Validación de nuevas tecnologías genómicas en la rutina clínica asistencial en pacientes con trastorno del espectro autista primario, mediante el abordaje combinado de microarrays y NGS

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Medicina. Fecha de Lectura: 17-11-2022El trastorno del espectro autista (TEA) son un conjunto de alteraciones del neurodesarrollo caracterizadas por deficiencias en la comunicación, interacción social, conductas repetitivas y restrictivas. El descubrimiento de la implicación genética en la etiología del TEA ha convertido a esta afección en un fuerte candidato para las pruebas de diagnóstico basadas en el genoma. El uso de microarrays de SNP (Single Nucleotide Polymorphim) y CGH (Comparative Genome Hybridization) (en adelante CMA, del inglés chromosomal microarray analysis) ha demostrado ser un método rápido y eficaz para detectar micro y grandes deleciones/duplicaciones asociadas con el TEA, mientras que la NGS (next generation sequencing) es de utilidad para la detección de variantes en la secuencia de diferentes genes en pacientes TEA. Presentamos en este trabajo de Tesis Doctoral la implementación del uso de los CMA y panel personalizado de NGS para autismo (AutismSeq), como prueba de primer nivel, para pacientes con TEA en un período prospectivo de cuatro años en la rutina clínica de un hospital terciario. La cohorte está compuesta por 212 individuos, mayores de 3 años, que cumplían con los criterios diagnósticos del DSM-V para TEA. Usando KaryoArray®, un CMA de diseño propio en casos prospectivos de TEA, encontramos 96 CNVs en 212 individuos (45,2%), de los que 34 pacientes (35%) tienen deleciones y 62 pacientes (65%) duplicaciones. Veintisiete de los 212 pacientes eran portadores de CNVs patogénicas o posiblemente patogénicas (pathogenic/likely pathogenic; P/LP), respectivamente, lo que representa alrededor del 12,7% de la muestra. Un 21,2% (45/212) eran portadores de reordenamientos benignos y 24 de 212 (11,32%) eran portadores de variantes de significado incierto (VUS, del inglés, variant of uncertain significance). Por otro lado, el panel NGS (AutismSeq) de diseño propio, resultó ser una herramienta con buena eficacia diagnóstica en la rutina asistencial, donde detectamos 11 mutaciones patogénicas (23%) y 13 VUS (27%) en los 48 pacientes seleccionados, que previamente habían sido negativos en los estudios con CMA y otros estudios. Nuestros resultados demuestran la heterogeneidad genética y clínica de los individuos con TEA, y la dificultad actual de su diagnóstico molecular. También muestra que CMA y el panel de NGS podrían ser muy útiles para diagnosticar los casos en pacientes con autismo esencial/primario y son tecnologías coste-efectivas. Adicionalmente, se ha propuesto la implementación de un algoritmo diagnóstico que facilite la toma de decisiones en los pacientes TEA en la rutina asistencialAutism spectrum disorder (ASD) is a set of neurodevelopmental disorders characterized by deficiencies in communication, social interaction, repetitive and restrictive behaviors. The discovery of genetic involvement in the etiology of ASD has made this condition a strong candidate for genome-based diagnostic tests. The use of SNP(Single Nucleotide Polymorphim) y CGH (Comparative Genome Hybridization) microarrays (hereinafter CMA, chromosomal microarray analysis) has proven to be a fast and efficient method to detect micro and large deletions/duplications associated with ASD, while the NGS (next generation sequencing) is useful for the detection of variants in the sequence of different genes in ASD patients. We present in this Doctoral Thesis the implementation of the use of the CMA and personalized NGS panel for autism (AutismSeq), as a first level test, for patients with ASD in a prospective period of four years in the clinical routine of a tertiary hospital. The cohort is composed of 212 individuals, older than 3 years, who met the DSM-V diagnostic criteria for ASD. Using KaryoArray®, a CMA of our own design in prospective cases of ASD, we found 96 CNVs in 212 individuals (45.2%), of which 34 patients (35%) had deletions and 62 patients (65%) had duplications. Twenty-seven of the 212 patients were carriers of pathogenic or possibly pathogenic (P/LP) CNVs, respectively, representing about 12.7% of the sample. A 21.2% (45/212) were carriers of benign rearrangements and 24 of 212 (11.32%) were carriers of variants of uncertain significance (VUS). On the other hand, the NGS panel (AutismSeq) of our own design turned out to be a tool with good diagnostic efficacy in routine care, where we detected 11 pathogenic mutations (23%) and 13 VUS (27%) in the 48 selected patients, which they had previously been negative in CMA and other studies. Our results demonstrate the great genetic and clinical heterogeneity of individuals with ASD, and the current difficulty of its molecular diagnosis. Our study also shows that CMA and NGS panel could be very useful to diagnose cases in patients with essential/primary autism, and it is cost-effective. Additionally, the implementation of a diagnostic algorithm that facilitates decision-making in ASD patients in routine care has been propose

Deep Phenotyping and Genetic Characterization of a Cohort of 70 Individuals With 5p Minus Syndrome.

