Maternal Allele Research Articles

Difficulties in the differential diagnosis of Angelman’s syndrome

Angelman syndrome is a rare neurogenetic disease caused by the loss of the function of the maternal allele of the UBE3A gene on chromosome 15 (site 15q11.2–q13) and is characterized by severe mental retardation, lack of speech, epilepsy, microcephaly and a characteristic facial phenotype with a unique behavior in the form of frequent laughter. The combination of microcephaly, epilepsy, speechlessness and mental retardation poses a problem for differential diagnosis with many genetic diseases presenting with similar symptoms. Epileptic encephalopathy due to CDKL5 gene mutation and Rett syndrome have the greatest similarity. The hallmark of Angelman syndrome are laughter attacks and specific EEG changes. The authors have presented a table of the differential diagnosis of Angelman syndrome with some phenotypically similar genetic syndromes, indicating the most significant distinguishing features, which should facilitate for the pediatrician and neurologist the diagnostic path of establishing the correct diagnosis.

A region-dependent allele-biased expression of Dopa decarboxylase in mouse brain.

Genomic imprinting is an epigenetic event in which genes are expressed only from either the paternal or maternal allele. Dopa decarboxylase (Ddc), is an imprinted gene that encodes an enzyme which catalyzes the conversion of L-dopa to dopamine. Although Ddc has been reported to be paternally expressed in embryonic and neonatal hearts, its expression pattern in the brain has been controversial. To visualize Ddc-expressing neurons, we established a knock-in mouse carrying a humanized Kusabira orange 1 (hKO1) reporter cassette at the Ddc locus (Ddc-hKO1). The expression of Ddc-hKO1 was detected in all known Ddc-positive cells in the brains of embryonic, neonatal, adult, and aged mice. We further developed an efficient purification method for Ddc-hKO1-positive neurons using a cell sorter. RNA sequencing analysis confirmed the enrichment of dopaminergic, serotonergic and cholinergic neurons in Ddc-hKO1-positive cell population recovered using this method. A detailed analysis of Ddc-hKO1 paternally and maternally derived heterozygous mice combined with immunostaining revealed that Ddc was preferentially expressed from the maternal allele in ventral tegmented area (VTA), substantia nigra pars compacta (SNc), and retrorubral field (RRF); while it was expressed from both alleles in dorsal raphe nucleus (DR). These results indicate that Ddc exhibit an allele-specific expression pattern in different brain regions, presumably reflecting the diverse regulatory mechanisms of imprinting.

Addition of an affected family member to a previously ascertained autosomal recessive nonsyndromic hearing loss pedigree and systematic phenotype-genotype analysis of splice-site variants in MYO15A

Pathogenic variants in MYO15A are known to cause autosomal recessive nonsyndromic hearing loss (ARNSHL), DFNB3. We have previously reported on one ARNSHL family including two affected siblings and identified MYO15A c.5964+3G > A and c.8375 T > C (p.Val2792Ala) as the possible deafness-causing variants. Eight year follow up identified one new affected individual in this family, who also showed congenital, severe to profound sensorineural hearing loss. By whole exome sequencing, we identified a new splice-site variant c.5531+1G > C (maternal allele), in a compound heterozygote with previously identified missense variant c.8375 T > C (p.Val2792Ala) (paternal allele) in MYO15A as the disease-causing variants. The new affected individual underwent unilateral cochlear implantation at the age of 1 year, and 5 year follow-up showed satisfactory speech and language outcomes. Our results further indicate that MYO15A-associated hearing loss is good candidates for cochlear implantation, which is in accordance with previous report. In light of our findings and review of the literatures, 58 splice-site variants in MYO15A are correlated with a severe deafness phenotype, composed of 46 canonical splice-site variants and 12 non-canonical splice-site variants.

