Multitrait genome association analysis identifies new susceptibility genes for human anthropometric variation in the GCAT cohort.

BACKGROUND: Heritability estimates have revealed an important contribution of SNP variants for most common traits; however, SNP analysis by single-trait genome-wide association studies (GWAS) has failed to uncover their impact. In this study, we applied a multitrait GWAS approach to discover additional factor of the missing heritability of human anthropometric variation. METHODS: We analysed 205 traits, including diseases identified at baseline in the GCAT cohort (Genomes For Life- Cohort study of the Genomes of Catalonia) (n=4988), a Mediterranean adult population-based cohort study from the south of Europe. We estimated SNP heritability contribution and single-trait GWAS for all traits from 15 million SNP variants. Then, we applied a multitrait-related approach to study genome-wide association to anthropometric measures in a two-stage meta-analysis with the UK Biobank cohort (n=336 107). RESULTS: Heritability estimates (eg, skin colour, alcohol consumption, smoking habit, body mass index, educational level or height) revealed an important contribution of SNP variants, ranging from 18% to 77%. Single-trait analysis identified 1785 SNPs with genome-wide significance threshold. From these, several previously reported single-trait hits were confirmed in our sample with LINC01432 (p=1.9×10-9) variants associated with male baldness, LDLR variants with hyperlipidaemia (ICD-9:272) (p=9.4×10-10) and variants in IRF4 (p=2.8×10-57), SLC45A2 (p=2.2×10-130), HERC2 (p=2.8×10-176), OCA2 (p=2.4×10-121) and MC1R (p=7.7×10-22) associated with hair, eye and skin colour, freckling, tanning capacity and sun burning sensitivity and the Fitzpatrick phototype score, all highly correlated cross-phenotypes. Multitrait meta-analysis of anthropometric variation validated 27 loci in a two-stage meta-analysis with a large British ancestry cohort, six of which are newly reported here (p value threshold <5×10-9) at ZRANB2-AS2, PIK3R1, EPHA7, MAD1L1, CACUL1 and MAP3K9. CONCLUSION: Considering multiple-related genetic phenotypes improve associated genome signal detection. These results indicate the potential value of data-driven multivariate phenotyping for genetic studies in large population-based cohorts to contribute to knowledge of complex traits.

Large-scale screening of circulating microRNAs in individuals with HIV-1 mono-infections reveals specific liver damage signatures.

Human immunodeficiency virus type 1 (HIV-1)-induced inflammation and/or long-term antiretroviral drug toxicity may contribute to the evolution of liver disease. We investigated circulating plasma microRNAs (miRNAs) as potential biomarkers of liver injury in patients mono-infected with HIV-1. We performed large-scale deep sequencing analyses of small RNA level on plasma samples from patients with HIV-1 mono-infection that had elevated or normal levels of alanine aminotransferase (ALT) or focal nodular hyperplasia (FNH). Hepatitis C virus (HCV) mono-infected patients were also studied. Compared to healthy donors, patients with HIV-1 or HCV mono-infections showed significantly altered (fold change >2, adjusted p < 0.05) level of 25 and 70 miRNAs, respectively. Of the 25 altered miRNAs found in patients with HIV-1, 19 were also found in patients mono-infected with HCV. Moreover, 13 of the 14 most up-regulated miRNAs (range: 9.3-3.4-fold increase) in patients with HCV mono-infections were also up-regulated in patients with HIV-1 mono-infections. Importantly, most of these miRNAs significantly and positively correlated with ALT and aspartate aminotransferase (AST) levels, and liver fibrosis stage (p < 0.05). MiR-122-3p and miR-193b-5p were highly up-regulated HIV-1 mono-infected patients with elevated ALT or FNH, but not in HIV-1 patients with normal levels of ALT. These results reveal that HIV-1 infections impacted liver-related miRNA levels in the absence of an HCV co-infection, which highlights the potential of miRNAs as biomarkers for the progression of liver injury in HIV-1 infected patients.

Double-factor preimplantation genetic diagnosis: monogenic and cytogenetic diagnoses analyzing a single blastomere.

OBJECTIVE: Enhancing implantation rates in preimplantation genetic diagnosis (PGD) cycles is still a challenging aspect to address. As aneuploidy can be one of the factors influencing the low implantation rates obtained, the aim of this work was to combine monogenic analysis with comprehensive aneuploidy screening (double factor) in order to transfer the selected (healthy and euploid) embryos in the same in-vitro fertilization (IVF) cycle. METHOD: In the present double-factor PGD (DF-PGD) approach, a single blastomere was biopsied from each embryo, and the whole genome amplification DNA product obtained was successfully used for both monogenic analysis and metaphase comparative genomic hybridization cytogenetic screening. The developed DF-PGD was applied to 62 embryos from seven families at risk for monogenic-inherited diseases in a total of seven IVF-DF-PGD cycles. RESULTS: While 68.2% of the diagnosed embryos were healthy for the monogenic diseases, only 43.3% of them were chromosomally normal considering aneuploidies and/or segmental chromosome imbalances. Six out of seven families had transferrable embryos according to DF-PGD results. Two healthy babies were born from the 11 selected embryo transfers. CONCLUSION: In families at risk for monogenic diseases, the DF-PGD is a useful tool to select healthy and potentially viable embryos for transfer, according to their chromosome complement.

