Rare disease Background: Translocations are the most common type of chromosomal structural anomal... more Rare disease 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 Reports: 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.
<p>Human genomic DNA from a male subject (AMELY) is serially diluted with the female DNA (A... more <p>Human genomic DNA from a male subject (AMELY) is serially diluted with the female DNA (AMELX) in concentrations of 10%, 1%, 0.1%, and 0.01% AMELY (r<sup>2</sup>>0.99).</p
<p><sup><b>a</b></sup>Bold nucleotides denote gene specific sequenc... more <p><sup><b>a</b></sup>Bold nucleotides denote gene specific sequences.</p><p><sup><b>b</b></sup>Diehl et al 2006.</p><p><sup><b>c</b></sup>ChrX: 11296878–11296965</p><p><sup><b>d</b></sup>ChrY: 6869861–68619954</p><p>All the primers and probes used in the study.</p
Spontaneous oocyte activation (SOA) is a recently classified phenomenon characterized by the pres... more 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. 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. 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. 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.
The discovery of cell-free fetal DNA (cfDNA) circulating in the maternal blood has provided new o... more 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 ne-cessitate 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 plas-ma by a bead-based emulsion PCR technology known as BEAMing (beads, emulsion, am-plification, 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 conven-tional PCR then by BEAMing. The positive beads either for AMELX or AMELY gene se-quences were counted by a f...
Purpose: Male infertility remains poorly understood at the molecular level. We aimed in this stud... more Purpose: Male infertility remains poorly understood at the molecular level. We aimed in this study to investigate the yield of a "genomics first" approach to male infertility. Methods: Patients with severe oligospermia and nonobstructive azoospermia were investigated using exome sequencing (ES) in parallel with the standard practice of chromosomal analysis. Results: In 285 patients, 10.5% (n = 30) had evidence of chromosomal aberrations while nearly a quarter (n = 69; 24.2%) had a potential monogenic form of male infertility. The latter ranged from variants in genes previously reported to cause male infertility with or without other phenotypes in humans (24 patients; 8.4%) to those in novel candidate genes reported in this study (37 patients; 12.9%). The 33 candidate genes have biological links to male germ cell development including compatible mouse knockouts, and a few (TERB1 [CCDC79], PIWIL2, MAGEE2, and ZSWIM7) were found to be independently mutated in unrelated patients in our cohort. We also found that male infertility can be the sole or major phenotypic expression of a number of genes that are known to cause multisystemic manifestations in humans (n = 9 patients; 3.1%). Conclusion: The standard approach to male infertility overlooks the significant contribution of monogenic causes to this important clinical entity.
Developmental and/or epileptic encephalopathies (DEEs) are a group of devastating genetic disorde... more 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 12 individuals from 10 families presenting with a severe form of intractable epilepsy, severe developmental delay, progressive microcephaly and visual disturbance. 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. We show that the absence of the shorter isoform leads to a reduction of functional UGP2 enzyme in brain cell types, leading to altered glycogen metabolism, upregulated unfolded protein response and premature neuronal differentiation, as modelled during pluripotent stem cell differentiation in vitro. In ...
The discovery of cell-free fetal DNA (cfDNA) circulating in the maternal blood has provided new o... more 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.
, in charge of providing PGD service for monogenic diseases and chromosomal disorders, supervisin... more , in charge of providing PGD service for monogenic diseases and chromosomal disorders, supervising more than 300 PGD cycles for single gene disorders and chromosomal aberrations.
We have developed a sequencing-based PGS method that allows for the detection of gross chromosoma... more We have developed a sequencing-based PGS method that allows for the detection of gross chromosomal abnormalities. MATERIALS AND METHODS: The sequencing library preparation is based on a novel transposase-based tagging protocol. The sequencing libraries were sequenced with multiplexed, 36 cycle single read sequencing runs on an IlluminaÒ MiSeq. Up to 12 samples were combined per sequencing run. After alignment, only perfectly and uniquely aligning reads were kept for tag counting. The reference genome was divided into variable sized bins depending on genomic context and the number of reads that map to each bin was counted. The read count per bin is subsequently correlated with copy number. Turnaround time from cell biopsy to analyzed data was less than 12 hours. RESULTS: In order to determine the limit of resolution, single cell, two cell and three cell aliquots of tissue culture cell lines with known aberrations were assayed. 12Mb events were readily detectable even at the single cell input level. The protocol was subsequently applied to single cells of cleavage-stage embryos that had previously been characterized by FISH. Monosomies of chromosome 13 and 14 were confirmed in one embryo and trisomy of chromosome 13 was confirmed in another. Additional aberrations were also detected that were previously unknown due to the non-comprehensive nature of FISH. CONCLUSION: PGS by MPS is a rapid, comprehensive screening method that allows for the detection of subchromosomal abnormalities in single cell biopsies.
Klinefelter syndrome (KS) is the most commonly diagnosed sex chromosome disorder in males, with a... more Klinefelter syndrome (KS) is the most commonly diagnosed sex chromosome disorder in males, with an annual incidence of 1 in 600 to 1000 [1]. In the majority (80%) of cases, the karyotype is 47,XXY in all body cells, whereas the remaining patients (20%) display a mosaic XY/XXY pattern. Rarely, multiple-line mosaics can also be found [2].
Sodium leak channel, nonselective (NALCN) is a voltage-independent and cation-nonselective channe... more 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.