Beckwith-Wiedemann syndrome with long QT caused by a deletion involving KCNQ1 but not KCNQ1OT1:TSS-DMR

Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder with characteristic features, such as overgrowth, macroglossia, and exomphalos. Hypomethylation of the KCNQ1OT1:TSS-differentially methylated region (DMR) on the 11p15.5 imprinted region is the most common etiology of BWS. KCNQ1 on 11p15.5 is expressed from the maternally inherited allele in most tissues, but is biparentally expressed in the heart, and maternal KCNQ1 transcription is required to establish the maternal DNA imprint in the KCNQ1OT1:TSS-DMR. Loss of function variants in KCNQ1 result in long QT syndrome type 1 (LQT1). To date, eight patients with BWS due to KCNQ1 splice variants or structural abnormalities involving KCNQ1 but not the KCNQ1OT1:TSS-DMR have been reported (KCNQ1-BWS), and four of them had LQT1. We report a Japanese boy with BWS and LQT1 presenting with extreme hypomethylation of the KCNQ1OT1:TSS-DMR caused by a de novo 215-kb deletion including KCNQ1 but not the KCNQ1OT1:TSS-DMR on the maternal allele. He was born by emergency cesarean section due to suspicion of placental abruption at 30 weeks of gestation. His birth weight and length were +1.6 SD and +1.0 SD, respectively. His placental weight was +3.9 SD, and histological examination of his placenta was consistent with mesenchymal dysplasia. He had BWS clinical features, including macroglossia, ear creases and pits, body asymmetry, and rectus abdominis muscle dehiscence, and BWS was therefore diagnosed. LQT1 was first noticed at three months in a preoperative examination for lingual frenectomy. The summarized data of our patient and the previously reported eight patients in KCNQ1-BWS showed more frequent and earlier preterm births and smaller sized birth weight in KCNQ1-BWS cases than those with BWS caused by epimutation of the KCNQ1OT1:TSS-DMR. In addition, in five of nine patients with KCNQ1-BWS, LQT1 was detected, and two of them were identified at school age. In our patient and in another single case with LQT1, the LQT1 was not detected early despite neonatal ECG monitoring. For BWS patients with extreme hypomethylation of the KCNQ1OT1:TSS-DMR, searching for CNVs involving KCNQ1 and mutation screening for KCNQ1 should be considered together with periodic ECG monitoring. (338/500 words)

Molar pregnancy unveiled by DNA profiling: a rare forensic case study

BackgroundForensic DNA analysis is one of the most advanced tools in the criminal investigation. It is used successfully in solving offenses involving rape, paternity disputes, murder or attempt to murder, and dacoity as well as identification of mutilated body remains. DNA profiling is used to determine paternity in sexual offense cases where abortion takes place and the product of conception can be anywhere from 6 to 10 weeks of gestation to 8 months. In the present case, a tissue sample stated as a vesicular mole and blood samples of the mother and suspected father were submitted to the DNA division of our laboratory for paternity analysis.ResultsGenotyping results revealed a single allele at all the tested short tandem repeat (STR) loci. The allele obtained at each locus was common with the suspected father. Such type of genotype was very rare and not observed earlier; therefore, repeated analysis was done and the same genotype was obtained every time. DNA profiling revealed all the alleles in the vesicular mole to be of paternal origin only, devoid of any maternal alleles. After referring to books on gynecology, it was confirmed that the genotype obtained was of hydatidiform mole.ConclusionsIn this POCSO Act case, the product of conception (about 1.5 months old) was termed a vesicular mole, and blood samples of the mother and suspected father were sent for the DNA paternity test. STR profiling of the product of conception sample displayed no maternal tissue contamination and non-inheritance of maternal alleles, showing the case to be of molar pregnancy also called hydatidiform mole, a very rare phenomenon in the forensic scenario. After thorough analysis, the case was reported and it was the first of its kind to be reported in a forensic laboratory in Maharashtra, India.

Endosperm and Maternal-specific expression of EIN2 in the endosperm affects endosperm cellularization and seed size in Arabidopsis.

