Identification of a De Novo Xq26.2 Microduplication Encompassing FIRRE Gene in a Child with Intellectual Disability.

Long non-coding RNAs (lncRNAs), defined as transcripts of >200 nucleotides not translated into protein, have been involved in a wide range of regulatory functions. Their dysregulations have been associated with diverse pathological conditions such as cancer, schizophrenia, Parkinson's, Huntington's, Alzheimer's diseases and Neurodevelopmental Disorders (NDDs), including autism spectrum disorders (ASDs). We report on the case of a five-year-old child with global developmental delay carrying a de novo microduplication on chromosome Xq26.2 region characterized by a DNA copy-number gain spanning about 147 Kb (chrX:130,813,232-130,960,617; GRCh37/hg19). This small microduplication encompassed the exons 2-12 of the functional intergenic repeating RNA element (FIRRE) gene (chrX:130,836,678-130,964,671; GRCh37/hg19) that encodes for a lncRNA involved in the maintenance of chromatin repression. The association of such a genetic alteration with a severe neurodevelopmental delay without clear dysmorphic features and congenital abnormalities indicative of syndromic condition further suggests that small Xq26.2 chromosomal region microduplications containing the FIRRE gene may be responsible for clinical phenotypes mainly characterized by structural or functioning neurological impairment.

A novel mosaic 1q32.1 microduplication identified through Chromosome Microarray Analysis: narrowing the smallest critical region including KDM5B gene found associated with neurodevelopmetal disorders.

Microduplications involving 1q32.1 chromosomal region have been rarely reported in literature. Patients with these microduplications suffer from intellectual disability, developmental delay and a number of dysmorphic features, although no clear karyotype/phenotype correlation has yet been determined. In this case report we describe two monochorionic-diamniotic twins with intellectual disability, abnormality of coordination and dysmorphic features associated with a de novo 280 kb mosaic microduplication of 1q32.1 chromosomal region, identified using a Chromosome Microarray Analysis (CMA) and confirmed by quantitative PCR analysis. The duplicated region encompassed entirely three OMIM genes KDM5B (*605393), KLHL12 (*614522), RABIF (*603417) and involved partially SYT2 (*600104). This unique case report allows to redefine the critical 1q32.1 microduplicated region implicated in the ethiopathogenesis of intellectual disability and developmental delay. Furthermore, it suggests that KDM5B gene can have a pivotal role in the development of neurodevelopmental disorders through its demethylase activity.

Improved GMP compliant approach to manipulate lipoaspirates, to cryopreserve stromal vascular fraction, and to expand adipose stem cells in xeno-free media.

BACKGROUND: The stromal vascular fraction (SVF) derived from adipose tissue contains adipose-derived stromal/stem cells (ASC) and can be used for regenerative applications. Thus, a validated protocol for SVF isolation, freezing, and thawing is required to manage product administration. To comply with Good Manufacturing Practice (GMP), fetal bovine serum (FBS), used to expand ASC in vitro, could be replaced by growth factors from platelet concentrates. METHODS: Throughout each protocol, GMP-compliant reagents and devices were used. SVF cells were isolated from lipoaspirates by a standardized enzymatic protocol. Cells were cryopreserved in solutions containing different albumin or serum and dimethylsulfoxide (DMSO) concentrations. Before and after cryopreservation, we analyzed: cell viability (by Trypan blue); immunophenotype (by flow cytometry); colony-forming unit-fibroblast (CFU-F) formation; and differentiation potential. ASC, seeded at different densities, were expanded in presence of 10% FBS or 5% supernatant rich in growth factors (SRGF) from platelets. The differentiation potential and cell transformation grade were tested in expanded ASC. RESULTS: We demonstrated that SVF can be obtained with a consistent yield (about 185 × 103 cells/ml lipoaspirate) and viability (about 82%). Lipoaspirate manipulation after overnight storage at +4 °C reduced cell viability (-11.6%). The relative abundance of ASC (CD34+CD45-CD31-) and endothelial precursors (CD34+CD45-CD31+) in the SVF product was about 59% and 42%, respectively. A period of 2 months cryostorage in autologous serum with added DMSO minimally affected post-thaw SVF cell viability as well as clonogenic and differentiation potentials. Viability was negatively affected when SVF was frozen at a cell concentration below 1.3 × 106 cells/ml. Cell viability was not significantly affected after a freezing period of 1 year. Independent of seeding density, ASC cultured in 5% SRGF exhibited higher growth rates when compared with 10% FBS. ASC expanded in both media showed unaltered identity (by flow cytometry) and were exempt from genetic lesions. Both 5% SRGF- and 10% FBS-expanded ASC efficiently differentiated to adipocytes, osteocytes, and chondrocytes. CONCLUSIONS: This paper reports a GMP-compliant approach for freezing SVF cells isolated from adipose tissue by a standardized protocol. Moreover, an ASC expansion method in controlled culture conditions and without involvement of animal-derived additives was reported.

Gonadal agenesis with hypoplastic paramesonephric ducts (PMNDs) derivatives in dizygotic twins.

The co-occurrence of gonadal agenesis alongside hypoplastic derivatives of the paramesonephric ducts has rarely been observed. PATIENT(S): 16-year-old dizygotic twin sisters were referred to our department because of primary amenorrhea. X-ray, bone densitometry, ultrasonography, pelvic MRI and measurement of pituitary, ovary, and thyroid hormones were performed. Both twins showed hypergonadotropic hypogonadism, bilateral gonadal agenesis, fallopian tube, uterus, and vaginal hypoplasia but normal kidney and urinary tract structures and skeletal system. Analysis of Q-banded chromosomes in peripheral blood for the search for centromeric X-chromosome DNA and SRY gene was normal as well as the molecular analysis of FMR1, GDF9, and BMP15 genes. Estradiol gel was administered for one year followed by estroprogestin treatment. Both twins growth increased; breast development was stimulated and first menses occurred. Deregulation in the expression of the various HOX genes along the axis of the developing reproductive tract in a determinate time of development may be one of the mechanisms involved in the origin of this complex and rare association.

Heterozygous variant at nucleotide position 875+11A>T in exon 6A cystic fibrosis transmembrane conductance regulator gene induces 852del22 mutation false-positivity by line probe assay.

OBJECTIVE: To explain the lack of genotype-phenotype correlation observed in a patient double heterozygous for the 852del22 and F508del mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. DESIGN: Case report. SETTING: Medical laboratory department. PATIENT(S): A 42-year-old asymptomatic patient underwent genetic screening for in vitro fertilization (IVF). INTERVENTION(S): CFTR genetic screening (commercial kit aimed at detecting 57 mutations), segregation analysis, evaluation of the polymerase chain reaction (PCR) products using a denaturing high performance liquid chromatography (DHPLC), and sequence analysis. MAIN OUTCOME MEASURE(S): To avoid diagnostic errors and improve genetic counseling. RESULT(S): Segregation analysis allowed us to establish that the mutations were in trans. Analysis of the PCR products using a DHPLC apparatus showed a heteroduplex formation indicative of a heterozygous variant in exon 6A. Direct sequencing characterized the heterozygous variant as an A to T transversion at nucleotide position 875+11. Therefore, the change of one single nucleotide in a portion surrounding the 852del22 mutation facilitated the aspecific interaction between the commercial oligonucleotide probe and the amplified genomic DNA, which explains the 852del22 mutation false molecular positivity that was detected by the line probe assay. CONCLUSION(S): The individualization of 852del22 mutation by a standard genetic panel should be confirmed by more extensive analyses.