Chromosome-5p minus syndrome (5p-Sd, OMIM #123450) formerly known as Cri du Chat syndrome results from the loss of genetic material at the distal region of the short arm of chromosome 5. It is a neurodevelopmental disorder of genetic cause. So far, about 400 patients have been reported worldwide. Individuals affected by this syndrome have large phenotypic heterogeneity. However, a specific phenotype has emerged including global developmental delay, microcephaly, delayed speech, some dysmorphic features, and a characteristic and monochromatic high-pitch voice, resembling a cat's cry. We here describe a cohort of 70 patients with clinical features of 5p- Sd characterized by means of deep phenotyping, SNP arrays, and other genetic approaches. Individuals have a great clinical and molecular heterogeneity, which can be partially explained by the existence of additional significant genomic rearrangements in around 39% of cases. Thus, our data showed significant statistical differences between subpopulations (simple 5p deletions versus 5p deletions plus additional rearrangements) of the cohort. We also determined significant "functional" differences between male and female individuals.S

Cognitive–Behavioral Profile in Pediatric Patients with Syndrome 5p-; Genotype–Phenotype Correlationships

(1) Background: 5p minus Syndrome (S5p-) is a neurodevelopmental disorder caused by a deletion in the short arm of chromosome 5. Among the phenotypic characteristics of S5p-, the most characteristic and representative element is a monochromatic cry with a high-pitched tone reminiscent of a cat’s meow. Individuals may also show great phenotypic heterogeneity and great genetic variability. Regarding cognitive–behavioral aspects of the syndrome, the studies are scarce and do not establish a general profile of the main cognitive–behavioral particularities that this syndrome presents. The main objective of this work was to describe the development profile of a cohort of 45 children with 5p minus Syndrome, concerning the biomedical, genetic, cognitive, and behavioral aspects. Establishing putative genotype–phenotype (cognitive–behavioral profiles) relationships in our cohort, from an interdisciplinary approach. (2) Methods: A selection of instruments of measures was selected for neuropsychological assessment (3) Results: In general, children with S5p- have a higher cognitive level than a communicative and motor level. Language difficulties, especially expressive ones, influence the frequency and severity of the most frequent behavioral problems in S5p. The most significant problem behavior of children with S5p-, especially girls, is self-harm. Compulsive behavior, limited preferences, and interest in monotony are significantly more frequent in subjects with better cognitive levels. We also find a significant correlation between the size of the loss of genetic material on 5p and the cognitive level of the subjects. (4) Conclusions: We described for the first time, the cognitive–behavioral profile of a cohort of minors with S5p-. Remarkably, it was found that language, especially of an expressive nature, modulates the most frequent behavioral aspects in subjects with lower cognitive levels, so it is essential to develop verbal or alternative communication strategies adjusted to these individuals

Cognitive–behavioral profile in pediatric patients with syndrome 5p-; genotype–phenotype correlationships

Publicación derivada de un trabajo de investigación desde un equipo interdisciplinar entre áreas ciencias médicas, ciencias sociales y ciencias del comportamiento al tratarse de una población objeto de estudio considerada enfermedad rara, que requiere de un abordaje interdisciplinar de cara a ir avanzando en la identificación características de la misma desde distintas dimensiones que influyan en su desarrollo.(1) Background: 5p minus Syndrome (S5p-) is a neurodevelopmental disorder caused by a deletion in the short arm of chromosome 5. Among the phenotypic characteristics of S5p-, the most characteristic and representative element is a monochromatic cry with a high-pitched tone reminiscent of a cat’s meow. Individuals may also show great phenotypic heterogeneity and great genetic variability. Regarding cognitive–behavioral aspects of the syndrome, the studies are scarce and do not establish a general profile of the main cognitive–behavioral particularities that this syndrome presents. The main objective of this work was to describe the development profile of a cohort of 45 children with 5p minus Syndrome, concerning the biomedical, genetic, cognitive, and behavioral aspects. Establishing putative genotype–phenotype (cognitive–behavioral profiles) relationships in our cohort, from an interdisciplinary approach. (2) Methods: A selection of instruments of measures was selected for neuropsychological assessment (3) Results: In general, children with S5p- have a higher cognitive level than a communicative and motor level. Language difficulties, especially expressive ones, influence the frequency and severity of the most frequent behavioral problems in S5p. The most significant problem behavior of children with S5p-, especially girls, is self-harm. Compulsive behavior, limited preferences, and interest in monotony are significantly more frequent in subjects with better cognitive levels. We also find a significant correlation between the size of the loss of genetic material on 5p and the cognitive level of the subjects. (4) Conclusions: We described for the first time, the cognitive–behavioral profile of a cohort of minors with S5p-. Remarkably, it was found that language, especially of an expressive nature, modulates the most frequent behavioral aspects in subjects with lower cognitive levels, so it is essential to develop verbal or alternative communication strategies adjusted to these individuals.Depto. de Estudios EducativosDepto. de Investigación y Psicología en EducaciónFac. de EducaciónTRUEpu

GATA2 null mutation associated with incomplete penetrance in a family with Emberger syndrome

Introduction: GATA2 mutations are associated with several conditions, including Emberger syndrome which is the association of primary lymphedema with hematological anomalies and an increased risk for myelodysplasia and leukemia. Objective: To describe a family with Emberger syndrome with incomplete penetrance. Methods: A DNA sequencing of GATA2 gene was performed in the parents and offspring (5 individuals in total). Results: The family consisted of 5 individuals with a GATA2 null mutation (c.130G>T, p.Glu44*); three of them were affected (2 of which were deceased) while 2 remained unaffected at the age of 40 and 13 years old. The three affected siblings (2 boys and 1 girl) presented with lymphedema of the lower limbs, recurrent warts, epistaxis and recurrent infections. Two died due to hematological abnormalities (AML and pancytopenia). In contrast, the two other family members who carry the same mutation (the mother and one brother) have not presented any symptoms and their blood tests remain normal. Discussion: Incomplete penetrance may indicate that GATA2 haploinsufficiency is not enough to produce the phenotype of Emberger syndrome. Therefore, in cases where incomplete penetrance or high variable expressivity is described, whole exome or genome sequencing would be useful in order to identify specific gene interactions that drastically modify the phenotype. In addition, Skewed gene expression by an epigenetic mechanism of gene regulation should also be considered