Heterozygous Females Research Articles

Cone dysfunction in ARR3-mutation-associated early-onset high myopia: an electrophysiological study

BackgroundMyopia-26, a Mendelian form of early-onset high-myopia (eoHM) caused by mutations in the X-chromosomal ARR3 gene and predominantly affecting females, curiously, may provide an alternative route of investigation to unveil retinal mechanisms underlying pathological eye growth. We conducted a case-control cross-sectional prospective electrophysiological study in genetically characterized Myopia-26 patients (ARR3 heterozygous symptomatic females) compared with high myopes harboring intact ARR3 alleles and one carrier hemizygous male.ResultsParticipants were 26 volunteers: 10 healthy control females (E-CTRL, mean age = 31.5 ± 8.8 years), one healthy control male, one carrier male of the mutant ARR3 allele and 14 female eoHM patients (mean age = 27.0 ± 13.1 years) divided in two groups: seven without (M-CTRL) and seven with (MYP-26) genetic alteration in the ARR3 gene. The clinical evaluation included complete eye screening and full-field electroretinograms (ERGs) recorded from both eyes under mydriasis. Spherical equivalent was comparable (mean=-9.55 ± 2.46 and − 10.25 ± 3.22 for M-CTRL and MYP-26, respectively) and best corrected visual acuity (BCVA) was significantly different between M-CTRL and MYP-26 (1.0 vs. 0.406 ± 0.253, respectively). E-CTRL and M-CTRL showed similar light-adapted flash and flicker ERG amplitudes; however, the prior values were reduced by ~ 35% (a- and b-waves alike), the latter by ~ 55% in the MYP-26 group (F(2, 45) > 21.821, p < 0.00001). Dark-adapted a-wave amplitudes were slightly reduced (by ~ 20%) in all myopic patients compared to E-CTRL, irrespective of the ARR3 genotype (E-CTRL vs. eoHM, p = 0.038).ConclusionsThe cone dysfunction observed in Myopia-26 patients is specifically linked to the mutation of ARR3, and is not the consequence of eoHM, i.e. elongation of the eye. It may play a role in myopic refractive error development through a yet unconfirmed pathomechanism.

Buffy coat removal to mitigate reporting false normal G6PD phenotype results in patients undergoing rasburicase therapy for tumor lysis syndrome

Abstract Glucose-6-phosphate dehydrogenase (G6PD) is the primary source of NADPH in red blood cells (RBCs). NADPH reduces reactive oxygen species (ROS) thereby protecting RBCs from oxidative stress-mediated hemolytic anemia (HA). Individuals with partial or full G6PD deficiency are therefore susceptible to acute HA provoked by medications that generate high ROS loads in vivo, e.g., rasburicase. Rasburicase is an enzyme-based therapeutic used to degrade uric acid (UA) in patients with tumor lysis syndrome (TLS); however, catalysis of UA generates H2O2, a potent ROS, which poses a risk of HA in individuals who are deficient in G6PD activity. Screening G6PD activity prior to rasburicase administration is clinically indicated in ethnic groups with high rates of G6PD deficiency, although reports of HA in rasburicase-treated patients have been reported across low prevalence ethnic groups as well. TLS occurs most commonly during the treatment of highly proliferative neoplasms, such as leukemia and lymphoma. Since WBCs contain significant G6PD activity, an elevated WBC count in these patients can cause the false assurance of low HA risk due to a falsely normal G6PD activity. We sought to establish and evaluate a novel method to mitigate WBC contamination of G6PD measurements in patients undergoing rasburicase therapy. In this study, we first developed and validated an innovative method (“piercing method”) to minimize the interference of WBCs in G6PD measurements. The piercing method demonstrated acceptable accuracy (slope: 1.008; bias: -6.22%) and precision (1.4% CV between 9.9 to 20.7 U/g Hb) and was effective at eliminating a broad range of WBC types and concentrations from whole blood samples (N = 85, tested range = 0.31 – 413.05 K/μL, 93% reduction in WBCs). We compared G6PD activity in rasburicase-treated patients (N = 39) with varying WBC counts before and after buffy coat removal using the piercing method. Average percent change in G6PD activity (post-pierce – pre-pierce) between patients with and without elevated WBCs was substantial (45% versus 12%). In comparing G6PD activity in patients before and after the initiation of rasburicase therapy, there is no evidence that in vivo-generated ROS from rasburicase affect G6PD measurement in vitro in an assay that relies on monitoring absorbance of NADPH (22.2 U/g Hb versus 22.9 U/g Hb, p=0.721). Results demonstrate how WBCs increase risk for false elevation in G6PD activity in patients being considered for rasburicase therapy. At greatest risk are patients with partial G6PD deficiency, e.g., heterozygous females, where G6PD from WBCs could mask deficiency. Literature describing testing of G6PD in specimens with elevated WBCs is lacking. This study shows that the piercing method provides accurate G6PD results in samples with elevated WBCs from patients at risk of TLS, who might develop HA after starting rasburicase therapy.

