The impact of maternal age on gene expression during the GV to MII transition in euploid human oocytes.

STUDY QUESTION: Are there age-related differences in gene expression during the germinal vesicle (GV) to metaphase II (MII) stage transition in euploid human oocytes? SUMMARY ANSWER: A decrease in mitochondrial-related transcripts from GV to MII oocytes was observed, with a much greater reduction in MII oocytes with advanced age. WHAT IS KNOWN ALREADY: Early embryonic development is dependent on maternal transcripts accumulated and stored within the oocyte during oogenesis. Transcriptional activity of the oocyte, which dictates its ultimate developmental potential, may be influenced by age and explain the reduced competence of advanced maternal age (AMA) oocytes compared with the young maternal age (YMA). Gene expression has been studied in human and animal oocytes; however, RNA sequencing could provide further insights into the transcriptome profiling of GV and in vivo matured MII euploid oocytes of YMA and AMA patients. STUDY DESIGN, SIZE, DURATION: Fifteen women treated for infertility in a single IVF unit agreed to participate in this study. Five GV and 5 MII oocytes from 6, 21-26 years old women (YMA cohort) and 5 GV and 6 MII oocytes from 6, 41-44 years old women (AMA cohort) undergoing IVF treatment were donated. The samples were collected within a time frame of 4 months. RNA was isolated and deep sequenced at the single-cell level. All donors provided either GV or MII oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cumulus dissection from donated oocytes was performed 38 h after hCG injection, denuded oocytes were inserted into lysis buffer supplemented with RNase inhibitor. The samples were stored at -80°C until further use. Isolated RNA from GV and MII oocytes underwent library preparation using an oligo deoxy-thymidine (dT) priming approach (SMART-Seq v4 Ultra Low Input RNA assay; Takara Bio, Japan) and Nextera XT DNA library preparation assay (Illumina, USA) followed by deep sequencing. Data processing, quality assessment and bioinformatics analysis were performed using source-software, mainly including FastQC, HISAT2, StringTie and edgeR, along with functional annotation analysis, while scploid R package was employed to determine the ploidy status. MAIN RESULTS AND THE ROLE OF CHANCE: Following deep sequencing of single GV and MII oocytes in both YMA and AMA cohorts, several hundred transcripts were found to be expressed at significantly different levels. When YMA and AMA MII oocyte transcriptomes were compared, the most significant of these were related to mitochondrial structure and function, including biological processes, mitochondrial respiratory chain complex I assembly and mitochondrial translational termination (false discovery rate (FDR) 6.0E-10 to 1.2E-7). These results indicate a higher energy potential of the YMA MII cohort that is reduced with ageing. Other biological processes that were significantly higher in the YMA MII cohort included transcripts involved in the translation process (FDR 1.9E-2). Lack of these transcripts could lead to inappropriate protein synthesis prior to or upon fertilisation of the AMA MII oocytes. LARGE SCALE DATA: The RNA sequencing data were deposited in the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo), under the accession number: GSE164371. LIMITATIONS, REASONS FOR CAUTION: The relatively small sample size could be a reason for caution. However, the RNA sequencing results showed homogeneous clustering with low intra-group variation and five to six biological replicates derived from at least three different women per group minimised the potential impact of the sample size. WIDER IMPLICATIONS OF THE FINDINGS: Understanding the effects of ageing on the oocyte transcriptome could highlight the mechanisms involved in GV to MII transition and identify biomarkers that characterise good MII oocyte quality. This knowledge has the potential to guide IVF regimes for AMA patients. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Medical Research Council (MRC Grant number MR/K020501/1).

Dysmorphology services: a snapshot of current practices and a vision for the future.

Dysmorphology concerns the recognition and management of rare, multiple anomaly syndromes. Genomic technologies and software for gestalt recognition will re-shape dysmorphology services. In order to reflect on a model of the service in the post-genomic era, we compared the utility of dysmorphology consultations in two Mediterranean cities, Athens, Greece and Afula, Israel (MDS), the Manchester Centre for Genomic Medicine, a UK service with dysmorphology expertise (UKDS) and the DYSCERNE, digital service (DDS). We show that it is more likely that chromosome microarray analysis will be performed if suggested in the UKDS rather than in the MDS; this, most probably reflects the difference of access to genetic testing following funding limitations in the MDS. We also show that in terms of achieved diagnosis, the first visit to a dysmorphology clinic is more significant than a follow-up. We show that a confirmed syndrome diagnosis significantly decreases the requests for other, non-genetic, laboratory investigations. Conversely, it increases the requests for reviews by other specialists and, most significantly (t-test: 8.244), it increases further requests for screening for possible associated complications. This is the first demonstration of the demands, on a health service, following the diagnosis of a dysmorphic condition.

