RESUMEN
Structural variants (SVs) pose a challenge to detect and interpret, but their study provides novel biological insights and molecular diagnosis underlying rare diseases. The aim of this study was to resolve a 9p24 rearrangement segregating in a family through five generations with a congenital heart defect (congenital pulmonary and aortic valvular stenosis and pulmonary artery stenosis), by applying a combined genomic analysis. The analysis involved multiple techniques, including karyotype, chromosomal microarray analysis (CMA), FISH, genome sequencing (GS), RNA-seq, and optical genome mapping (OGM). A complex 9p24 SV was hinted at by CMA results, showing three interspersed duplicated segments. Combined GS and OGM analyses revealed that the 9p24 duplications constitute a complex SV, on which a set of breakpoints matches the boundaries of the CMA duplicated sequences. The proposed structure for this complex rearrangement implies three duplications associated with an inversion of ~ 2 Mb region on chromosome 9 and a SINE element insertion at the more distal breakpoint. Interestingly, this genomic structure of rearrangement forms a chimeric transcript of the KANK1/DMRT1 loci, which was confirmed by both RNA-seq and Sanger sequencing on blood samples from 9p24 rearrangement carriers. Altogether with breakpoint amplification and FISH analysis, this combined approach allowed a deep characterization of this complex rearrangement. Although the genotype-phenotype correlation remains elusive from the molecular mechanism point of view, this study identified a large genomic rearrangement at 9p24 segregating with a familial congenital heart defect, revealing a genetic biomarker that was successfully applied for embryo selection, changing the reproductive perspective of affected individuals.
Asunto(s)
Cromosomas , Variaciones en el Número de Copia de ADN , Humanos , Inversión Cromosómica , Secuencia de Bases , Células Germinativas , Proteínas del Citoesqueleto/genética , Proteínas Adaptadoras Transductoras de Señales/genéticaRESUMEN
Robinow syndrome is characterized by a triad of craniofacial dysmorphisms, disproportionate-limb short stature, and genital hypoplasia. A significant degree of phenotypic variability seems to correlate with different genes/loci. Disturbances of the noncanonical WNT-pathway have been identified as the main cause of the syndrome. Biallelic variants in ROR2 cause an autosomal recessive form of the syndrome with distinctive skeletal findings. Twenty-two patients with a clinical diagnosis of autosomal recessive Robinow syndrome were screened for variants in ROR2 using multiple molecular approaches. We identified 25 putatively pathogenic ROR2 variants, 16 novel, including single nucleotide variants and exonic deletions. Detailed phenotypic analyses revealed that all subjects presented with a prominent forehead, hypertelorism, short nose, abnormality of the nasal tip, brachydactyly, mesomelic limb shortening, short stature, and genital hypoplasia in male patients. A total of 19 clinical features were present in more than 75% of the subjects, thus pointing to an overall uniformity of the phenotype. Disease-causing variants in ROR2, contribute to a clinically recognizable autosomal recessive trait phenotype with multiple skeletal defects. A comprehensive quantitative clinical evaluation of this cohort delineated the phenotypic spectrum of ROR2-related Robinow syndrome. The identification of exonic deletion variant alleles further supports the contention of a loss-of-function mechanism in the etiology of the syndrome.
