Generation of induced pluripotent stem cells (iPSCs) by retroviral transduction of skin fibroblasts from four patients suffering Williams-Beuren syndrome (7q11.23 deletion).

Skin fibroblasts were obtained from four patients with Williams-Beuren syndrome (WBS) carrying the typical 1.5 Mb or 1.8 Mb deletion at the 7q11.23 genomic region. Induced pluripotent stem cells (iPSCs) were generated by retroviral infection of fibroblasts with polycystronic vectors. The generated iPSC clones ESi059A, ESi060B and ESi068A had the 1.5 Mb deletion of 7q11.23 and ESi069A the 1.8 Mb, with no novel additional genomic alterations, stable karyotype, expressed pluripotency markers and could differentiate towards the three germ layers in vitro via embryoid body formation and in vivo by teratoma formation. WBS patient's lines are a valuable resource for in vitro modelling of WBS.

Derivation of induced pluripotent stem cells (iPSCs) by retroviral transduction of skin fibroblasts from four patients suffering 7q11.23 microduplication syndrome.

Skin fibroblasts were obtained from four patients with 7q11.23 microduplication syndrome carrying the reciprocal rearrangement of Williams-Beuren syndrome at the 7q11.23 genomic region. Induced pluripotent stem cells (iPSCs) were generated by retroviral infection of fibroblasts with polycystronic vectors. The generated iPSC clones ESi058B, ESi057B, ESi070A and ESi071A had the 7q11.23 duplication with no additional genomic alterations, a stable karyotype, expressed pluripotency markers and could differentiate towards the three germ layers in vitro via embryoid body formation and in vivo by teratoma formation. Patient's derived iPSCs are a valuable resource for in vitro modeling of 7q11.23 microduplication syndrome. Resource Table.

Genetic and epigenetic methylation defects and implication of the ERMN gene in autism spectrum disorders.

Autism spectrum disorders (ASD) are highly heritable and genetically complex conditions. Although highly penetrant mutations in multiple genes have been identified, they account for the etiology of <1/3 of cases. There is also strong evidence for environmental contribution to ASD, which can be mediated by still poorly explored epigenetic modifications. We searched for methylation changes on blood DNA of 53 male ASD patients and 757 healthy controls using a methylomic array (450K Illumina), correlated the variants with transcriptional alterations in blood RNAseq data, and performed a case-control association study of the relevant findings in a larger cohort (394 cases and 500 controls). We found 700 differentially methylated CpGs, most of them hypomethylated in the ASD group (83.9%), with cis-acting expression changes at 7.6% of locations. Relevant findings included: (1) hypomethylation caused by rare genetic variants (meSNVs) at six loci (ERMN, USP24, METTL21C, PDE10A, STX16 and DBT) significantly associated with ASD (q-value <0.05); and (2) clustered epimutations associated to transcriptional changes in single-ASD patients (n=4). All meSNVs and clustered epimutations were inherited from unaffected parents. Resequencing of the top candidate genes also revealed a significant load of deleterious mutations affecting ERMN in ASD compared with controls. Our data indicate that inherited methylation alterations detectable in blood DNA, due to either genetic or epigenetic defects, can affect gene expression and contribute to ASD susceptibility most likely in an additive manner, and implicate ERMN as a novel ASD gene.

MLL2 mutation detection in 86 patients with Kabuki syndrome: a genotype-phenotype study.

Recently, pathogenic variants in the MLL2 gene were identified as the most common cause of Kabuki (Niikawa-Kuroki) syndrome (MIM#147920). To further elucidate the genotype-phenotype correlation, we studied a large cohort of 86 clinically defined patients with Kabuki syndrome (KS) for mutations in MLL2. All patients were assessed using a standardized phenotype list and all were scored using a newly developed clinical score list for KS (MLL2-Kabuki score 0-10). Sequencing of the full coding region and intron-exon boundaries of MLL2 identified a total of 45 likely pathogenic mutations (52%): 31 nonsense, 10 missense and four splice-site mutations, 34 of which were novel. In five additional patients, novel, i.e. non-dbSNP132 variants of clinically unknown relevance, were identified. Patients with likely pathogenic nonsense or missense MLL2 mutations were usually more severely affected (median 'MLL2-Kabuki score' of 6) as compared to the patients without MLL2 mutations (median 'MLL2-Kabuki score' of 5), a significant difference (p < 0.0014). Several typical facial features such as large dysplastic ears, arched eyebrows with sparse lateral third, blue sclerae, a flat nasal tip with a broad nasal root, and a thin upper and a full lower lip were observed more often in mutation positive patients.

