene found within) and occurrence in the Database of Genomic Variants (DGV).

*<p>CNV minimum size.</p

Array-CGH study: Comparison between patients and controls of the mean number and mean extension of CNVs (A) as well as the number of all subjects bearing more than one CNV (B).

<p>sd = standard deviation. OR = odds ratio. CI = confidence interval. <i>p</i>1 refers to the mean number of CNV/subject. <i>p</i>2 refers to the mean DNA change/subject.</p

List of the 33 control-specific (not found in idiopathic patients) CNVs detected by array-CGH and their description according to type, gene location (<i>NO</i> = no gene found within) and occurrence in the Database of Genomic Variants (DGV).

*<p>CNV minimum size.</p