Asunto(s)
Anomalías Craneofaciales , Enanismo , Deformidades Congénitas de las Extremidades , Receptores Huérfanos Similares al Receptor Tirosina Quinasa , Anomalías Urogenitales , Anomalías Craneofaciales/diagnóstico , Anomalías Craneofaciales/genética , Enanismo/diagnóstico , Enanismo/genética , Genes Recesivos , Humanos , Deformidades Congénitas de las Extremidades/diagnóstico , Deformidades Congénitas de las Extremidades/genética , Masculino , Fenotipo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Anomalías Urogenitales/diagnóstico , Anomalías Urogenitales/genéticaRESUMEN
We describe monozygotic twin girls with genetic variation at two separate loci resulting in a blended phenotype of Prader-Willi syndrome and Pitt-Hopkins syndrome. These girls were diagnosed in early infancy with Prader-Willi syndrome, but developed an atypical phenotype, with apparent intellectual deficiency and lack of obesity. Array-comparative genomic hybridization confirmed a de novo paternal deletion of the 15q11.2q13 region and exome sequencing identified a second mutational event in both girls, which was a novel variant c.145+1G>A affecting a TCF4 canonical splicing site inherited from the mosaic mother. RNA studies showed that the variant abolished the donor splicing site, which was accompanied by activation of an alternative non-canonical splicing-site which then predicts a premature stop codon in the following exon. Clinical re-evaluation of the twins indicated that both variants are likely contributing to the more severe phenotypic presentation. Our data show that atypical clinical presentations may actually be the expression of blended clinical phenotypes arising from independent pathogenic events at two loci.
Asunto(s)
Hiperventilación/genética , Discapacidad Intelectual/genética , Patología Molecular , Síndrome de Prader-Willi/genética , Factor de Transcripción 4/genética , Adolescente , Secuencia de Bases/genética , Niño , Deleción Cromosómica , Cromosomas Humanos Par 15/genética , Hibridación Genómica Comparativa , Exoma/genética , Facies , Femenino , Humanos , Hiperventilación/diagnóstico , Hiperventilación/fisiopatología , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/fisiopatología , Obesidad/diagnóstico , Obesidad/genética , Obesidad/fisiopatología , Fenotipo , Síndrome de Prader-Willi/diagnóstico , Síndrome de Prader-Willi/fisiopatología , Gemelos MonocigóticosRESUMEN
We investigated complex genomic rearrangements (CGRs) consisting of triplication copy-number variants (CNVs) that were accompanied by extended regions of copy-number-neutral absence of heterozygosity (AOH) in subjects with multiple congenital abnormalities. Molecular analyses provided observational evidence that in humans, post-zygotically generated CGRs can lead to regional uniparental disomy (UPD) due to template switches between homologs versus sister chromatids by using microhomology to prime DNA replication-a prediction of the replicative repair model, MMBIR. Our findings suggest that replication-based mechanisms might underlie the formation of diverse types of genomic alterations (CGRs and AOH) implicated in constitutional disorders.
Asunto(s)
Variaciones en el Número de Copia de ADN/genética , Reparación del ADN/genética , Replicación del ADN/genética , Reordenamiento Génico/genética , Pérdida de Heterocigocidad/genética , Modelos Genéticos , Disomía Uniparental/genética , Secuencia de Bases , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Países Bajos , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ADNRESUMEN
The 17p13.1 microdeletion syndrome is a recently described genomic disorder with a core clinical phenotype of intellectual disability, poor to absent speech, dysmorphic features, and a constellation of more variable clinical features, most prominently microcephaly. We identified five subjects with copy-number variants (CNVs) on 17p13.1 for whom we performed detailed clinical and molecular studies. Breakpoint mapping and retrospective analysis of published cases refined the smallest region of overlap (SRO) for microcephaly to a genomic interval containing nine genes. Dissection of this phenotype in zebrafish embryos revealed a complex genetic architecture: dosage perturbation of four genes (ASGR1, ACADVL, DVL2, and GABARAP) impeded neurodevelopment and decreased dosage of the same loci caused a reduced mitotic index in vitro. Moreover, epistatic analyses in vivo showed that dosage perturbations of discrete gene pairings induce microcephaly. Taken together, these studies support a model in which concomitant dosage perturbation of multiple genes within the CNV drive the microcephaly and possibly other neurodevelopmental phenotypes associated with rearrangements in the 17p13.1 SRO.