Seed size is related to plant evolution and crop yield and is affected by genetic mutations, imprinting, and genome dosage. Imprinting is a widespread epigenetic phenomenon in mammals and flowering plants. ETHYLENE INSENSITIVE2 (EIN2) encodes a membrane protein that links the ethylene perception to transcriptional regulation. Interestingly, during seed development EIN2 is maternally expressed in Arabidopsis and maize, but the role of EIN2 in seed development is unknown. Here, we show that EIN2 is expressed specifically in the endosperm, and the maternal-specific EIN2 expression affects temporal regulation of endosperm cellularization. As a result, seed size increases in the genetic cross using the ein2 mutant as the maternal parent or in the ein2 mutant. The maternal-specific expression of EIN2 in the endosperm is controlled by DNA methylation but not by H3K27me3 or by ethylene and several ethylene pathway genes tested. RNA-seq analysis in the endosperm isolated by laser-capture microdissection show upregulation of many endosperm-expressed genes such as AGAMOUS-LIKEs (AGLs) in the ein2 mutant or when the maternal EIN2 allele is not expressed. EIN2 does not interact with DNA and may act through ETHYLENE INSENSITIVE3 (EIN3), a DNA-binding protein present in sporophytic tissues, to activate target genes like AGLs, which in turn mediate temporal regulation of endosperm cellularization and seed size. These results provide mechanistic insights into endosperm and maternal-specific expression of EIN2 on endosperm cellularization and seed development, which could help improve seed production in plants and crops.

Allele-specific differential regulation of monoallelically expressed autosomal genes in the cardiac lineage

Each mammalian autosomal gene is represented by two alleles in diploid cells. To our knowledge, no insights have been made in regard to allele-specific regulatory mechanisms of autosomes. Here we use allele-specific single cell transcriptomic analysis to elucidate the establishment of monoallelic gene expression in the cardiac lineage. We find that monoallelically expressed autosomal genes in mESCs and mouse blastocyst cells are differentially regulated based on the genetic background of the parental alleles. However, the genetic background of the allele does not affect the establishment of monoallelic genes in differentiated cardiomyocytes. Additionally, we observe epigenetic differences between deterministic and random autosomal monoallelic genes. Moreover, we also find a greater contribution of the maternal versus paternal allele to the development and homeostasis of cardiac tissue and in cardiac health, highlighting the importance of maternal influence in male cardiac tissue homeostasis. Our findings emphasize the significance of allele-specific insights into gene regulation in development, homeostasis and disease.

Conserved noncoding sequences and de novo Mutator insertion alleles are imprinted in maize.

Genomic imprinting is an epigenetic phenomenon in which differential allele expression occurs in a parent-of-origin-dependent manner. Imprinting in plants is tightly linked to transposable elements (TEs), and it has been hypothesized that genomic imprinting may be a consequence of demethylation of TEs. Here, we performed high-throughput sequencing of ribonucleic acids from four maize (Zea mays) endosperms that segregated newly silenced Mutator (Mu) transposons and identified 110 paternally expressed imprinted genes (PEGs) and 139 maternally expressed imprinted genes (MEGs). Additionally, two potentially novel paternally suppressed MEGs are associated with de novo Mu insertions. In addition, we find evidence for parent-of-origin effects on expression of 407 conserved noncoding sequences (CNSs) in maize endosperm. The imprinted CNSs are largely localized within genic regions and near genes, but the imprinting status of the CNSs are largely independent of their associated genes. Both imprinted CNSs and PEGs have been subject to relaxed selection. However, our data suggest that although MEGs were already subject to a higher mutation rate prior to their being imprinted, imprinting may be the cause of the relaxed selection of PEGs. In addition, although DNA methylation is lower in the maternal alleles of both the maternally and paternally expressed CNSs (mat and pat CNSs), the difference between the two alleles in H3K27me3 levels was only observed in pat CNSs. Together, our findings point to the importance of both transposons and CNSs in genomic imprinting in maize.