Recurrent copy number variations as risk factors for autism spectrum disorders: analysis of the clinical implications.

Chromosomal microarray analysis (CMA) is currently considered a first-tier diagnostic assay for the investigation of autism spectrum disorders (ASD), developmental delay and intellectual disability of unknown etiology. High-resolution arrays were utilized for the identification of copy number variations (CNVs) in 195 ASD patients of Greek origin (126 males, 69 females). CMA resulted in the detection of 65 CNVs, excluding the known polymorphic copy number polymorphisms also found in the Database of Genomic Variants, for 51/195 patients (26.1%). Parental DNA testing in 20/51 patients revealed that 17 CNVs were de novo, 6 paternal and 3 of maternal origin. The majority of the 65 CNVs were deletions (66.1%), of which 5 on the X-chromosome while the duplications, of which 7 on the X-chromosome, were rarer (22/65, 33.8%). Fifty-one CNVs from a total of 65, reported for our cohort of ASD patients, were of diagnostic significance and well described in the literature while 14 CNVs (8 losses, 6 gains) were characterized as variants of unknown significance and need further investigation. Among the 51 patients, 39 carried one CNV, 10 carried two CNVs and 2 carried three CNVs. The use of CMA, its clinical validity and utility was assessed.

miR-15a and miR-24-1 as putative prognostic microRNA signatures for pediatric pilocytic astrocytomas and ependymomas.

In the current setting, we attempted to verify and validate miRNA candidates relevant to pediatric primary brain tumor progression and outcome, in order to provide data regarding the identification of novel prognostic biomarkers. Overall, 26 resected brain tumors were studied from children diagnosed with pilocytic astrocytomas (PAs) (n = 19) and ependymomas (EPs) (n = 7). As controls, deceased children who underwent autopsy and were not present with any brain malignancy were used. The experimental approach included microarrays covering 1211 miRNAs. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to validate the expression profiles of miR-15a and miR-24-1. The multiparameter analyses were performed with MATLAB. Matching differentially expressed miRNAs were detected in both PAs and EPs, following distinct comparisons with the control cohort; however, in several cases, they exhibited tissue-specific expression profiles. On correlations between miRNA expression and EP progression or outcome, miR-15a and miR-24-1 were found upregulated in EP relapsed and EP deceased cases when compared to EP clinical remission cases and EP survivors, respectively. Taken together, following several distinct associations between miRNA expression and diverse clinical parameters, the current study repeatedly highlighted miR-15a and miR-24-1 as candidate oncogenic molecules associated with inferior prognosis in children diagnosed with ependymoma.

SMA prenatal diagnosis: a modified protocol to help differentiation between deletions and gene conversion.

In SMA, unusual findings such as deletions restricted only to SMN1 exon 8, inspite of honozygous SMN1 exons 7-8 deletions in the family, may obscure final diagnosis. Application of a modified PCR procedure allowed discrimination between a deletion or a gene conversion event in a case of prenatal diagnosis.

De novo interstitial duplication of the 15q11.2-q14 PWS/AS region of maternal origin: Clinical description, array CGH analysis, and review of the literature.