Asunto(s)
Anomalías Múltiples/genética , Dosificación de Gen/genética , Discapacidad Intelectual/genética , Microcefalia/genética , Acil-CoA Deshidrogenasa de Cadena Larga/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas Reguladoras de la Apoptosis , Receptor de Asialoglicoproteína/genética , Secuencia de Bases , Línea Celular , Puntos de Rotura del Cromosoma , Deleción Cromosómica , Cromosomas Humanos Par 17/genética , Proteínas Dishevelled , Citometría de Flujo , Humanos , Inmunohistoquímica , Proteínas Asociadas a Microtúbulos/genética , Datos de Secuencia Molecular , Fosfoproteínas/genética , Estudios Retrospectivos , Análisis de Secuencia de ADN , Síndrome de Smith-Magenis , Síndrome , Pez CebraRESUMEN
We investigated 67 breakpoint junctions of gene copy number gains in 31 unrelated subjects. We observed a strikingly high frequency of small deletions and insertions (29%) apparently originating from polymerase slippage events, in addition to frameshifts and point mutations in homonucleotide runs (13%), at or flanking the breakpoint junctions of complex copy number variants. These single-nucleotide variants were generated concomitantly with the de novo complex genomic rearrangement (CGR) event. Our findings implicate low-fidelity, error-prone DNA polymerase activity in synthesis associated with DNA repair mechanisms as the cause of local increase in point mutation burden associated with human CGR.
Asunto(s)
Roturas del ADN , Variaciones en el Número de Copia de ADN/genética , Reparación del ADN/genética , Replicación del ADN/genética , Reordenamiento Génico/genética , Secuencia de Bases , Mutación del Sistema de Lectura , Variación Genética , Genotipo , Humanos , Análisis de Secuencia de ADN , Eliminación de SecuenciaRESUMEN
BACKGROUND/AIMS: Approximately four million Africans were taken as slaves to Brazil, where they interbred extensively with Amerindians and Europeans. We have previously shown that while most White Brazilians carry Y chromosomes of European origin, they display high proportions of African and Amerindian mtDNA lineages, because of sex-biased genetic admixture. METHODS: We studied the Y chromosome and mtDNA haplogroup structure of 120 Black males from Sao Paulo, Brazil. RESULTS: Only 48% of the Y chromosomes, but 85% of the mtDNA haplogroups were characteristic of sub-Saharan Africa, confirming our previous observation of sexually biased mating. We mined literature data for mtDNA and Y chromosome haplogroup frequencies for African native populations from regions involved in Atlantic Slave Trade. Principal Components Analysis and Bayesian analysis of population structure revealed no genetic differentiation of Y chromosome marker frequencies between the African regions. However, mtDNA examination unraveled considerable genetic structure, with three clusters at Central-West Africa, West Africa and Southeast Africa. A hypothesis is proposed to explain this structure. CONCLUSION: Using these mtDNA data we could obtain for the first time an estimate of the relative ancestral contribution of Central-West (0.445), West (0.431) and Southeast Africa (0.123) to African Brazilians from Sao Paulo. These estimates are consistent with historical information.
Asunto(s)
Geografía , Filogenia , África/etnología , Brasil , Cromosomas Humanos Y , ADN Mitocondrial/genética , Humanos , Indígenas Sudamericanos , MasculinoRESUMEN
OBJECTIVE: To investigate the evolutionary and demographic history of the Gaucho, a distinct population of southern Brazil, relating it to their culture, to assess possible parallel continuity. METHODS: Six binary polymorphisms, an Alu insertion polymorphism (YAP) and 12 short tandem repeat loci in the non-recombining region of the Y-chromosome, as well as the sequence of the first hypervariable segment (HVS-I) of the mitochondrial DNA (mtDNA) control region were studied in 150 unrelated males born in the Pampa region of Rio Grande do Sul. RESULTS: Comparison of the results with the other Brazilian and Uruguayan populations, as well as with their putative ancestors, indicated a stronger male Spanish influence than that observed elsewhere in Brazil, a former Portuguese colony. Extensive mtDNA analyses of their Amerindian component gave clear indications of the presence there of material from extinct (Charrua), as well as extant (Guarani) tribes. CONCLUSIONS: The genetic analyses contributed in a significant way to reveal that the known cultural continuity between pre- and post-Columbian Pampa populations was also accompanied by an extraordinary genetic continuity.