The Lonely Life of a Champion Tree, Aesculus glabra

Seedlings derived from two Ohio buckeyes (Aesculus glabra Willd.) trees, the National Champion growing in Illinois, USA, and the Ohio State Champion were sampled. The National Champion grows at the northern limits of the species’ native range. The Ohio State Champion grows in Huron County, Ohio, well within the eastern range of the species. We also sampled 40 adult trees growing in Ohio and Illinois. All trees were genotyped at six microsatellite loci. We found that 42 of the 44 sampled seedlings (95%) from the National Champion tree, collected over two seasons, exhibited only maternal alleles at all six microsatellite loci, indicating they were produced by self-fertilization. In contrast, all seedlings from the Ohio state champion tree (N = 48) exhibited non-maternal alleles, indicating they were produced by outcrossing. Our results suggest that when outcross pollen is not available, A. glabra will self-fertilize, but does so rarely or never when outcross pollen is available. Seed germination and early survival were similar for progeny of both champions, but seedlings from the National Champion show lower growth rates and higher mortality during a spring frost, possibly due to inbreeding depression. There was little evidence for genetic structure between trees sampled in Ohio and Illinois.

Circulating maternal chimeric cells have an impact on the outcome of biliary atresia.

IntroductionWe aimed to quantify the DNA of maternal chimeric (MC) cells in the peripheral blood of the BA patients and investigated the impact on the outcome.MethodsPatients with progressive jaundice because of no bile flow, which necessitated liver transplantation, or who showed inadequate bile flow with or without episodes of cholangitis and progressive hepatic fibrosis and portal hypertension were classified into the poor group. Those with adequate bile flow with completely normal liver function tests beyond 2 years were classified into the good group. The qPCR were separately carried out in buffy coat samples and plasma samples, targeting the non-inherited maternal HLA alleles in the DNA samples.ResultsMC-DNA was present in the buffy coat (10–328 gEq per 106 host cells) in seven patients. There was no MC-DNA in the remaining five patients. MC-DNA (214–15,331 gEq per 106 host cells) was observed in the plasma of five patients. The quantity of MC-DNA in the buffy coat showed a significant difference between the two prognostic groups (p = 0.018), whereas there was no significant difference in the quantity of MC-DNA in plasma (p = 0.205). MC-DNA in the buffy coat was significantly associated with the outcome (p = 0.028), whereas MC-DNA in the plasma did not influence the outcome (p = 0.56).ConclusionsPoor outcomes in BA were correlated with circulating maternal chimeric lymphocytes.

HLA sharing and maternal HLA expression in couples with recurrent pregnancy loss in Taiwan

ObjectiveThe aim of this study is to investigate the frequency and distribution of human HLA sharing and maternal HLA allele expression in couples with recurrent pregnancy loss in Taiwan. Materials and methodsWe retrospectively reviewed couples experienced two or more pregnancy loss before 20th weeks of gestation from March 2014 to November 2020 having HLA determination. Fertile individuals with one or more live-birth offspring receiving HLA allele determination during the same period were included as the control group. The distribution and frequency of HLA sharing were analyzed and presented by descriptive statistics. Fisher Exact Test were used to analyze specific maternal and paternal HLA allele comparing individuals with RPL to fertile group. P-value < 0.05 was thought to be statistically significant. Results72 couples were enrolled from March 2014 to November 2020. Regarding the distribution of HLA sharing, HLA sharing between females and their male partners less and equal to 2 pairs were found in 40.3% of the couples. HLA sharing greater and equal to 3 pairs are found in 59.8% couples. HLA sharing was most frequently found in alleles HLA-A02, A11, DQ07, C07 and B60 in descending order. There was a significant lower expression of HLA-B13 in women with RPL compared to women who had successful pregnancy (p = 0.0234). Compared infertile men with fertile men cohort, the frequency of HLA-DR04 (p = 0.0438, OR 2.444, 95% CI 1.0251–5.8287), HLA-DR12 (p = 0.001, OR 30.85, 95% CI 4.0296–236.19) and HLA-15 (p = 0.0357, OR 9.354, 95% CI 1.1610–75.37) were found to be significantly higher in men with RPL. On the contrary, HLA-DR07 (p = 0.0085, OR 0.124, 95% CI 0.0264–0.587) and HLA-DR10 (p = 0.0395, OR 0.048, 95% CI 0.0027–0.8641) were found to be significantly lower in men with RPL. ConclusionWe found a tendency to recurrent pregnancy loss in couples with more than 2 pairs of HLA sharing. The similarity of HLA sharing, the expression of maternal HLA-B13 allele and paternal HLA-DR alleles in Taiwanese couples might play a role in recurrent pregnancy loss.

