C14orf132 gene is possibly related to extremely low birth weight.

BACKGROUND: Despite extensive research the genetic component of extremely low birth weight (ELBW) in newborns has remained obscure. RESULTS: The aim of the case study was to identify candidate gene(s) causing ELBW in newborns and hypotrophy in infants. A family of four was studied: mother, father and two ELBW-phenotype children. Studies were made of the medical conditions of the second child at birth and post-partum - peculiar phenotype, micro-anomalies, recurrent infections, suspicion of autoimmune hepatitis, multifactorial encephalopathy and suspected metabolic and chromosomal abnormalities. Whole genome single nucleotide polymorphism (SNP) genotyping array was used to investigate the genomic rearrangements in both affected children using peripheral blood DNA samples. Whole blood transcriptome was assessed by using RNA sequencing (RNA-seq) in all four family members. RNA-seq identified a single gene - C14orf132 (chromosome 14 open reading frame 132) differentially expressed, with the level of the transcript significantly lower in the blood samples of the children. Copy number variant (CNV) analysis did not reveal any pathogenic CNVs in the region of C14orf132 gene of both affected children. CONCLUSION: We demonstrated the importance of combining whole genome CNV and transcriptome analysis in identification of the candidate gene(s) in case studies. We propose the C14orf132 gene expression to be associated with the ELBW-phenotype. C14orf132 gene is a novel long non-coding RNA (lincRNA) with unknown function, which might be associated with the pre- and early postnatal developmental delay through the altered gene expression.

A novel amplification strategy for genotyping with liquid chromatography-electrospray ionization mass spectrometry.

Among numerous available genotyping techniques, mass spectrometry (MS) based methods play a major role in providing high quality genotype data at reasonable costs for research and diagnostics, e.g. for pharmacogenetic applications. Ion-pair reversed-phase liquid chromatography hyphenated to electrospray ionization time-of-flight MS (ICEMS) is, for example, a powerful instrument that allows a direct characterization of complex mixtures of polymerase chain reaction (PCR) amplified DNA fragments. Current limitations of PCR-ICEMS genotyping are mainly concerned with the multiplex PCR set-up. Assay development often requires time-consuming primer design and intensive optimization of PCR conditions. To overcome this restraint, a robust amplification strategy originally combined with arrayed primer extension genotyping was transferred and adapted to ICEMS genotyping. The modifications involved limitation of the primer length, application of two universal sequences and amplification with an appropriate DNA polymerase. To demonstrate the applicability of the novel amplification strategy for ICEMS, a 23-plex pharmacogenetic genotyping assay was developed. After slight optimization steps, an efficient and quantitatively balanced amplification of all targeted markers was achieved, resulting in a convenient characterization of the multiplexed PCR fragments with ICEMS. Expenditure of time, costs and hands-on work associated with assay design and optimization was dramatically lowered compared to previous multiplex PCR-ICEMS assays. The developed 23-plex assay was applied in a pharmacogenetic study including 284 individuals (genotype call rate 99.0%). A total of 399 SNPs were retyped by Sanger sequencing (concordance rate 99.8%). The PCR-ICEMS assay turned out to be an accurate, reliable, cost-effective and a ready-to-use tool for pharmacogenetic genotyping.

Comparison of DNA extraction methods for multiplex polymerase chain reaction.

We compared six DNA extraction methods for obtaining DNA from whole blood and saliva for use in multiplex polymerase chain reaction (PCR) assays. The aim was to evaluate saliva sampling as an alternative to blood sampling to obtain DNA for molecular diagnostics, genetic genealogy, and research purposes. The DNA quantity, DNA purity (A(260/280)), PCR inhibition ratio, and mitochondrial DNA/genomic DNA ratio were measured to compare the extraction methods. The different extraction methods resulted in variable DNA quantity and purity, but there were no significant differences in the efficiency of multiplex PCR and oligomicroarray signals after single-base extension on the arrayed primer extension 2 (APEX-2).

MTHFR and MSX1 contribute to the risk of nonsyndromic cleft lip/palate.