Asunto(s)
Cultura , Flujo Génico , Polimorfismo Genético , Brasil/etnología , Cromosomas Humanos Y/genética , ADN Mitocondrial/genética , Efecto Fundador , Humanos , Indígenas Sudamericanos , MasculinoRESUMEN
The Y chromosome carries several spermatogenesis genes distributed in three regions: AZFa, AZFb and AZFc. Microdeletions in these regions have been seen in 10% of sterile males with azoospermia or oligozoospermia, the most frequent of them being characterized by a complete deletion of AZFc region. A partial AZFc deletion named gr/gr has been singled out as a risk factor for spermatogenic failure. However, other authors have diagnosed it as a polymorphic deletion with no clinical relevance. We decided to investigate the association of gr/gr deletion and infertility in Brazilian males. We analysed 350 individuals (110 azoospermic, 122 fertile and 118 presumably fertile) and observed 12 g/gr deletions: five in infertile men (4.5%), three among fertile males (2.5%) and four in probably fertile individuals (3.4%). These differences were not statistically significant. Then, we decided to ascertain whether the clinical impact of the gr/gr deletion was associated with the type of Y chromosome. We have identified Y-chromosome haplogroups using 22 unique event polymorphisms (UEPs). Among the individuals with the gr/gr deletion, we found haplogroups R, K*, F*, E1, E3b2 and E3b*, all of which are common in white Brazilian males, and none revealed a particular association with infertility. Taken together, these results show no evidence of association between the occurrence of gr/gr deletion and male infertility.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Y/genética , Infertilidad Masculina/genética , Eliminación de Secuencia/genética , Brasil , Análisis Mutacional de ADN , Sitios Genéticos , Genética de Población , Haplotipos/genética , Humanos , Masculino , Oligospermia/genética , Polimorfismo de Nucleótido Simple , Proteínas de Plasma Seminal/genética , Espermatogénesis/genéticaRESUMEN
Several technologically sophisticated high-throughput techniques have been recently developed for the study of human single nucleotide polymorphisms and the diagnosis of point mutations in human diseases. However, there is also a need for simple and inexpensive techniques suitable for clinical services and small research laboratories. Minisequencing meets the latter requirements. It is simple, non-radioactive and can be easily multiplexed by adding oligonucleotide tails of increasing size to the sequencing oligonucleotide primers. To optimize the minisequencing protocol, we designed a test multiplex system capable of typing simultaneously 12 different human autosomal single nucleotide polymorphisms. We discovered that the quality of minisequencing primers and the careful selection of the tail sequences were especially critical for success. This optimized protocol permits rapid genotyping at low cost and can serve as a blueprint for the creation of multiplex minisequencing systems suitable to virtually any typing application in population studies and medical genetics.
Asunto(s)
Cartilla de ADN/genética , Mutación Puntual/genética , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ADN/métodos , Genotipo , HumanosRESUMEN
The Y chromosome carries several genes involved in spermatogenesis, which are distributed in three regions in the euchromatic part of the long arm, called AZFa (azoospermia factor a), AZFb, and AZFc. Microdeletions in these regions have been seen in 10-15% of sterile males with azoospermia or severe oligozoospermia. The relatively high de novo occurrence of these microdeletion events might be due to particular chromosome arrangements associated with certain Y chromosome haplogroups. To test whether there is any association between Y chromosome types and male infertility, we studied a sample of 84 Japanese oligozoospermic or azoospermic males. The patients were analyzed for the presence of Yq microdeletions and also typed with a battery of unique event polymorphisms (UEPs) to define their Y haplogroups. Six of the infertile patients presented likely pathological microdeletions detectable with the sequence tagged sites (STS) markers used. There was no significant association between Y chromosome haplogroups and the microdeletions. We also compared the Y haplogroup frequencies in our subset sample of 51 idiopathic azoospermia patients with 57 fertile control Japanese males, and did not observe any significant differences. Contrary to previous reports, our data suggest that Y microdeletions and other molecular events causally associated with male infertility in Japan occur independently of the Y chromosome background.