New insights into the criteria of functional heterozygosity of the Apis mellifera complementary sex determining gene-Discovery of a functional allele pair differing by a single amino acid.

The complementary sex determiner (csd) gene is responsible for controlling the sex-determination molecular switch in western honey bees (Apis mellifera): bees that are heterozygous for csd develop into females, whereas bees that are hemizygous or homozygous develop into males. The homozygous diploid males are destroyed at an early stage of their development. It has been proposed that the minimal number of amino acid differences between two csd alleles needed to fully determine femaleness is five and it has also been shown that smaller differences may result in forming an evolutionary intermediate that is not fully capable of female determination, but has increased fitness compared to the homozygous genotype. In this study, we have implemented a terminal restriction length polymorphism-based method of identifying and distinguishing paternal alleles in a given bee colony and assigning them to a particular maternal allele in order to gather information on large number of functional csd pairs and also to identify, to some extent, genotypes that are underrepresented or absent in bee colonies. The main finding of this study is the identification of a fully functional genotype consisting of csd alleles that differed from each other by a one amino acid position. The individuals carrying this genotype expressed only female-specific transcripts of feminizer and double-sex genes. By comparing the sequences differences between the csd pair identified in our study with those described earlier, we conclude that functional heterozygosity of the csd gene is dependent not only on the number of the amino acid differences but also on the sequence context and position of the change. The discovery of a functional allele pair differing by a single amino acid also implies that the generation of a new csd specificity may also occur during a single mutation step with no need for evolutionary intermediates accumulating further mutations.

Clinical Spectrum and Tumour Risk Analysis in Patients with Beckwith-Wiedemann Syndrome Due to CDKN1C Pathogenic Variants.

Simple SummaryBeckwith–Wiedemann syndrome (BWS) is an overgrowth disorder caused by imprinting or genetic alterations at the 11p15.5 locus. BWS is considered a spectrum disorder (BWSp) with an increased neoplasm incidence. CDKN1C variants have been reported in 5–10% of patients, with a higher incidence in familial cases. In this study, we examined the clinical and molecular features of all cases of BWSp identified by the Spanish Overgrowth Registry Initiative with CDKN1C variants, ascertained by Sanger sequencing or next-generation sequencing. We present 21 cases, 19 of which were classified as classical BWS and 1 that developed a mediastinal ganglioneuroma. Our study supports the high heterogeneity of the clinical features of BWSp and adds evidence on tumour development in this BWSp molecular subgroup. Genotype–phenotype correlation studies of patients with suspected BWS are essential for improving the diagnosis and assessing whether its cause can be directly related to the BWS clinical spectrum in the few cases that develop tumours.Beckwith–Wiedemann syndrome spectrum (BWSp) is an overgrowth disorder caused by imprinting or genetic alterations at the 11p15.5 locus. Clinical features include overgrowth, macroglossia, neonatal hypoglycaemia, omphalocele, hemihyperplasia, cleft palate, and increased neoplasm incidence. The most common molecular defect observed is hypomethylation at the imprinting centre 2 (KCNQ1OT1:TSS DMR) in the maternal allele, which accounts for approximately 60% of cases, although CDKN1C pathogenic variants have been reported in 5–10% of patients, with a higher incidence in familial cases. In this study, we examined the clinical and molecular features of all cases of BWSp identified by the Spanish Overgrowth Registry Initiative with pathogenic or likely pathogenic CDKN1C variants, ascertained by Sanger sequencing or next-generation sequencing, with special focus on the neoplasm incidence, given that there is scarce knowledge of this feature in CDKN1C-associated BWSp. In total, we evaluated 21 cases of BWSp with CDKN1C variants; 19 were classified as classical BWS according to the BWSp scoring classification by Brioude et al. One of our patients developed a mediastinal ganglioneuroma. Our study adds evidence that tumour development in patients with BWSp and CDKN1C variants is infrequent, but it is extremely relevant to the patient’s follow-up and supports the high heterogeneity of BWSp clinical features associated with CDKN1C variants.