The 15q11-q13 PWS/AS critical region involves genes that are characterized by genomic imprinting. Multiple repeat elements within the region mediate rearrangements, including interstitial duplications, interstitial triplications, and supernumerary isodicentric marker chromosomes, as well as the deletions that cause Prader-Willi syndrome (PWS) and Angelman syndrome (AS). Recently, duplications of maternal origin concerning the same critical region have been implicated in autism spectrum disorders (ASD). We present a 6-month-old girl carrying a de novo duplication of maternal origin of the 15q11.2-q14 PWS/AS region (17.73 Mb in size) [46,XX,dup(15)(q11.2-q14)] detected with a high-resolution microarray-based comparative genomic hybridization (array-CGH). The patient is characterized by severe hypotonia, obesity, microstomia, long eyelashes, hirsutism, microretrognathia, short nose, severe psychomotor retardation, and multiple episodes of drug-resistant epileptic seizures, while her brain magnetic resonance imaging (MRI) documented partial corpus callosum dysplasia. In our patient the duplicated region is quite large extending beyond the Prader-Willi-Angelman critical region (PWACR), containing a number of genes that have been shown to be involved in ASD, exhibiting a severe phenotype, beyond the typical PWS/AS clinical manifestations. Reporting of similar well-characterized clinical cases with clearly delineated breakpoints of the duplicated region will clarify the contribution of specific genes to the phenotype.

Long term follow up of a woman with classic form of Ehlers-Danlos syndrome associated with rare manifestations and review of the literature.

We present the case of a 46 year-old woman with the classic type of Ehlers-Danlos syndrome who developed the rare manifestations of colon diverticula and mitral valve prolapse. We emphasize on clinical features and complications associated to this type of the syndrome, which gradually developed in our patient. A review of the literature referring to the epidemiology, molecular basis and manifestations of the classic type Ehlers-Danlos syndrome is also discussed.

PGD for X-linked and gender-dependent disorders using a robust, flexible single-tube PCR protocol.

X-linked genetic diseases include a wide range of disorders such as the dystrophinopathies. Additionally in some rare genetic diseases, severity of expression is gender dependent. Prevention of such disorders usually involves prenatal diagnosis and termination of affected pregnancies, while preimplantation genetic diagnosis (PGD) represents a specialized alternative that avoids pregnancy termination. To preclude the rejection of unaffected male embryos that cannot be differentiated from those affected when using fluorescence in-situ hybridization, a flexible protocol based on multiplex fluorescence polymerase chain reaction (PCR) was standardized and validated for gender determination in single cells, which can potentially incorporate any disease-specific locus. The final panel of nine loci included four loci on the Y chromosome, two on the X chromosome plus up to three microsatellite markers to either support the gender diagnosis or to further monitor extraneous contamination. The protocol, standardized on single lymphocytes, established a PCR efficiency of >93% for all loci with maximum allele dropout rates of 4%. Microsatellite analysis excluded external contamination and confirmed biallelic inheritance. Proof of principle for the simplicity and flexibility of the assay was demonstrated through its application to clinical PGD cycles for lipoid congenital adrenal hyperplasia, which presents a more severe clinical course in males, and Duchenne muscular dystrophy.

Unilateral microtia in an infant with trisomy 18 mosaicism.

Trisomy 18 is the second most frequent autosomal aneuploidy, after Down's syndrome, in humans. It causes severe congenital abnormalities and mental retardation although phenotypic features, clinical manifestations and prognosis vary occasionally. In cases oftrisomy 18 mosaicism, as in every chromosomal mosaicism, the spectrum of clinical characteristics extends from pathological to almost normal. We report a 9 months old female infant who has been referred to the Genetics Department for evaluation because of unilateral severe microtia, aplasia of mastoid abscess and hemifacial palsy and inlet type intraventricular defect with pulmonary hypertension. Chromosomal investigation revealed a mosaic trisomy 18 [46,XX/47,XX+18] in proportion of 52% and 48% respectively. Microtia/anotia is present in 1.46-4.36/10,000 live births in the general population while the combination of microtia/anotia with trisomy 18 has been reported in very few cases in the relevant bibliography.

Association of repeat polymorphisms in the estrogen receptors alpha, beta (ESR1, ESR2) and androgen receptor (AR) genes with the occurrence of breast cancer.