Recent studies suggest that multiple interacting loci, with possible additional environmental factors, influence the risk for nonsyndromic oral clefts, one of the most common birth defects in humans. Advances in high-throughput genotyping technology allow the testing of multiple markers, simultaneously, in many candidate genes. We tested for associations between 176 haplotype-tagging single nucleotide polymorphisms (SNPs) in 18 candidate genes/loci and nonsyndromic clefts in a case-control study in an Estonian sample (153 patients, 205 controls). The most significant associations with nonsyndromic cleft lip with or without cleft palate (CL/P) were found for SNPs in MSX1, MTHFR, and PVRL2, including several common haplotypes in the MTHFR and MSX1 genes. The strongest association was observed for rs6446693 in the MSX1 region, which remained statistically significant after Bonferroni correction. The strongest association with nonsyndromic cleft palate (CP) was found for the SNP rs11624283 in the JAG2 gene. Epistatic interactions were observed for SNPs within PVRL2, between BCL3 and EDN1, and between IRF6 and MSX1 genes. This study provides further evidence implicating MSX1 and MTHFR in the etiology of nonsyndromic CL/P across different populations.

In vitro fertilization does not increase the incidence of de novo copy number alterations in fetal and placental lineages.

Although chromosomal instability (CIN) is a common phenomenon in cleavage-stage embryogenesis following in vitro fertilization (IVF)1-3, its rate in naturally conceived human embryos is unknown. CIN leads to mosaic embryos that contain a combination of genetically normal and abnormal cells, and is significantly higher in in vitro-produced preimplantation embryos as compared to in vivo-conceived preimplantation embryos4. Even though embryos with CIN-derived complex aneuploidies may arrest between the cleavage and blastocyst stages of embryogenesis5,6, a high number of embryos containing abnormal cells can pass this strong selection barrier7,8. However, neither the prevalence nor extent of CIN during prenatal development and at birth, following IVF treatment, is well understood. Here we profiled the genomic landscape of fetal and placental tissues postpartum from both IVF and naturally conceived children, to investigate the prevalence and persistence of large genetic aberrations that probably arose from IVF-related CIN. We demonstrate that CIN is not preserved at later stages of prenatal development, and that de novo numerical aberrations or large structural DNA imbalances occur at similar rates in IVF and naturally conceived live-born neonates. Our findings affirm that human IVF treatment has no detrimental effect on the chromosomal constitution of fetal and placental lineages.

rs10732516 polymorphism at the IGF2/H19 locus associates with genotype-specific effects on placental DNA methylation and birth weight of newborns conceived by assisted reproductive technology.

Background: Assisted reproductive technology (ART) has been associated with low birth weight of fresh embryo transfer (FRESH) derived and increased birth weight of frozen embryo transfer (FET)-derived newborns. Owing to that, we focused on imprinted insulin-like growth factor 2 (IGF2)/H19 locus known to be important for normal growth. This locus is regulated by H19 imprinting control region (ICR) with seven binding sites for the methylation-sensitive zinc finger regulatory protein (CTCF). A polymorphism rs10732516 G/A in the sixth binding site for CTCF, associates with a genotype-specific trend to the DNA methylation. Due to this association, 62 couples with singleton pregnancies derived from FRESH (44 IVF/18 ICSI), 24 couples from FET (15 IVF/9 ICSI), and 157 couples with spontaneously conceived pregnancies as controls were recruited in Finland and Estonia for genotype-specific examination. DNA methylation levels at the H19 ICR, H19 DMR, and long interspersed nuclear elements in placental tissue were explored by MassARRAY EpiTYPER (n = 122). Allele-specific changes in the methylation level of H19 ICR in placental tissue (n = 26) and white blood cells (WBC, n = 8) were examined by bisulfite sequencing. Newborns' (n = 243) anthropometrics was analyzed by using international growth standards. Results: A consistent trend of genotype-specific decreased methylation level was observed in paternal allele of rs10732516 paternal A/maternal G genotype, but not in paternal G/maternal A genotype, at H19 ICR in ART placentas. This hypomethylation was not detected in WBCs. Also genotype-specific differences in FRESH-derived newborns' birth weight and head circumference were observed (P = 0.04, P = 0.004, respectively): FRESH-derived newborns with G/G genotype were heavier (P = 0.04) and had larger head circumference (P = 0.002) compared to newborns with A/A genotype. Also, the placental weight and birth weight of controls, FRESH- and FET-derived newborns differed significantly in rs10732516 A/A genotype (P = 0.024, P = 0.006, respectively): the placentas and newborns of FET-derived pregnancies were heavier compared to FRESH-derived pregnancies (P = 0.02, P = 0.004, respectively). Conclusions: The observed DNA methylation changes together with the phenotypic findings suggest that rs10732516 polymorphism associates with the effects of ART in a parent-of-origin manner. Therefore, this polymorphism should be considered when the effects of environmental factors on embryonic development are studied.