Sex-biased and parental allele-specific gene regulation by KDM6A

BackgroundKDM6A is a demethylase encoded by a gene with female-biased expression due to escape from X inactivation. Its main role is to facilitate gene expression through removal of the repressive H3K27me3 mark, with evidence of some additional histone demethylase-independent functions. KDM6A mutations have been implicated in congenital disorders such as Kabuki Syndrome, as well as in sex differences in cancer.MethodsKdm6a was knocked out using CRISPR/Cas9 gene editing in F1 male and female mouse embryonic stem cells (ES) derived from reciprocal crosses between C57BL6 x Mus castaneus. Diploid and allelic RNA-seq analyses were done to compare gene expression between wild-type and Kdm6a knockout (KO) clones. The effects of Kdm6a KO on sex-biased gene expression were investigated by comparing gene expression between male and female ES cells. Changes in H3K27me3 enrichment and chromatin accessibility at promoter regions of genes with expression changes were characterized by ChIP-seq and ATAC-seq followed by diploid and allelic analyses.ResultsWe report that Kdm6a KO in male and female embryonic stem (ES) cells derived from F1 hybrid mice cause extensive gene dysregulation, disruption of sex biases, and specific parental allele effects. Among the dysregulated genes are candidate genes that may explain abnormal developmental features of Kabuki syndrome caused by KDM6A mutations in human. Strikingly, Kdm6a knockouts result in a decrease in sex-biased expression and in preferential downregulation of the maternal alleles of a number of genes. Most promoters of dysregulated genes show concordant epigenetic changes including gain of H3K27me3 and loss of chromatin accessibility, but there was less concordance when considering allelic changes.ConclusionsOur study reveals new sex-related roles of KDM6A in the regulation of developmental genes, the maintenance of sex-biased gene expression, and the differential expression of parental alleles.

Dopamine-transporter heterozygous rats carrying maternal wild-type allele are more vulnerable to the development of compulsive behavior.

Compulsivity is defined as an unstoppable tendency toward repetitive and habitual actions, which are reiterated despite negative consequences. Polydipsia is induced preclinically by intermittent reward, leading rodents to ingest large amounts of fluids. We focused on the role of dopamine transporter (DAT) and inheritance factors in compulsive behavior. Our sample consisted of DAT heterozygous (HET) rats with different genetic inheritance (MAT-HET, born from WT-dams×KO-fathers; MIX-HET, born from HET-dams × KO-fathers). As controls, we used both wild-type (WT) rats and their socially-isolated (WTi) siblings. We ran the schedule-induced polydipsia (SIP) protocol, to induce compulsive behavior; then the Y-maze and marble-burying tests, to verify its actual development. Only MAT-HET (who inherited the functional DAT allele from the WT mother) is vulnerable to developing compulsive behavior. MAT-HET rats drank increasingly more water during SIP; they showed significant perseverance in the Y-maze test and exhibited compulsive actions in the marble-burying test. Interestingly, compulsive behaviors of MAT-HET rats correlated with expression ex vivo of different genes in different areas. Regarding the prefrontal cortex (PFC), D2R correlatedwith Y-maze "perseverance" in addition to BDNF; considering the amygdala (AMY), both D3R and OXTR correlated with SIP "licks." Indeed, compulsivity may be linked to D2R and BDNF in PFC, while extreme anxiety in MAT-HET rats may be associated with D3R and OXTR in the AMY. These results confirm some similarities between MAT-HET and DAT-KO subjects, and link the epigenetic context of the DAT gene to the development of compulsive behavior.

