The Genetic Landscape of Inherited Retinal Diseases in a Mexican Cohort: Genes, Mutations and Phenotypes.

In this work, we aimed to provide the genetic diagnosis of a large cohort of patients affected with inherited retinal dystrophies (IRDs) from Mexico. Our data add valuable information to the genetic portrait in rare ocular diseases of Mesoamerican populations, which are mostly under-represented in genetic studies. A cohort of 144 unrelated probands with a clinical diagnosis of IRD were analyzed by next-generation sequencing using target gene panels (overall including 346 genes and 65 intronic sequences). Four unsolved cases were analyzed by whole-exome sequencing (WES). The pathogenicity of new variants was assessed by in silico prediction algorithms and classified following the American College of Medical Genetics and Genomics (ACMG) guidelines. Pathogenic or likely pathogenic variants were identified in 105 probands, with a final diagnostic yield of 72.9%; 17 cases (11.8%) were partially solved. Eighteen patients were clinically reclassified after a genetic diagnostic test (17.1%). In our Mexican cohort, mutations in 48 genes were found, with ABCA4, CRB1, RPGR and USH2A as the major contributors. Notably, over 50 new putatively pathogenic variants were identified. Our data highlight cases with relevant clinical and genetic features due to mutations in the RAB28 and CWC27 genes, enrich the novel mutation repertoire and expand the IRD landscape of the Mexican population.

Increasing the Genetic Diagnosis Yield in Inherited Retinal Dystrophies: Assigning Pathogenicity to Novel Non-canonical Splice Site Variants.

AIMS: We aimed to validate the pathogenicity of genetic variants identified in inherited retinal dystrophy (IRD) patients, which were located in non-canonical splice sites (NCSS). METHODS: After next generation sequencing (NGS) analysis (target gene panels or whole exome sequencing (WES)), NCSS variants were prioritized according to in silico predictions. In vivo and in vitro functional tests were used to validate their pathogenicity. RESULTS: Four novel NCSS variants have been identified. They are located in intron 33 and 34 of ABCA4 (c.4774-9G>A and c.4849-8C>G, respectively), intron 2 of POC1B (c.101-3T>G) and intron 3 of RP2 (c.884-14G>A). Functional analysis detected different aberrant splicing events, including intron retention, exon skipping and intronic nucleotide addition, whose molecular effect was either the disruption or the elongation of the open reading frame of the corresponding gene. CONCLUSIONS: Our data increase the genetic diagnostic yield of IRD patients and expand the landscape of pathogenic variants, which will have an impact on the genotype-phenotype correlations and allow patients to opt for the emerging gene and cell therapies.

Aberrant Splicing Events Associated to CDH23 Noncanonical Splice Site Mutations in a Proband with Atypical Usher Syndrome 1.

AIMS: The aim of this study was the genetic diagnosis by next generation sequencing (NGS) of a patient diagnosed with Usher syndrome type 2 and the functional evaluation of the identified genetic variants to establish a phenotype-genotype correlation. METHODS: Whole exome sequencing (WES) analysis identified two heterozygous intronic variants in CDH23, a gene responsible of Usher syndrome type 1. Evaluation of the putative splicing effects was performed in vivo, in whole blood samples, and in vitro, by transfection of midigene constructs in HEK293T cells. RESULTS: Two intronic variants were identified in intron 45 of CDH23-one novel, c.6050-15G>A, and the other, c.6050-9G>A, already reported as a noncanonical splice site (NCSS) mutation-with partial functional characterization. In vivo and in vitro analyses showed aberrant transcripts by the addition of 13 and 7 nucleotides to exon 46, respectively. Transcript degradation by nonsense mediated decay (NMD) in blood cells could only be prevented by cycloheximide treatment. Midigene constructs showed that the two variants contributed to exon skipping and generated aberrantly spliced transcripts. CONCLUSIONS: A combination of in vivo and in vitro assays provided a comprehensive view of the physiological effects of NCSS variants, which in this case led to a clinical reassignment of the proband as affected with atypical USH1 syndrome.