Using RNA sequencing for identifying gene imprinting and random monoallelic expression in human placenta.

Given the possible critical importance of placental gene imprinting and random monoallelic expression on fetal and infant health, most of those genes must be identified, in order to understand the risks that the baby might meet during pregnancy and after birth. Therefore, the aim of the current study was to introduce a workflow and tools for analyzing imprinted and random monoallelic gene expression in human placenta, by applying whole-transcriptome (WT) RNA sequencing of placental tissue and genotyping of coding DNA variants in family trios. Ten family trios, each with a healthy spontaneous single-term pregnancy, were recruited. Total RNA was extracted for WT analysis, providing the full sequence information for the placental transcriptome. Parental and child blood DNA genotypes were analyzed by exome SNP genotyping microarrays, mapping the inheritance and estimating the abundance of parental expressed alleles. Imprinted genes showed consistent expression from either parental allele, as demonstrated by the SNP content of sequenced transcripts, while monoallelically expressed genes had random activity of parental alleles. We revealed 4 novel possible imprinted genes (LGALS8, LGALS14, PAPPA2 and SPTLC3) and confirmed the imprinting of 4 genes (AIM1, PEG10, RHOBTB3 and ZFAT-AS1) in human placenta. The major finding was the identification of 4 genes (ABP1, BCLAF1, IFI30 and ZFAT) with random allelic bias, expressing one of the parental alleles preferentially. The main functions of the imprinted and monoallelically expressed genes included: i) mediating cellular apoptosis and tissue development; ii) regulating inflammation and immune system; iii) facilitating metabolic processes; and iv) regulating cell cycle.

Evaluation of the 124-plex SNP typing microarray for forensic testing.

Human identification systems such as criminal databases, forensic DNA testing and genetic genealogy require reliable and cost-effective genotyping of autosomal, mitochondrial and Y chromosome markers from different biological materials, including venous blood and saliva. Although many such assays are available, few systems are capable of simultaneously detecting all three targets in a single reaction. Employing the APEX-2 principle, we have characterized a novel 124-plex assay, using specific primer extension, universal primer amplification and single base extension on an oligonucleotide array. The assay has been designed for simultaneous genotyping of SNPs from the single copy loci (46 autosomal and 29 Y chromosomal markers) side by side with SNPs from the mitochondrial genome (49 markers) that appears in up to thousands of copies per cell in certain tissue types. All the autosomal SNPs (from the SNPforID Consortium) included in the multiplex assay are unlinked and are distributed widely across autosomes, enabling genetic fingerprints to be distinguished. Mitochondrial DNA and Y chromosome polymorphisms that define haplogroups common in European populations are included to allow for maternity and paternity testing and for the analysis of genetic genealogies. After assay optimization we estimated the accuracy (99.83%) and call rate (99.66%) of the protocol on 17 mother-father-child/children families and five internal control DNAs. In addition, 79 unrelated Estonian and Swedish DNA samples were genotyped and the accuracy of mtDNA and Y chromosome haplogroup inference by the multiplex method was assessed using conventional genotyping methods and direct sequencing.