Abstract 1597: Characterization of genomic diversity in high grade serous ovarian and triple negative breast cancer at single cell resolution

Abstract Copy number alterations and structural variants are associated with disease progression, therapeutic response, and metastasis in human cancers, yet the extent and mechanisms driving continued genomic instability remain poorly understood. We generated more than 20,000 single-cell whole genomes from 25 high-grade serous ovarian and triple-negative breast cancers across a range of mutational subtypes. Through the development of new computational methods, we resolved alterations into cancer haplotypes, providing fine-grained resolution of genomic events in single-cells and exposing extensive cell-to-cell genomic variation. We identified 3 key processes that contribute to this diversity. Firstly, we observed that parallel copy number events - whereby different cancer cells harbor chromosomal aberrations with identical total copy number but different combinations of maternal and paternal alleles - were present in &amp;gt; 80% of the tumors. This is consistent with extensive convergent evolution in these tumors. Secondly, we identified considerable cell-to-cell variability in the amplitude of high-level amplifications, often impacting oncogenes. Single-cell transcriptomes and immunohistochemistry from the same samples showed that this genomic diversity led to phenotypic differences between cells. Finally, we observed copy number break-point variability consistent with progressive genomic diversification, which we termed serriform structural variation (SSV). Both SSV’s and variable high-level amplifications were enriched in foldback inversion (FBI) bearing tumors relative to homologous recombination deficient tumors, highlighting that FBI tumors have greater levels of structural instability. Our study sheds new light on the evolution of genomically unstable tumors with implications for cancer evolution, therapeutic targeting, and patient stratification. Citation Format: Marc J. Williams, Tyler Funnell, Ciara H. O'Flanagan, Andrew McPherson, Samuel Aparicio, Sohrab P. Shah. Characterization of genomic diversity in high grade serous ovarian and triple negative breast cancer at single cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1597.

Maternal FMR1 alleles expansion in newborns during transmission: a prospective cohort study.

The CGG repeats in the 5' untranslated region of the fragile X mental retardation 1 gene (FMR1) gene shows increased instability upon maternal transmission. Maternal FMR1 intermediate (45-54 repeats) and premutation (PM: 55-<200 repeats) alleles usually expand to full mutation (>200 repeats) alleles in offspring and consequently, cause fragile X syndrome (FXS) in them. In a prospective cohort study, Pakistani pregnant women in prenatal care were first screened for FMR1 expanded alleles. In the follow-up, pregnancy outcomes in women carrying FMR1 expanded alleles were recorded and their newborn offspring were also screened for FXS. In a total of 1950 pregnant women, 89 (4.6%) were detected carriers for FMR1 expanded alleles; however, rates of detection of expanded alleles were found significantly high in women with a history of FXS. In addition, miscarriages and birth of affected newborns with FXS were significantly more common in women carrying large size PM alleles and had a history of FXS (P = 0.0494 and P = 0.0494, respectively). The current study provides the first evidence of screening Pakistani pregnant women for FMR1 expanded alleles in prenatal care. Moreover, the miscarriage was also detected as a clinical predictor for FXS. Offspring would have a higher risk of developing FXS due to maternal FMR1 alleles expansions during transmission. This is the first prospective cohort study in Pakistan for finding FMR1 allelic status of pregnant women and their newborn offspring in follow-up. The robust offspring risk for FXS estimated in this study may be valuable information for genetic counseling of women carriers for FMR1 expanded alleles. The family history and miscarriage were detected as effective indicators for FXS carrier screening in Pakistani women.

Optimized Droplet Digital PCR Assay on Cell-Free DNA Samples for Non-Invasive Prenatal Diagnosis: Application to Beta-Thalassemia.