Triplication of the PCDH19 Gene as a Novel Disease Mechanism Leading to Epileptic Encephalopathy Resembling Loss-of-Function Pathogenic Variants

Background/Objectives: Developmental and epileptic encephalopathy 9 (DEE9) (MIM #300088) affects heterozygous females and males with somatic pathogenic variants, while male carriers with hemizygous PCDH19 pathogenic variants are clinically unaffected. There are hundreds of pathogenic single nucleotide variants in the PCDH19 gene reported in the literature, which lead to the loss of function of the PCDH19 protein. To date, no phenotypes associated with overexpression or copy number gains have been described in this gene. Methods and results: We present a female patient with a de novo triplication in the Xq21.3–q22.1 chromosomal region, which includes the PCDH19 gene, which implies an unbalanced dose gain. This patient displayed a phenotype of epileptic encephalopathy compatible with DEE9. By comparison, another male patient with a similar duplication showed mild developmental delay and autism but never developed epilepsy. Conclusions: Here, we propose the dose gain of PCDH19 as a new pathogenic mechanism that results in a phenotype similar to that found in patients with loss-of-function variants in PCDH19, when present in a heterozygous state.

6784 A Case of Euthyroid Hyperthyroxinemia due to Thyroid Binding Globulin Excess Syndrome

Abstract Disclosure: A.S. Iyer: None. Background: Congenital thyroid binding globulin (TBG) excess is a rare phenomenon associated with elevated serum total T3 and total T4 concentrations but normal serum concentrations of free thyroid hormones, with clinically euthyroid patients. While TBG excess can be caused by pregnancy, estrogen-secreting tumors, and estrogen therapy, congenital TBG excess follows an X-linked pattern, with hemizygous males most affected, and heterozygous females showing a spectrum of disease due to random inactivation of one X chromosome. Clinical Case: A 39-year-old Caucasian female presented as a referral from the Gastroenterology clinic due to concern for thyroid resistance syndrome. Symptoms included chronic constipation (improving with Miralax®), gradual weight gain of 8 pounds in one year, fatigue, and palpitations. She reported that her total thyroid hormone levels have always been elevated, with other thyroid labs remaining within normal limits, along with family history of similar labs noted in her two sons, father, and paternal grandmother. Upon initial encounter, TSH was 1.637 (0.430-3.550 uIU/mL), total T4 15.4 (4.8-11.7 ug/dL), total T3 231 (80-200 ng/dL), free T4 0.97 (0.70-1.25 ng/dL), free T3 3.2 (1.7-3.7 pg/mL), and albumin 4.2 (3.5-5.0 g/dL). The patient was not on oral contraceptive pills and she denied any pregnancies in the last three years. Thyroid ultrasound showed a normal-appearing homogeneous thyroid gland with a small cyst with colloid and no cervical lymphadenopathy. Initial differential diagnosis included TBG excess syndrome versus familial dysalbuminemic hyperthyroxinemia. Permission was obtained from the patient to access one of her son’s medical records from age 8-weeks old when he was also evaluated for abnormal thyroid labs. TSH was 2.144 (0.350-6.500 uIU/mL), total T4 28.2 (5.0-12.0 ug/dL), free T4 1.55 (0.8-1.8 ng/dL), and free T3 4.0 pg/mL (no reference available). Thyroid binding globulin was found to be elevated at 81 (12-26 mcg/mL) in her son and found to be elevated to 78.0 (13.5-30.9 mcg/mL) in this patient, confirming a diagnosis of TBG excess syndrome. Conclusions: TBG excess syndrome should be considered in the differential of patients with high total T3 and total T4 levels with normal levels of free T3, free T4, and TSH, especially in individuals who have a known family history and who have minimal or nonspecific symptoms. Definitive diagnosis will make further testing and treatment unnecessary. However, if these patients develop hypo- or hyperthyroidism, then use of TSH and free levels of T4 and T3 is necessary to direct management and avoid confusion. Reference: Pappa, T., and S. Refetoff. “Thyroid hormone transport proteins: Thyroxine-binding globulin, transthyretin, and albumin.” Reference Module in Neuroscience and Biobehavioral Psychology, 2017, https://doi.org/10.1016/b978-0-12-809324-5.03494-5. Presentation: 6/2/2024