Patterns of somatic uniparental disomy identify novel tumor suppressor genes in colorectal cancer.

Colorectal cancer (CRC) is characterized by specific patterns of copy number alterations (CNAs), which helped with the identification of driver oncogenes and tumor suppressor genes (TSGs). More recently, the usage of single nucleotide polymorphism arrays provided information of copy number neutral loss of heterozygosity, thus suggesting the occurrence of somatic uniparental disomy (UPD) and uniparental polysomy (UPP) events. The aim of this study is to establish an integrative profiling of recurrent UPDs/UPPs and CNAs in sporadic CRC. Our results indicate that regions showing high frequencies of UPD/UPP mostly coincide with regions typically involved in genomic losses. Among them, chromosome arms 3p, 5q, 9q, 10q, 14q, 17p, 17q, 20p, 21q and 22q preferentially showed UPDs/UPPs over genomic losses suggesting that tumor cells must maintain the disomic state of certain genes to favor cellular fitness. A meta-analysis using over 300 samples from The Cancer Genome Atlas confirmed our findings. Several regions affected by recurrent UPDs/UPPs contain well-known TSGs, as well as novel candidates such as ARID1A, DLC1, TCF7L2 and DMBT1. In addition, VCAN, FLT4, SFRP1 and GAS7 were also frequently involved in regions of UPD/UPP and displayed high levels of methylation. Finally, sequencing and fluorescence in situ hybridization analysis of the gene APC underlined that a somatic UPD event might represent the second hit to achieve biallelic inactivation of this TSG in colorectal tumors. In summary, our data define a profile of somatic UPDs/UPPs in sporadic CRC and highlights the importance of these events as a mechanism to achieve the inactivation of TSGs.

Comprehensive preimplantation genetic screening and sperm deoxyribonucleic acid fragmentation from three males carrying balanced chromosome rearrangements.

OBJECTIVE: To assess whether preimplantation genetic screening can successfully identify cytogenetically normal embryos in couples carrying balanced chromosome rearrangements in addition to increased sperm DNA fragmentation. DESIGN: Comprehensive preimplantation genetic screening was performed on three couples carrying chromosome rearrangements. Sperm DNA fragmentation was assessed for each patient. SETTING: Academic center. PATIENT(S): One couple with the male partner carrying a chromosome 2 pericentric inversion and two couples with the male partners carrying a Robertsonian translocation (13:14 and 14:21, respectively). INTERVENTION(S): A single blastomere from each of the 18 cleavage-stage embryos obtained was analysed by metaphase comparative genomic hybridization. Single- and double-strand sperm DNA fragmentation was determined by the alkaline and neutral Comet assays. MAIN OUTCOME MEASURE(S): Single- and double-strand sperm DNA fragmentation values and incidence of chromosome imbalances in the blastomeres were analyzed. RESULT(S): The obtained values of single-strand sperm DNA fragmentation were between 47% and 59%, and the double-strand sperm DNA fragmentation values were between 43% and 54%. No euploid embryos were observed in the couple showing the highest single-strand sperm DNA fragmentation. However, euploid embryos were observed in the other two couples: embryo transfer was performed, and pregnancy was achieved by the couple showing the lowest sperm DNA fragmentation values. CONCLUSION(S): Preimplantation genetic screening enables the detection of euploid embryos in couples affected by balanced chromosome rearrangements and increased sperm DNA fragmentation. Even though sperm DNA fragmentation may potentially have clinical consequences on fertility, comprehensive preimplantation genetic screening allows for the identification and transfer of euploid embryos.

Oligonucleotide arrays vs. metaphase-comparative genomic hybridisation and BAC arrays for single-cell analysis: first applications to preimplantation genetic diagnosis for Robertsonian translocation carriers.

Comprehensive chromosome analysis techniques such as metaphase-Comparative Genomic Hybridisation (CGH) and array-CGH are available for single-cell analysis. However, while metaphase-CGH and BAC array-CGH have been widely used for Preimplantation Genetic Diagnosis, oligonucleotide array-CGH has not been used in an extensive way. A comparison between oligonucleotide array-CGH and metaphase-CGH has been performed analysing 15 single fibroblasts from aneuploid cell-lines and 18 single blastomeres from human cleavage-stage embryos. Afterwards, oligonucleotide array-CGH and BAC array-CGH were also compared analysing 16 single blastomeres from human cleavage-stage embryos. All three comprehensive analysis techniques provided broadly similar cytogenetic profiles; however, non-identical profiles appeared when extensive aneuploidies were present in a cell. Both array techniques provided an optimised analysis procedure and a higher resolution than metaphase-CGH. Moreover, oligonucleotide array-CGH was able to define extra segmental imbalances in 14.7% of the blastomeres and it better determined the specific unbalanced chromosome regions due to a higher resolution of the technique (≈ 20 kb). Applicability of oligonucleotide array-CGH for Preimplantation Genetic Diagnosis has been demonstrated in two cases of Robertsonian translocation carriers 45,XY,der(13;14)(q10;q10). Transfer of euploid embryos was performed in both cases and pregnancy was achieved by one of the couples. This is the first time that an oligonucleotide array-CGH approach has been successfully applied to Preimplantation Genetic Diagnosis for balanced chromosome rearrangement carriers.