SMN2 copy number predicts acute or chronic spinal muscular atrophy but does not account for intrafamilial variability in siblings.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder that affects motor neurons. It is caused by mutations in the survival motor neuron gene 1 (SMN1). The SMN2 gene, which is the highly homologous SMN1 copy that is present in all the patients, is unable to prevent the disease. An SMN2 dosage method was applied to 45 patients with the three SMA types (I-III) and to four pairs of siblings with chronic SMA (II-III) and different phenotypes. Our results confirm that the SMN2 copy number plays a key role in predicting acute or chronic SMA. However, siblings with different SMA phenotypes show an identical SMN2 copy number and identical markers, indicating that the genetic background around the SMA locus is insufficient to account for the intrafamilial variability. In our results, age of onset appears to be the most important predictor of disease severity in affected members of the same family. Given that SMN2 is regarded as a target for potential pharmacological therapies in SMA, the identification of genetic factors other than the SMN genes is necessary to better understand the pathogenesis of the disease in order to implement additional therapeutic approaches.

Detection of novel mutations in the SMN Tudor domain in type I SMA patients.

The authors present a complete SMN gene analysis in four type I unrelated spinal muscular atrophy patients who retained one copy of the SMN1 gene. Two intragenic point mutations were identified in exon 3 (I116F, Q136E), affecting a very conserved region with the Tudor domain of SMN1. The remaining two patients showed no alterations in the SMN1 coding sequences although a transcription defect was detected in one of them, corroborating the existence of non-functional SMN1 genes.

Survival and respiratory decline are not related to homozygous SMN2 deletions in ALS patients.

The presence of the SMN2 deletion in 124 patients with ALS was investigated. Eleven patients had the homozygous deletion of SMN2 (8.8%) in comparison with 20 of 200 (10%) of the healthy control population. No significant differences in sex, age at onset, initial symptoms, form of inheritance, decline in ventilatory function, or survival time were found between patients with and without the deletion. The hypothesis that SMN2 is a prognostic factor in sporadic or familial ALS was not confirmed in this study.

Prenatal diagnosis for risk of spinal muscular atrophy.

OBJECTIVES: Prenatal diagnosis of spinal muscular atrophy is usually performed in high risk couples by detection of a homozygous deletion in the survival motor neurone gene (SMN1). However, other relatives at risk of being carriers very often request genetic counselling and the possibility of prenatal diagnosis. The aim of this study was to validate a SMN1 gene quantitative test to help the couples formed by one spinal muscular atrophy carrier and a partner of the general population (1/200 potential risk) to achieve a less ambiguous risk result for the pregnancy. DESIGN: Spinal muscular atrophy carrier studies in at-risk individuals. SETTING: Department of Genetics and Gynaecology and Obstetrics in a large university hospital. POPULATION: Seventy-nine obligate carriers (more than one affected child with deletion in the offspring) and 58 non-carriers (relatives of spinal muscular atrophy families defined by marker studies) were tested to set up a quantitative analysis. The method was applied in different situations in 126 members from 34 families with spinal muscular atrophy patients. METHODS: DNA studies of the SMNI gene by marker analysis and quantitative assay. MAIN OUTCOME MEASURES: To determine double (non-carrier) or single dose (carrier) of exon 7 of the SMN1 gene in relatives of spinal muscular atrophy patients. Bayesian calculation of risk. RESULTS: The sensitivity and specificity of the method were 96% and 100%, respectively. Studies on different couples with an a priori risk of 1/200 allowed us to reduce the final risk to 1/5000 or to increase it to 1/4. CONCLUSIONS: The quantitative method can be used to achieve a less ambiguous risk in pregnancies with a 1/200 risk and in families where no sample is available to study the index case. Screening of gamete donors when the recipient is a known carrier should also be considered.

Characterisation of SMN hybrid genes in Spanish SMA patients: de novo, homozygous and compound heterozygous cases.

Autosomal recessive spinal muscular atrophy (SMA) is classified, by age of onset and maximal motor milestones achieved, into type I (severe form), type II (intermediate form) and type III (mild/moderate form). SMA is caused by mutations in the survival motor neuron telomeric gene (SMN1) and a centromeric functional copy of this gene (SMN2) exists, both genes being located at 5q13. Homozygous deletion of exons 7 and 8 of SMN1 has been detected in approx 85% of Spanish SMA patients regardless of their phenotype. Nineteen cases with the sole deletion of exon 7 but not exon 8 (2 cases of type I, 13 cases of type II, four cases of type III) were further analysed for the presence of SMN2-SMN1 hybrid genes. We detected four different hybrid structures. Most of the patients were carriers of a hybrid structure: centromeric intron 6- centromeric exon 7- telomeric exon 8 (CCT), with or without neuronal apoptosis-inhibitor protein (NAIP). In two patients, a different hybrid structure, viz. telomeric intron 6- centromeric exon 7- telomeric exon 8 (TCT), was detected with or without NAIP. A phenotype-genotype correlation comparing the different structures of the hybrid alleles was delineated. Type I cases in our series are attributable to intrachromosomal deletion with a smaller number of SMN2 copies. Most cases with hybrid genes are type II occurring by a combination of a classical deletion in one chromosome and a hybrid gene in the other. Type III cases are closely associated with homozygozity or compound heterozygozity for hybrid genes resulting from two conversion events and have more copies of hybrid genes and SMN2 than type I or II cases.