X chromosome inactivation does not necessarily determine the severity of the phenotype in Rett syndrome patients.

Rett syndrome (RTT) is a severe neurological disorder usually caused by mutations in the MECP2 gene. Since the MECP2 gene is located on the X chromosome, X chromosome inactivation (XCI) could play a role in the wide range of phenotypic variation of RTT patients; however, classical methylation-based protocols to evaluate XCI could not determine whether the preferentially inactivated X chromosome carried the mutant or the wild-type allele. Therefore, we developed an allele-specific methylation-based assay to evaluate methylation at the loci of several recurrent MECP2 mutations. We analyzed the XCI patterns in the blood of 174 RTT patients, but we did not find a clear correlation between XCI and the clinical presentation. We also compared XCI in blood and brain cortex samples of two patients and found differences between XCI patterns in these tissues. However, RTT mainly being a neurological disease complicates the establishment of a correlation between the XCI in blood and the clinical presentation of the patients. Furthermore, we analyzed MECP2 transcript levels and found differences from the expected levels according to XCI. Many factors other than XCI could affect the RTT phenotype, which in combination could influence the clinical presentation of RTT patients to a greater extent than slight variations in the XCI pattern.

Characterization of large deletions of the MECP2 gene in Rett syndrome patients by gene dosage analysis.

BACKGROUND: Rett syndrome (RTT) is a developmental disorder with an early onset and X-linked dominant inheritance pattern. It is first recognized in infancy and is seen almost always in girls, but it may be seen in boys on rare occasions. Typical RTT is caused by de novo mutations of the gene MECP2 (OMIM*300005), and atypical forms of RTT can be caused by mutations of the CDKL5 (OMIM*300203) and FOXG1 (OMIM*164874) genes. METHODS: Approximately 5% of the mutations detected in MECP2 are large rearrangements that range from exons to the entire gene. Here, we have characterized the deletions detected by multiplex ligation-dependent probe amplification (MLPA) in the gene MECP2 of 21 RTT patients. Breakpoints were delineated by DNA-qPCR until the amplification of the deleted allele by long-PCR was possible. RESULTS: This methodology enabled us to characterize deletions ranging from 1,235 bp to 85 kb, confirming the partial or total deletion of the MECP2 gene in all these patients. Additionally, our cases support the evidence claiming that most of these breakpoints occur in some restricted regions of the MECP2 gene. CONCLUSION: These molecular data together with the clinical information enable us to propose a genotype-phenotype correlation, which is essential for providing genetic counseling.

The utility of Next Generation Sequencing for molecular diagnostics in Rett syndrome.

Rett syndrome (RTT) is an early-onset neurodevelopmental disorder that almost exclusively affects girls and is totally disabling. Three genes have been identified that cause RTT: MECP2, CDKL5 and FOXG1. However, the etiology of some of RTT patients still remains unknown. Recently, next generation sequencing (NGS) has promoted genetic diagnoses because of the quickness and affordability of the method. To evaluate the usefulness of NGS in genetic diagnosis, we present the genetic study of RTT-like patients using different techniques based on this technology. We studied 1577 patients with RTT-like clinical diagnoses and reviewed patients who were previously studied and thought to have RTT genes by Sanger sequencing. Genetically, 477 of 1577 patients with a RTT-like suspicion have been diagnosed. Positive results were found in 30% by Sanger sequencing, 23% with a custom panel, 24% with a commercial panel and 32% with whole exome sequencing. A genetic study using NGS allows the study of a larger number of genes associated with RTT-like symptoms simultaneously, providing genetic study of a wider group of patients as well as significantly reducing the response time and cost of the study.

FOXG1, un nuevo gen responsable de la forma cognítiva del síndrome de Rett / FOXG1, a new gene responsible for the congenital form of Rett syndrome

Introducción. El síndrome de Rett (SR) es un trastorno del neurodesarrollo que afecta casi exclusivamente a niñas. Laidentificación de mutaciones en los genes MECP2 y CDKL5 aporta una confirmación genética del diagnóstico clínico. El genFOXG1 se perfila como un nuevo responsable de la variante congénita del SR.Caso clínico. Se describe la primera paciente española con variante atípica (congénita) del SR con mutación en el genFOXG1, y se compara con 12 pacientes publicados en la bibliografía; se proponen criterios clínicos sugestivos de alteracionesen el FOXG1. La paciente fue remitida a los 6 meses por retraso global, hipotonía axial, microcefalia y fenotipo peculiar.Resonancia magnética cerebral: hipoplasia del cuerpo calloso, atrofia frontal y ventriculomegalia. Aparición de estereotipiasmano-boca a los 12 meses que orientan hacia el diagnóstico clínico de variante atípica del SR, forma congénita;mejoría progresiva del contacto visual e interés por el medio. Infecciones respiratorias frecuentes y síndrome de apneaobstructiva del sueño. A los 5 años existe un control parcial del tono axial, prensión manual, buen contacto y balbuceo,sin epilepsia ni alteraciones conductuales. El estudio de MECP2 y deleciones subteloméricas no mostró alteraciones; seidentificaron dos polimorfismos en el gen CDKL5 y una mutación patogénica en el FOXG1 (c.624C>G p.Tyr203X).Conclusiones. El 92% de pacientes con mutaciones en el gen FOXG1 presenta la forma congénita del SR con una gravehipotonía generalizada, microcefalia adquirida precoz (–3 a –6 desviaciones estándares) y fenotipo peculiar. Ante undiagnóstico de SR sin alteración en los genes MECP2 y CDKL5, especialmente en la variante congénita, debe investigarse elgen FOXG1. El diagnóstico molecular confirma el diagnóstico clínico y aporta un consejo genético a la familia (AU)

Introduction. Rett syndrome (RS) is a neurodevelopmental disorder that affects girls almost exclusively. The identificationof mutations in the MECP2 and CDKL5 genes offers genetic confirmation of the clinical diagnosis. The FOXG1 gene appearsto be a novel cause of the congenital variant of RS.Case report. We describe the first Spanish patient with the atypical (congenital) variant of RS with mutation of the FOXG1gene and the case is compared with 12 patients previously reported in the literature; clinical criteria that suggest alterationsin FOXG1 are proposed. The patient was referred at the age of 6 months due to overall retardation, axial hypotonia,microcephaly and a peculiar phenotype. Magnetic resonance imaging of the brain revealed hypoplasia of the corpuscallosum, frontal atrophy and ventriculomegaly. The appearance of hand-to-mouth stereotypic movements at 12 monthspointed the clinical diagnosis towards an atypical variant of RS, the congenital form; there was progressive improvementof visual contact and interest in her surroundings. Frequent respiratory infections and obstructive sleep apnoea syndrome.At the age of 5 years there was partial control over the axial tone, grasping with the hands, good contact and babbling,without epilepsy or behavioural disorders. The MECP2 and subtelomeric deletion study did not reveal any alterations; twopolymorphisms were identified in the CDKL5 gene and a pathogenic mutation was found in FOXG1 (c.624C>G p.Tyr203X).Conclusions. It has been shown that 92% of patients with mutations in the FOXG1 gene present the congenital form of RSwith severe generalised hypotonia, early acquired microcephaly (–3 to –6 standard deviations) (AU)