Glucose-6-phosphate dehydrogenase deficiency detection using fluorocytometric assay: Evaluation after 1 year of clinical implementation.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common enzymopathy that affects red blood cells (RBCs) and renders them susceptible to oxidative stress. G6PD deficiency can cause hemolytic anemia, especially after exposure to certain drugs or infections. The diagnosis of G6PD deficiency is usually based on spectrophotometric measurement of enzyme activity, but this method has limitations in heterozygous females and in patients with other hematological disorders. In this study, we evaluated the use of flow cytometry as an alternative method for detecting G6PD deficiency in 514 samples (265 females and 249 males) from a clinical laboratory. We compared the results of flow cytometry with those of spectrophotometry and molecular analysis, and assessed the performance of flow cytometry in different subgroups of patients. We found that flow cytometry was able to identify G6PD deficiency in most cases, with high sensitivity and specificity. Flow cytometry also allowed the quantification of the percentage of G6PD-deficient RBCs, which varied among heterozygous females due to X-chromosome inactivation. Moreover, flow cytometry detected several cases of G6PD deficiency that were missed by spectrophotometry, especially in heterozygous females with normal or subnormal enzyme activity. However, flow cytometry also showed some false negative results, mainly in patients with sickle cell disease. Therefore, flow cytometry is a reliable and efficient tool for screening G6PD deficiency, but some precautions should be taken in interpreting the results in patients with other hematological conditions.

Clinical and Molecular Aspects of Craniofrontonasal Syndrome due to Contiguous Gene Deletion Involving AWAT2, EFNB1, EDA, OTUD6A, and PJA1 Genes

AbstractCraniofrontonasal syndrome (CFNS; Online Mendelian Inheritance in Man [OMIM] 340110) is an infrequent X linked disorder characterized by specific facial features and digital abnormalities with or without visceral anomalies. There is a peculiar paradoxical difference in severity of the phenotype in heterozygous females compared to hemizygous males. Here, we present a case where the mother, with clinical features of the syndrome, had terminated her previous pregnancy as the fetus had partial agenesis of the corpus callosum. Exome sequencing of the mother revealed no pathogenic variants related to the phenotype. Chromosomal microarray revealed 1.3-Mb pathogenic heterozygous deletion in chromosome X encompassing the Xq13.1 region with five OMIM genes, including EFNB1 gene related to craniofrontonasal syndrome. Detailed phenotyping of the parents and exact genetic etiology with molecular mechanism is important to arrive at a definitive diagnosis crucial for genetic counseling and definitive prenatal testing.