Genetic variation in genes involved in estrogen biosynthesis, metabolism and signal transduction have been suggested to play a role in breast cancer. To determine the possible contribution of genetic variation in the ESR1 (ER-alpha), ESR2 (ER-beta) and AR genes in breast cancer risk the -1174(TA)(7-27), c. 1092+3607(CA)(10-26) and c. 172(CAG)(6-40) repeat variants were studied in a case-control study of 79 women with sporadic breast cancer and 155 controls. No significant difference was observed in the frequency distribution of -1174(TA)(7-27) in the ESR1 gene between patients and controls, while a significant difference was observed for repeat polymorphisms c. 1092+3607(CA)(10-26) in the ESR2 gene and c. 172(CAG)(6-40) in the AR gene (p0.0001). A significantly decreased odds ratio (OR) for breast cancer risk was observed in individuals having the LL and the SL genotypes for both the ESR2 (OR=0.010, 95% CI 0.003-0.036, p<0.001; OR=0.013, 95% CI 0.004-0.040, p<0.0001, respectively) and the AR gene (OR=0.040, 95% CI 0.011-0.138, p<0.0001; OR=0.189, 95% CI 0.10-0.359, p<0.0001, respectively), compared to SS genotype. The protective effect of these genotypes remained evident even after adjustment for various risk factors (BMI, age, age at menarche and menopause, family history). In conclusion, an association for breast cancer risk between short (SS) alleles for the repeat variants of the ESR2 and AR genes was found in women of Greek descent.

10 years' experience in fragile X testing among mentally retarded individuals in Greece: a molecular and epidemiological approach.

Fragile X syndrome, the second most common genetic cause of mental retardation, is due to the expansion of a trinucleotide repeat (CGG)n within the first exon of the FMR-1 gene. Molecular genetic analysis provides accurate diagnosis and facilitates genetic counselling and prenatal testing. Screening for the fragile X mutation in a sample of 3,888 individuals in Greece is reported: 1,755 children with non-specific mental retardation, 1,733 parents and other family members and 400 normal individuals. Molecular analysis allowed for the identification and characterization of 52 fragile X families confirming the clinical diagnosis in 57 males and 4 females. Sixty-six female carriers (6 mentally retarded) and 4 normal transmitting males were also identified. Four severely retarded males and their mothers carried unmethylated premutations, while a moderately retarded girl had a deletion of approximately equal to 150 bp. Overall sizing of the CGG repeat produced an allele distribution of 6-58 CGG repeats (mean 28-30), similar to that in other Caucasian populations.

Coinheritance of mutated SMN1 and MECP2 genes in a child with phenotypic features of spinal muscular atrophy (SMA) type II and Rett syndrome.

Spinal muscular atrophy (SMA) is a neuromuscular autosomal recessive disease characterized by progressive muscle weakness and atrophy combined with motor neuron degeneration caused by mutations in the SMN 1 gene locus (5q11.2-13.2). Rett syndrome (RS) is an X-linked dominant neurodevelopmental disorder caused by mutations in MECP2 (Xq28) and characterized by normal development until 6-12 months of age, followed by regression with loss of acquired skills, gradual onset of microcephaly, stereotypic hand movements and psychomotor delay. We report a 6-year-old girl who, at 2 years of age, presented with hypotonia, psychomotor delay, amyotrophy and areflexia of the lower extremities. Molecular DNA analysis (PCR-RFLP's) for SMA type II revealed that both exons 7 and 8 of SMN 1 gene were deleted. Over the past 4 years, onset of stereotypic hand-washing movements, epileptic seizures, microcephaly, hyperventilation/breath-holding attacks and severe psychomotor delay raised the suspicion of the coexistence of RS. DNA analysis (DGGE and sequencing) identified the hotspot missense mutation R306C (c.916C>T) in exon 4 of the MECP2 gene. The coinheritance of SMA and RS, two rare monogenic syndromes in the same patient, has not been previously reported. Thorough clinical evaluation in combination with DNA analysis, allowed accurate diagnosis, providing valuable information for the genetic counseling of the family.

Saethre-Chotzen syndrome with severe developmental delay associated with deletion of chromosomic region 7p15 --> pter.

Saethre-Chotzen syndrome represents one of the most common types of craniosynostosis inherited as an autosomal dominant disorder while sporadic cases have also been reported. It is characterized by high penetrance and variable expressivity, leading to difficulties in clinical diagnosis. Some patients, who exhibit most of the diagnostic criteria of Saethre-Chotzen syndrome, have structural abnormalities of chromosome 7. The case of a 4 year old boy with notable dysmorphic features compatible with Saethre-Chotzen syndrome and severe developmental delay is described. Conventional and molecular cytogenetic analysis of peripheral blood samples from the patient and his parents revealed partial monosomy of chromosomal region 7p15 --> pter de novo. The TWIST gene, located on chromosome 7p21.1, is thought to be a negative transcriptional regulator involved in osteoblast differentiation and maturation and it is thought that haploinsufficiency of the gene can cause the disorder. The diagnosis of Saethre-Chotzen syndrome and the identification of the chromosomal abnormality in the patient facilitated genetic counseling of the family.

