Recurrent spontaneous oocyte activation causes female infertility.

PURPOSE: Spontaneous oocyte activation (SOA) is a recently classified phenomenon characterized by the presence of a single pronucleus immediately following oocyte retrieval, without the apparent involvement of sperm. SOA currently remains poorly understood in humans, with no clear genetic or pathological factor(s). Herein, we report two separate cases of recurrent spontaneous oocyte activation, investigating potential avenues to identify causative etiology. METHODS: Two patients with several cycles with SOA have undergone further genetic and embryologic investigation to reveal underlying causes for SOA and provide a treatment if possible. RESULTS: One case was a patient with recurrent pregnancy loss and the other was diagnosed as unexplained infertility. In the first case, 61 out of 69 oocytes retrieved exhibited SOA in five cycles while in the second case 44 out of 49 oocytes exhibited SOA in five cycles. Oocytes were injected with sperm; embryo development and presence of paternal contribution were investigated. No pregnancy is ensued following embryo transfer in both patients. Time-lapse imaging of embryogenesis from the second case did not reveal even momentary second pronucleus appearance. We also performed clinical whole exome sequencing for both patients but did not identify any disease-causing variant. CONCLUSION: Patients with SOA suffer from infertility. Our results indicate that more investigation is required to understand the etiology of SOA in humans concentrating on the molecular mechanisms that underpin regulation of oocyte activation and calcium dynamics need to be investigated to fully understand, and perhaps in the future rectify, recurrent SOA.

Loss of UGP2 in brain leads to a severe epileptic encephalopathy, emphasizing that bi-allelic isoform-specific start-loss mutations of essential genes can cause genetic diseases.

Developmental and/or epileptic encephalopathies (DEEs) are a group of devastating genetic disorders, resulting in early-onset, therapy-resistant seizures and developmental delay. Here we report on 22 individuals from 15 families presenting with a severe form of intractable epilepsy, severe developmental delay, progressive microcephaly, visual disturbance and similar minor dysmorphisms. Whole exome sequencing identified a recurrent, homozygous variant (chr2:64083454A > G) in the essential UDP-glucose pyrophosphorylase (UGP2) gene in all probands. This rare variant results in a tolerable Met12Val missense change of the longer UGP2 protein isoform but causes a disruption of the start codon of the shorter isoform, which is predominant in brain. We show that the absence of the shorter isoform leads to a reduction of functional UGP2 enzyme in neural stem cells, leading to altered glycogen metabolism, upregulated unfolded protein response and premature neuronal differentiation, as modeled during pluripotent stem cell differentiation in vitro. In contrast, the complete lack of all UGP2 isoforms leads to differentiation defects in multiple lineages in human cells. Reduced expression of Ugp2a/Ugp2b in vivo in zebrafish mimics visual disturbance and mutant animals show a behavioral phenotype. Our study identifies a recurrent start codon mutation in UGP2 as a cause of a novel autosomal recessive DEE syndrome. Importantly, it also shows that isoform-specific start-loss mutations causing expression loss of a tissue-relevant isoform of an essential protein can cause a genetic disease, even when an organism-wide protein absence is incompatible with life. We provide additional examples where a similar disease mechanism applies.

Mutations in NALCN cause an autosomal-recessive syndrome with severe hypotonia, speech impairment, and cognitive delay.

Sodium leak channel, nonselective (NALCN) is a voltage-independent and cation-nonselective channel that is mainly responsible for the leaky sodium transport across neuronal membranes and controls neuronal excitability. Although NALCN variants have been conflictingly reported to be in linkage disequilibrium with schizophrenia and bipolar disorder, to our knowledge, no mutations have been reported to date for any inherited disorders. Using linkage, SNP-based homozygosity mapping, targeted sequencing, and confirmatory exome sequencing, we identified two mutations, one missense and one nonsense, in NALCN in two unrelated families. The mutations cause an autosomal-recessive syndrome characterized by subtle facial dysmorphism, variable degrees of hypotonia, speech impairment, chronic constipation, and intellectual disability. Furthermore, one of the families pursued preimplantation genetic diagnosis on the basis of the results from this study, and the mother recently delivered healthy twins, a boy and a girl, with no symptoms of hypotonia, which was present in all the affected children at birth. Hence, the two families we describe here represent instances of loss of function in human NALCN.

Genome-wide karyomapping accurately identifies the inheritance of single-gene defects in human preimplantation embryos in vitro.

PURPOSE: Our aim was to compare the accuracy of family- or disease-specific targeted haplotyping and direct mutation-detection strategies with the accuracy of genome-wide mapping of the parental origin of each chromosome, or karyomapping, by single-nucleotide polymorphism genotyping of the parents, a close relative of known disease status, and the embryo cell(s) used for preimplantation genetic diagnosis of single-gene defects in a single cell or small numbers of cells biopsied from human embryos following in vitro fertilization. METHODS: Genomic DNA and whole-genome amplification products from embryo samples, which were previously diagnosed by targeted haplotyping, were genotyped for single-nucleotide polymorphisms genome-wide detection and retrospectively analyzed blind by karyomapping. RESULTS: Single-nucleotide polymorphism genotyping and karyomapping were successful in 213/218 (97.7%) samples from 44 preimplantation genetic diagnosis cycles for 25 single-gene defects with various modes of inheritance distributed widely across the genome. Karyomapping was concordant with targeted haplotyping in 208 (97.7%) samples, and the five nonconcordant samples were all in consanguineous regions with limited or inconsistent haplotyping results. CONCLUSION: Genome-wide karyomapping is highly accurate and facilitates analysis of the inheritance of almost any single-gene defect, or any combination of loci, at the single-cell level, greatly expanding the range of conditions for which preimplantation genetic diagnosis can be offered clinically without the need for customized test development.