Non-meiotic chromosome instability in human immature oocytes.

Aneuploidy has been a major issue in human gametes and is closely related to fertility problems, as it is known to be present in cleavage stage embryos and gestational losses. Pre-meiotic chromosome abnormalities in women have been previously described. The aim of this study is to assess the whole-chromosome complement in immature oocytes to find those abnormalities caused by mitotic instability. For this purpose, a total of 157 oocytes at the germinal vesicle or metaphase I stage, and discarded from IVF cycles, were analysed by CGH. Fifty-six women, between 18 and 45 years old (mean 32.5 years), including 32 IVF patients (25-45 years of age) and 24 IVF oocyte donors (18-33 years of age), were included in the study. A total of 25/157 (15.9%) of the oocytes analysed, obtained from three IVF clinics, contained chromosome abnormalities, including both aneuploidy (24/157) and structural aberrations (9/157). Independently of the maternal age, the incidence of abnormal oocytes which originated before meiosis is 15.9%, and these imbalances were found in 33.9% of the females studied. This work sheds light on the relevance of mitotic instability responsible for the generation of the abnormalities present in human oocytes.

Does the S phase have an impact on the accuracy of comparative genomic hybridization profiles in single fibroblasts and human blastomeres?

OBJECTIVE: To investigate if there is an association between single-cell replicative stage and the segmental chromosome imbalances detected by comparative genomic hybridization (CGH). DESIGN: First, 135 fibroblasts from cell-line GM03184 (Coriell) at three cell stages (G0/G1, S, and G2/M) were amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) or Sureplex and blindly analyzed by CGH. Second, 85 human blastomeres at the interphase and the metaphase stages, from 30 donated human cryopreserved embryos, were amplified by Sureplex and analyzed by CGH. SETTING: Academic center for reproductive medicine. PATIENT(S): None. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Incidence of aneuploidy and segmental imbalances detected at the different cell stages. RESULT(S): In DOP-PCR amplifications of fibroblasts, an increased incidence of segmental abnormalities was detected in the S phase. In Sureplex amplifications of fibroblasts and blastomeres, no differences were detected between the different cell stages. A significantly increased incidence of structural abnormalities was seen in the aneuploid blastomeres. CONCLUSION(S): The segmental imbalances detected after Sureplex amplification in 73.3% of the cryopreserved embryos analyzed are mainly nontransitory. They correspond to segmental imbalances present in the cells due to chromosome instability, rather than to replicative DNA segments.

Detection of unbalanced chromosome segregations in preimplantation genetic diagnosis of translocations by short comparative genomic hibridization.

OBJECTIVE: To apply a comprehensive chromosomal screening through short comparative genomic hybridization (CGH) in the preimplantation genetic diagnosis (PGD) of translocations. DESIGN: Clinical research study. SETTING: A PGD laboratory and two IVF clinics. PATIENT(S): Three Robertsonian translocation carriers, two reciprocal translocation carriers, and a double-translocation carrier. INTERVENTION(S): After using the short-CGH approach in the reanalysis of two unbalanced embryos, discarded from a PGD for a reciprocal translocation carrier, the same method was applied in the PGD of day-3 embryos of translocation carriers. MAIN OUTCOME MEASURE(S): Ability of short CGH to detect partial chromosomal abnormalities in unbalanced embryos, translocation segregation proportions, and proportion of embryos carrying chromosomal abnormalities not related to the translocations. RESULT(S): The short-CGH technique detected errors resulting from the meiotic segregation of the chromosomes involved in the translocations and other abnormalities affecting the remaining chromosomes. Alternate segregation was detected most frequently among Robertsonian translocation cases, whereas unbalanced chromosome segregations were found predominantly in reciprocal ones. Aneuploidy and structural chromosome errors were found more frequently in Robertsonian than in reciprocal translocation carriers. Application of short-CGH PGD achieved pregnancy in two cases. CONCLUSION(S): Short CGH is a reliable approach for PGD of translocations, as it is capable of detecting partial chromosome errors caused by unbalanced segregations simultaneously to the screening of all chromosomes, and it may improve the results after PGD for translocation carriers.

Comprehensive embryo analysis of advanced maternal age-related aneuploidies and mosaicism by short comparative genomic hybridization.

The short comparative genomic hybridization (short-CGH) method was used to perform a comprehensive cytogenetic study of isolated blastomeres from advanced maternal age embryos, discarded after fluorescent in situ hybridization (FISH) preimplantation genetic screening (PGS), detecting aneuploidies (38.5% of which corresponded to chromosomes not screened by 9-chromosome FISH), structural aberrations (31.8%), and mosaicism (77.3%). The short-CGH method was subsequently applied in one PGS, achieving a twin pregnancy.