Clinical manifestations in 17 Greek patients with Goldenhar syndrome.

Goldenhar (GS) syndrome is a well-recognised developmental disorder involving first and second branchial arches and characterized by considerable phenotypic variability. The present study presents clinical data on the morphologic features, hearing, ophthalmologic, orthopaedic, neurological, cardiovascular, genitourinary and gastrointestinal evaluation of 17 Greek patients (one pair of monozygotic twins) aged 20 days to 23 years with the clinical diagnosis of GS and with a normal karyotype. The most consistent findings were auricular defects (94%), followed by facial (76%) and ocular anomalies (65%), 70% unilateral, mainly right-sided. In the majority of our patients (90%) mandibular hypoplasia was ipsilateral to the dysplastic ear or the most severely affected ear in bilateral cases. Hearing loss, mainly conductive, was noted in 76% of our patients. Skeletal defects were evident in 23%, while cardiovascular, genitourinary and gastrointestinal in 18%, 23% and 12% respectively. The most frequent neurological manifestation was facial nerve paralysis (12%), while the incidence of mental retardation was higher (23%) than reported in the literature, presumably attributed to the severe hearing and vision loss. In a pair of monozygotic twins of our study discordance of clinical findings was noted. Precise evaluation of GS patients and multidisciplinary care management is necessary to avoid possible complications of many systems and to offer appropriate genetic counselling to the family.

MicroRNA expression profiles in pediatric dysembryoplastic neuroepithelial tumors.

Among noncoding RNAs, microRNAs (miRNAs) have been most extensively studied, and their biology has repeatedly been proven critical for central nervous system pathological conditions. The diagnostic value of several miRNAs was appraised in pediatric dysembryoplastic neuroepithelial tumors (DNETs) using miRNA microarrays and receiving operating characteristic curves analyses. Overall, five pediatric DNETs were studied. As controls, 17 samples were used: the FirstChoice Human Brain Reference RNA and 16 samples from deceased children who underwent autopsy and were not present with any brain malignancy. The miRNA extraction was carried out using the mirVANA miRNA Isolation Kit, while the experimental approach included miRNA microarrays covering 1211 miRNAs. Quantitative real-time polymerase chain reaction was performed to validate the expression profiles of miR-1909* and miR-3138 in all samples initially screened with miRNA microarrays. Our findings indicated that miR-3138 might act as a tumor suppressor gene when down-regulated and miR-1909* as a putative oncogenic molecule when up-regulated in pediatric DNETs compared to the control cohort. Subsequently, both miRNA signatures might serve as putative diagnostic biomarkers for pediatric DNETs.

Impact of cytogenetic and molecular cytogenetic studies on hematologic malignancies.

Conventional cytogenetic analysis of chromosome abnormalities in hematologic malignancies is hampered by the low mitotic index and poor quality of metaphases. A range of techniques based on fluorescence in situ hybridization (FISH) has greatly enhanced the identification of non-random translocations and deletions, pinpointing regions which contain genes involved in leukemogenesis. One of the main advantages of FISH is its ability to use non-dividing interphase cells as DNA targets, enabling the screening of large numbers of cells and providing access to a variety of cells with different hematopoetic activity. Furthermore, multicolor FISH (SKY, M-FISH and CGH microarrays) combines the screening potential of cytogenetics with the accuracy of molecular genetics, allowing the visualization of the entire human genome in 24 different colors.

Preimplantation genetic diagnosis in clinical practice.

Preimplantation genetic diagnosis (PGD) represents an alternative to prenatal diagnosis and allows selection of unaffected IVF embryos for establishing pregnancies in couples at risk for transmitting a genetic disorder.

Synthesized allosteric effectors of the hemoglobin molecule: a possible mechanism for improved erythrocyte oxygen release capability in hemoglobinopathy H disease.