First successful application of preimplantation genetic diagnosis and haplotyping for congenital hyperinsulinism.

Congenital hyperinsulinism is the most common cause of persistent hypoglycaemia in infancy. Early surgical intervention is usually required to prevent brain damage. The prevention of the transmission to the offspring is important in families carrying the mutated gene. Preimplantation genetic diagnosis (PGD) is an early genetic testing procedure for couples at risk of transmitting inherited diseases. A 36-year-old Saudi woman married to her first cousin with four affected children was referred for PGD. The hyperinsulinism disease was caused by a novel homozygous mutation in the KCNJ11 gene, an arginine 301 to proline (R301P) substitution.PGD was achieved by whole genome amplification followed by mutation detection combined with short tandem repeat identifier analysis in the first cycle and with haplotyping in the second cycle. The first and second cycles resulted in the births of healthy twin girls and a boy, respectively. As far as is known, this is the first application of PGD to hyperinsulinism. A feasible strategy including whole genome amplification followed by direct mutation detection combined with haplotyping is described.Utilizing haplotyping increases the efficiency of PGD diagnosis as well as confirming the genetic diagnosis. It reveals the parental origin of each inherited chromosome.

Male Infertility in Robertsonian Translocation: A Case Report.

BACKGROUND Translocations are the most common type of chromosomal structural anomalies. In balanced translocations, there is not an obvious loss of genetic material; they are usually phenotypically normal adults who present with reproductive issues. Male carriers of Robertsonian (ROB) translocation can have infertility and are shown to have abnormal semen analysis. Some patients have positive sperms in the ejaculate. Therefore, fertility management can be offered to couples to achieve pregnancy and delivery of healthy neonates. CASE REPORT We present 2 cases of 34- and 35-year-old males who presented to our tertiary care hospital because of primary infertility. Semen analysis showed nonobstructive cryptozoospermia and azoospermia, respectively. Genetic tests revealed ROB translocation (13;14). Fertility treatment was offered to both couples. CONCLUSIONS Males with ROB translocation can have positive sperms in the ejaculate. A multidisciplinary approach should be offered to the couples to help them achieve clinical pregnancy, reduce the risk of miscarriage, and increase the rates of delivery of healthy neonates.

Noninvasive Digital Detection of Fetal DNA in Plasma of 4-Week-Pregnant Women following In Vitro Fertilization and Embryo Transfer.

The discovery of cell-free fetal DNA (cfDNA) circulating in the maternal blood has provided new opportunities for noninvasive prenatal diagnosis (NIPD). However, the extremely low levels of cfDNA within a high background of the maternal DNA in maternal circulation necessitate highly sensitive molecular techniques for its reliable use in NIPD. In this proof of principle study, we evaluated the earliest possible detection of cfDNA in the maternal plasma by a bead-based emulsion PCR technology known as BEAMing (beads, emulsion, amplification, magnetics). Blood samples were collected from in vitro fertilization (IVF) patients at 2 to 6 weeks following embryo transfer (i.e., 4 to 8 week pregnancies) and plasma DNA was extracted. The genomic regions of both X and Y chromosome-specific sequences (AMELX and AMELY) were concurrently amplified in two sequential PCRs; first by conventional PCR then by BEAMing. The positive beads either for AMELX or AMELY gene sequences were counted by a flow cytometer. Our results showed that the pregnancies yielding boys had significantly higher plasma AMELY gene fractions (0.512 ± 0.221) than the ones yielding girls (0.028 ± 0.003) or non-pregnant women (0.020 ± 0.005, P= 0.0059). Here, we clearly demonstrated that the BEAMing technique is capable of reliably detecting cfDNA in the blood circulation of 4-week-pregnant women, which is only two weeks after the embryo transfer. BEAMing technique can also be used to early detect fetal DNA alterations in other pregnancy-associated disorders.

Preimplantation genetic diagnosis of Morquio disease.

OBJECTIVES: Morquio syndrome is an autosomal recessive disease and mutations in the N-acetylgalactosamine 6-sulfate sulfatase (GALNS) gene cause Morquio type A disease. Preimplantation genetic diagnosis (PGD), an early form of prenatal diagnosis for couples at risk of transmitting inherited diseases, was applied to prevent transmission of this disease. METHODS: A couple with three affected children, having homozygous W159C (p. Trp 159 Cys) mutation in GALNS gene, underwent in vitro fertilization (IVF) treatment and PGD. Mutation analyses from the embryos were performed following whole genome amplification of single blastomeres using multiple displacement amplification (MDA). RESULTS: Three embryos were diagnosed as normal and two were transferred on day 4. The cycle resulted in a pregnancy and a live birth of a carrier male infant. Genetic haplotyping analysis of the infant and the leftover MDA samples enabled us to determine which embryo was implanted. The discrepancy in results was explained by allele dropout (ADO) of the mutant allele from the MDA product. CONCLUSIONS: A feasible strategy for PGD of Morquio disease including whole genome amplification by MDA and the use of preimplantation genetic haplotyping is described. MDA product archiving will be useful for future investigations if needed.