Rare disease Background: Translocations are the most common type of chromosomal structural anomal... more Rare disease 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 Reports: 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.
<p>Human genomic DNA from a male subject (AMELY) is serially diluted with the female DNA (A... more <p>Human genomic DNA from a male subject (AMELY) is serially diluted with the female DNA (AMELX) in concentrations of 10%, 1%, 0.1%, and 0.01% AMELY (r<sup>2</sup>>0.99).</p
<p><sup><b>a</b></sup>Bold nucleotides denote gene specific sequenc... more <p><sup><b>a</b></sup>Bold nucleotides denote gene specific sequences.</p><p><sup><b>b</b></sup>Diehl et al 2006.</p><p><sup><b>c</b></sup>ChrX: 11296878–11296965</p><p><sup><b>d</b></sup>ChrY: 6869861–68619954</p><p>All the primers and probes used in the study.</p
Spontaneous oocyte activation (SOA) is a recently classified phenomenon characterized by the pres... more 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. 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. 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. 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.
The discovery of cell-free fetal DNA (cfDNA) circulating in the maternal blood has provided new o... more 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 ne-cessitate 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 plas-ma by a bead-based emulsion PCR technology known as BEAMing (beads, emulsion, am-plification, 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 conven-tional PCR then by BEAMing. The positive beads either for AMELX or AMELY gene se-quences were counted by a f...
Purpose: Male infertility remains poorly understood at the molecular level. We aimed in this stud... more Purpose: Male infertility remains poorly understood at the molecular level. We aimed in this study to investigate the yield of a "genomics first" approach to male infertility. Methods: Patients with severe oligospermia and nonobstructive azoospermia were investigated using exome sequencing (ES) in parallel with the standard practice of chromosomal analysis. Results: In 285 patients, 10.5% (n = 30) had evidence of chromosomal aberrations while nearly a quarter (n = 69; 24.2%) had a potential monogenic form of male infertility. The latter ranged from variants in genes previously reported to cause male infertility with or without other phenotypes in humans (24 patients; 8.4%) to those in novel candidate genes reported in this study (37 patients; 12.9%). The 33 candidate genes have biological links to male germ cell development including compatible mouse knockouts, and a few (TERB1 [CCDC79], PIWIL2, MAGEE2, and ZSWIM7) were found to be independently mutated in unrelated patients in our cohort. We also found that male infertility can be the sole or major phenotypic expression of a number of genes that are known to cause multisystemic manifestations in humans (n = 9 patients; 3.1%). Conclusion: The standard approach to male infertility overlooks the significant contribution of monogenic causes to this important clinical entity.
Developmental and/or epileptic encephalopathies (DEEs) are a group of devastating genetic disorde... more 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 12 individuals from 10 families presenting with a severe form of intractable epilepsy, severe developmental delay, progressive microcephaly and visual disturbance. 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. We show that the absence of the shorter isoform leads to a reduction of functional UGP2 enzyme in brain cell types, leading to altered glycogen metabolism, upregulated unfolded protein response and premature neuronal differentiation, as modelled during pluripotent stem cell differentiation in vitro. In ...
The discovery of cell-free fetal DNA (cfDNA) circulating in the maternal blood has provided new o... more 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.
, in charge of providing PGD service for monogenic diseases and chromosomal disorders, supervisin... more , in charge of providing PGD service for monogenic diseases and chromosomal disorders, supervising more than 300 PGD cycles for single gene disorders and chromosomal aberrations.
We have developed a sequencing-based PGS method that allows for the detection of gross chromosoma... more We have developed a sequencing-based PGS method that allows for the detection of gross chromosomal abnormalities. MATERIALS AND METHODS: The sequencing library preparation is based on a novel transposase-based tagging protocol. The sequencing libraries were sequenced with multiplexed, 36 cycle single read sequencing runs on an IlluminaÒ MiSeq. Up to 12 samples were combined per sequencing run. After alignment, only perfectly and uniquely aligning reads were kept for tag counting. The reference genome was divided into variable sized bins depending on genomic context and the number of reads that map to each bin was counted. The read count per bin is subsequently correlated with copy number. Turnaround time from cell biopsy to analyzed data was less than 12 hours. RESULTS: In order to determine the limit of resolution, single cell, two cell and three cell aliquots of tissue culture cell lines with known aberrations were assayed. 12Mb events were readily detectable even at the single cell input level. The protocol was subsequently applied to single cells of cleavage-stage embryos that had previously been characterized by FISH. Monosomies of chromosome 13 and 14 were confirmed in one embryo and trisomy of chromosome 13 was confirmed in another. Additional aberrations were also detected that were previously unknown due to the non-comprehensive nature of FISH. CONCLUSION: PGS by MPS is a rapid, comprehensive screening method that allows for the detection of subchromosomal abnormalities in single cell biopsies.
Klinefelter syndrome (KS) is the most commonly diagnosed sex chromosome disorder in males, with a... more Klinefelter syndrome (KS) is the most commonly diagnosed sex chromosome disorder in males, with an annual incidence of 1 in 600 to 1000 [1]. In the majority (80%) of cases, the karyotype is 47,XXY in all body cells, whereas the remaining patients (20%) display a mosaic XY/XXY pattern. Rarely, multiple-line mosaics can also be found [2].
Sodium leak channel, nonselective (NALCN) is a voltage-independent and cation-nonselective channe... more 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.
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