Patients with the nondeletion genotype of hemoglobinopathy H (HbH or beta4) disease have higher proportions of HbH and more severe tissue hypoxia than patients with the deletion genotype. Because these patients' red blood cells (RBCs) contain mainly two Hb species, HbH and HbA, the high proportion of HbA can be exploited by lowering its oxygen affinity; this would probably increase oxygen delivery to the RBCs and improve the patients' clinical phenotype. Allosteric effectors that induce a low-affinity Hb may be useful in this regard. We investigated the effect of a bezafibrate derivative, RSR-4, on the oxygen affinity of RBCs and purified hemolysates containing HbA and HbH. This allosteric effector crosses RBC membranes and binds reversibly to the alpha-chains of deoxy-Hb, decreasing hemoglobin oxygen affinity. The blood used was obtained from a patient with HbH disease (alphaTSaudi homozygote) whose HbH level was 33.5% as measured by high-performance liquid chromatography. Oxygen binding studies were performed in RBCs and purified hemolysates. RBCs incubated in the presence of 500 microM RSR-4 (2-[[[(3,5-dichloroanilino)-carbonyl]methyl]phenoxy]-2-methylpropi onic acid) in standard conditions (pH 7.4, 0.14 M NaCl, 37 degrees C) displayed an increase in their P50 value from 14.5 to 35.2 mm Hg. Oxygen binding studies in purified stripped hemolysates (pH 7.2, 0.1 M NaCl, 25 degrees C) showed that addition of both 500 microM RSR-4 and 1 mM of 2,3 diphosphoglycerate (DPG) led to an 11-fold decrease in oxygen affinity, whereas the addition of the natural effector DPG or RSR-4 alone produced a 2.7- and 5.7-fold decrease, respectively. In both cases, the oxygen equilibrium curves (OECs) were biphasic due to the presence of the noncooperative, high-oxygen-affinity HbH (beta4) component. After addition of RSR-4, the lower part of the OEC (corresponding to HbH) was not shifted compared with the upper part (corresponding to HbA). These results were confirmed by kinetic studies of CO recombination. Both experiments demonstrated that RSR-4 does not affect beta4 Hb. Our findings provide an experimental model for lowering the oxygen affinity of HbA in HbH-containing cells and suggest that the oxygen delivery capability of the latter would be thereby improved.

Abnormal mRNA splicing resulting from consensus sequence splicing mutations of ATP7B.

More than 200 Wilson disease (WD) disease-causing mutations have been defined to date. Missense mutations are largely prevalent while splice-site mutations are limited in number. Most reside in the splice donor or acceptor sites and only a minority are detected in splicing consensus sequences. Furthermore, only a few splicing mutations have been studied at the RNA level to date. In this study, using the RT-PCR method we performed the molecular characterization of four consensus splice-site mutations identified by DNA analysis in patients with WD. One of them, previously described 1707+3insT, occurred at position 3 in the donor splice site of intron 4, while the other three, 2122-8T>G, 2866-6T>G, and 3061-12T>A, are novel and occurred in the acceptor splice sites of introns 7, 12, and 13, respectively. Analysis revealed a prevalently abnormal splicing in the samples carrying the mutations compared to the normal controls. Comparison of RNA splicing with normal controls in liver and lymphocytes further suggests that abnormal splicing of the WD gene is also present and differentially regulated in normal tissues. The results produced in this study strongly suggest that DNA mutations residing in the consensus sequence of WD gene splice sites result in the WD phenotype by interfering with the production of the normal WD protein. Further studies are necessary to better quantify the amount of different transcripts produced by these mutations, and establish their correlation with the disease phenotype.

Haplotype and mutation analysis in Greek patients with Wilson disease.

In this study, we report the results of haplotype and mutation analysis of the ATP7B gene in Wilson disease (WD) patients of Greek origin. We have analysed 25 WD families and two single patients and characterised 94% of the WD chromosomes investigated. We have found 12 different molecular defects (three frameshifts, two splice site, two nonsense, five missense mutations), four of which are novel. Five of the mutations are widely prevalent accounting for 74% of the WD chromosomes analysed. These results may enable preclinical diagnosis in the large majority of WD patients of Greek descent, thereby improving genetic counselling and disease management.