Thalassemias are inherited blood disorders and by far one of the most common monogenic diseases globally. Beta-thalassemia has a particularly high prevalence in Cyprus, with the IVSI-110 G>A (HBB:c.93-21G>A) pathogenic variation representing almost 79% of the total carriers. The discovery that 3% to 20% of cell-free fetal DNA (cffDNA) is present in the maternal plasma allowed the development of non-invasive prenatal diagnosis (NIPD) of monogenic diseases, like beta-thalassemia, avoiding the risks of invasive procedures. However, the development of NIPD holds major technical challenges and has not yet reached the clinical setting. In this study, we apply droplet digital PCR (ddPCR) coupled with the relative variant dosage approach to develop a NIPD assay for IVSI-110 G>A beta-thalassemia. We have implemented an optimization process for ddPCR to address the challenges of ddPCR assays such as inconclusive rain droplets and thus increase the sensitivity and specificity of the assay. The established protocol was evaluated on 40 maternal plasma samples with a median gestational age of 10 weeks where both parents carried the same pathogenic variation. Thirty-three samples were correctly classified, 6 remained inconclusive, and 1 was misclassified. Our assay exhibited 97.06% accuracy (95% CI, 82.46-99.68), 100% sensitivity (95% CI, 76.84-100), and 95% specificity (95% CI, 75.13-99.87), demonstrating its efficiency for the non-invasive detection of both maternal and paternal alleles. We have developed an efficient, simple, and cost-effective ddPCR assay for the non-invasive determination of fetal genotype in couples at risk of IVSI-110 G>A beta-thalassemia, bringing NIPD of monogenic diseases closer to the diagnostic setting.

Inter and intra-specific crossability studies on Mangifera species

Inter-specific and inter-varietal crosses were attempted using Mangifera indica cv. Amrapali as a female parent and M. odorata and M. indica cv. Vanraj as male donor parents (2016–19). Initial fruit set after 7 days of pollination (DAP) was 81.48%, which reduced to 2.48% at harvest. However, in inter-specific crosses, initial fruit set was 79.89%, which reduced to 0.45% at the time of harvesting. Progenies obtained from inter-varietal and inter-specific crosses were subjected to confirmation of parentage using SSR markers. Eight polymorphic SSR markers were used to estimate the paternity, maternity and new alleles in the hybrids. Based on fragment analysis, the paternity, maternity and new alleles of 12 inter-specific and 17 inter-varietal hybrids were estimated by finding a similar number of alleles between the offsprings and parents. Among inter-specific hybrids, AO2, AO7, AO11, and AO12 showed ≥50% of paternal allele inheritance, while AO1, AO2, AO4, AO5, AO7, AO8, AO9, AO10 and AO12 showed ≥50% of maternal allele inheritance. Inter-specific hybrids such as AO1, AO3, AO5, AO9, AO10 and AO11 inherited new alleles. Among inter-varietal hybrids, AV1, AV2, AV4, AV5, AV6, AV10, AV11, AV12, AV13, AV14, AV15 and AV17 possessed ≥50% paternal allele, while, AV1, AV2, AV3, AV5, AV6, AV7, AV8, AV10, AV11 and AV15 had ≥50% maternal allele. Three inter-varietal hybrids, viz. AV8, AV9 and AV16 inherited new alleles. It is concluded that SSRs markers are useful in ascertaining the parentage of hybrids obtained from inter-specific and inter-varietal crosses in mango.

Epigenome editing reveals core DNA methylation for imprinting control in the Dlk1-Dio3 imprinted domain.

The Dlk1-Dio3 imprinted domain is controlled by an imprinting control region (ICR) called IG-DMR that is hypomethylated on the maternal allele and hypermethylated on the paternal allele. Although several genetic mutation experiments have shown that IG-DMR is essential for imprinting control of the domain, how DNA methylation itself functions has not been elucidated. Here, we performed both gain and loss of DNA methylation experiments targeting IG-DMR by transiently introducing CRISPR/Cas9 based-targeted DNA methylation editing tools along with one guide RNA into mouse ES cells. Altered DNA methylation, particularly at IG-DMR-Rep, which is a tandem repeat containing ZFP57 methylated DNA-binding protein binding motifs, affected the imprinting state of the whole domain, including DNA methylation, imprinted gene expression, and histone modifications. Moreover, the altered imprinting states were persistent through neuronal differentiation. Our results suggest that the DNA methylation state at IG-DMR-Rep, but not other sites in IG-DMR, is a master element to determine whether the allele behaves as the intrinsic maternal or paternal allele. Meanwhile, this study provides a robust strategy and methodology to study core DNA methylation in cis-regulatory elements, such as ICRs and enhancers.