Meier-Gorlin Syndrome: Clinical Misdiagnosis, Genetic Testing and Functional Analysis of ORC6 Mutations and the Development of a Prenatal Test.

Meier−Gorlin syndrome (MGS) is a rare genetic developmental disorder that causes primordial proportional dwarfism, microtia, the absence of or hypoplastic patellae and other skeletal anomalies. Skeletal symptoms overlapping with other syndromes make MGS difficult to diagnose clinically. We describe a 3-year-old boy with short stature, recurrent respiratory infections, short-rib dysplasia, tower head and facial dysmorphisms who was admitted to the Tomsk Genetic Clinic to verify a clinical diagnosis of Jeune syndrome. Clinical exome sequencing revealed two variants (compound heterozygosity) in the ORC6 gene: c.2T>C(p.Met1Thr) and c.449+5G>A. In silico analysis showed the pathogenicity of these two mutations and predicted a decrease in donor splicing site strength for c.449+5G>A. An in vitro minigene assay indicated that variant c.449+5G>A causes complete skipping of exon 4 in the ORC6 gene. The parents requested urgent prenatal testing for MGS for the next pregnancy, but it ended in a miscarriage. Our results may help prevent MGS misdiagnosis in the future. We also performed in silico and functional analyses of ORC6 mutations and developed a restriction fragment length polymorphism and haplotype-based short-tandem-repeat assay for prenatal genetic testing for MGS. These findings should elucidate MGS etiology and improve the quality of genetic counselling for affected families.

Prenatal Diagnosis of Small Supernumerary Marker Chromosome 10 by Array-Based Comparative Genomic Hybridization and Microdissected Chromosome Sequencing.

Interpreting the clinical significance of small supernumerary marker chromosomes (sSMCs) in prenatal diagnosis is still an urgent problem in genetic counselling regarding the fate of a pregnancy. We present a case of prenatal diagnosis of mosaic sSMC(10) in a foetus with a normal phenotype. Comprehensive cytogenomic analyses by array-based comparative genomic hybridization (aCGH), sSMC microdissection with next-generation sequencing (NGS) of microdissected library, fluorescence in situ hybridization (FISH) with locus-specific and telomere-specific DNA probes and quantitative real-time PCR revealed that sSMC(10) had a ring structure and was derived from the pericentromeric region of chromosome 10 with involvement of the 10p11.21-p11.1 and 10q11.21-q11.23 at 1.243 Mb and 7.173 Mb in size, respectively. We observed a difference in the length of sSMC(10) between NGS data of the DNA library derived from a single copy of sSMC(10), and aCGH results that may indicate instability and structural mosaicism for ring chromosomes in foetal cells. The presence of a 9 Mb euchromatin region in the analysed sSMC(10) did not lead to clinical manifestations, and a healthy girl was born at term. We suggest that the ring structure of sSMCs could influence sSMC manifestations and should be taken into account in genetic counselling during prenatal diagnosis.

A cookbook for DNase Hi-C.

BACKGROUND: The Hi-C technique is widely employed to study the 3-dimensional chromatin architecture and to assemble genomes. The conventional in situ Hi-C protocol employs restriction enzymes to digest chromatin, which results in nonuniform genomic coverage. Using sequence-agnostic restriction enzymes, such as DNAse I, could help to overcome this limitation. RESULTS: In this study, we compare different DNAse Hi-C protocols and identify the critical steps that significantly affect the efficiency of the protocol. In particular, we show that the SDS quenching strategy strongly affects subsequent chromatin digestion. The presence of biotinylated oligonucleotide adapters may lead to ligase reaction by-products, which can be avoided by rational design of the adapter sequences. Moreover, the use of nucleotide-exchange enzymes for biotin fill-in enables simultaneous labelling and repair of DNA ends, similar to the conventional Hi-C protocol. These improvements simplify the protocol, making it less expensive and time-consuming. CONCLUSIONS: We propose a new robust protocol for the preparation of DNAse Hi-C libraries from cultured human cells and blood samples supplemented with experimental controls and computational tools for the evaluation of library quality.

Allele-Specific Biased Expression of the CNTN6 Gene in iPS Cell-Derived Neurons from a Patient with Intellectual Disability and 3p26.3 Microduplication Involving the CNTN6 Gene.

Copy number variations (CNVs) of the human CNTN6 gene caused by megabase-scale microdeletions or microduplications in the 3p26.3 region are often the cause of neurodevelopmental disorders, including intellectual disability and developmental delay. Surprisingly, patients with different copy numbers of this gene display notable overlapping of neuropsychiatric symptoms. The complexity of the study of human neuropathologies is associated with the inaccessibility of brain material. This problem can be overcome through the use of reprogramming technologies that permit the generation of induced pluripotent stem (iPS) cells from fibroblasts and their subsequent in vitro differentiation into neurons. We obtained a set of iPS cell lines derived from a patient carrier of the CNTN6 gene duplication and from two healthy donors. All iPS cell lines displayed the characteristics of pluripotent cells. Some iPS cell lines derived from the patient and from healthy donors were differentiated in vitro by exogenous expression of the Ngn2 transcription factor or by spontaneous neural differentiation of iPS cells through the neural rosette stage. The obtained neurons showed the characteristics of mature neurons as judged by the presence of neuronal markers and by their electrophysiological characteristics. Analysis of allele-specific expression of the CNTN6 gene in these neuronal cells by droplet digital PCR demonstrated that the level of expression of the duplicated allele was significantly reduced compared to that of the wild-type allele. Importantly, according to the sequencing data, both copies of the CNTN6 gene, which were approximately 1 Mb in size, showed no any additional structural rearrangements.