Full Breastfeeding and Obesity in Children: A Prospective Study from Birth to 6 Years.

BACKGROUND: Obesity is a major public health crisis among both children and adults and contributes to significant physical, psychological, and economic burden. We aim to investigate the effect of duration of breastfeeding on excessive weight and obesity at 6 years of age. SUBJECTS/METHODS: Data on breastfeeding and child anthropometric measurements were collected in a birth-cohort study in Murcia, Spain (n = 350). Breastfeeding status and body mass index (BMI) were established according to WHO definitions. Other factors potentially related to children's weight were considered. Multiple log-linear and ordinal regressions were used to analyze the effects of breastfeeding on overweight and obesity when considering potential confounders. RESULTS: 33% and 17.3% of children in the study were of excess weight and obesity, respectively. Univariate predictors of BMI in children aged 6 were as follows: pregestational maternal BMI (kg/m2) (R2 = 0.127, p < 0.01); full breastfeeding (weeks) R2 = -0.035, p < 0.01); infant weight gain (kg) (R2 = 0.348, p < 0.01); and maternal alcohol consumption during pregnancy (g/day) (R2 = 0.266, p < 0.01) at age 6. In the ordinal logistic regression, full breastfeeding was associated with a significant decrease in obesity -0.052 (95% CI, -0.10 to -0.003). CONCLUSIONS: The delay of bottle feeding introduction may have a protective effect against obesity at 6 years of age. Our findings reinforce the need for greater support of breastfeeding and to promote a healthy environment and antipoverty interventions during pregnancy and infancy, alongside other strategies for obesity prevention.

The X-linked methyl binding protein gene Kaiso is highly expressed in brain but is not mutated in Rett syndrome patients.

Rett syndrome (RTT; OMIM 312750) is an X-linked dominant neurological disorder, which affects mostly females. It is associated with mutations of the MECP2 gene, codifying for a methyl-CpG DNA binding protein of the MBDs family, sharing the common Methyl Binding Domain. MeCP2 binds single methylated CpG pair and brings transcriptional silencing to the substrate DNA templates. However, around 5-10% of clinically well defined RTT patients do not show any mutations in this gene. Several hypotheses have been postulated to clarify the remaining unexplained RTT cases. We pointed our attention on Kaiso gene. This gene is localized in the Xq23 region and codifies for a protein acting as a methyl-CpG binding protein by using three zinc-finger domains: for this reason it is not strictly related to the MBD family of proteins, even if it may repress transcription of methylated genes as well. To investigate the potential association of Kaiso disfunction with pathogenesis of Rett syndrome, we approached the analysis at two different levels. Primarily, we performed an itemized murine brain expression analysis of Kaiso gene. Expression data and localization made it an excellent candidate as additional causative gene for MECP2 negative, classical RTT patients. On the bases of this data a detailed mutational analysis of 44 patients from Spanish, UK, and Italian archives has been performed to the coding region of Kaiso. No mutation was found while a very frequent polymorphism was identified and characterized. Our study suggests that this gene is not implicated in the RTT molecular pathogenesis, but additional analyses are needed to exclude it as causative gene for X-linked mental retardation disorders.

Hydrocephalus and Hirschsprung's disease with a mutation of L1CAM.

Abnormalities of the L1CAM gene, a member of the immunoglobulin gene superfamily of neural-cell adhesion molecules, are associated with X-linked hydrocephalus and some allelic disorders. Hirschsprung's disease (HSCR) is characterized by the absence of ganglion cells and the presence of hypertrophic nerve trunks in the distal bowel. There have been three reports of patients with X-linked hydrocephalus and HSCR with a mutation in the L1CAM gene. We report three more patients with similar conditions. We suspect that decreased L1CAM may be a modifying factor in the development of HSCR.