Overhauling dental implications in a child with rare craniofrontonasal syndrome: a case report

Craniofrontonasal syndrome is an extremely rare X-linked dominant genetic disorder characterized by features such as hypertelorism, craniosynostosis, ocular anomalies, a bifid nasal tip, and longitudinal ridging with splitting of the nails. Heterozygous females are more severely affected, presenting frontonasal dysplasia and coronal craniosynostosis (fusion of the coronal sutures), while males typically only exhibit hypertelorism. This case report describes the dental management of a 9-year-old female with craniofrontonasal syndrome who was referred to the department of paediatric and preventive dentistry for pain in the lower right and left back tooth region. The patient exhibited a clinical spectrum including microcephaly, short neck, axillary pterygium, clinodactyly of the toes, longitudinal ridging with split nails, pectus excavatum, and underdeveloped female genitalia. Extraoral examination revealed hypertelorism, flat nasal bridge, low-set ears, strabismus, antimongoloid slant, and an indistinct philtrum. Intraoral examination showed a high palatal vault, crowding of the upper and lower arches during the mixed dentition period, multiple dental caries, and poor oral hygiene. An orthopantomogram confirmed delayed dental development. Dental treatment was carried out on a dental chair system using adequate behaviour management techniques with short appointments, keeping in mind the reduced cooperative ability of the child. The importance of this report stems from the limited dental literature available on the syndrome. Preventive measures along with home care instructions were emphasized to support professional care.

Severe clinical phenotypes of heterozygous females with X-linked chronic granulomatous disease

Female X-linked chronic granulomatous disease (XL-CGD) carriers may develop severe clinical disease including infections with CGD-defining pathogens and inflammatory disorders. Similar to males with XL-CGD, female carriers warrant ongoing evaluation and prophylaxis where indicated.

Genotype-first analysis in an unselected health system-based population reveals variable phenotypic severity of COL4A5 variants.

Our knowledge of X-linked Alport Syndrome [AS] comes mostly from selected cohorts with more severe disease. We examined the phenotypic spectrum of X-linked AS in males and females with a genotype-based approach using data from the Geisinger MyCode DiscovEHR study, an unselected health system-based cohort with exome sequencing and electronic health records. Patients with COL4A5 variants reported as pathogenic (P) or likely pathogenic (LP) in ClinVar, or protein-truncating variants (PTVs), were each matched with up to 5 controls without COL4A3/4/5 variants by sociodemographics, diabetes diagnosis, and year of first outpatient encounter. AS-related phenotypes included dipstick hematuria, bilateral sensorineural hearing loss (BSHL), proteinuria, decreased eGFR, and ESKD. Out of 170,856 patients, there were 29 hemizygous males (mean age 52.0 y [SD 20.0]) and 55 heterozygous females (mean age 59.3 y [SD 18.8]) with a COL4A5 P/LP variant, including 48 with the hypomorphic variant p.Gly624Asp. Overall, penetrance (having any AS phenotypic feature) was highest for non-p.Gly624Asp P/LP variants (males: 94%, females: 85%), intermediate for p.Gly624Asp (males: 77%, females: 69%), compared to controls (males: 32%; females: 50%). The proportion with ESKD was highest for males with P/LP variants (44%), intermediate for males with p.Gly624Asp (15%) and females with P/LP variants (10%), compared to controls (males: 3%, females 2%). Only 47% of individuals with COL4A5 had completed albuminuria screening, and a minority were taking renin-angiotensin aldosterone system (RAAS) inhibitors. Only 38% of males and 16% of females had a known diagnosis of Alport syndrome or thin basement membrane disease. In an unselected cohort, we show increased risks of AS-related phenotypes in men and women compared to matched controls, while showing a wider spectrum of severity than has been described previously and variability by genotype. Future studies are needed to determine whether early genetic diagnosis can improve outcomes in Alport Syndrome.

An egg-sabotaging mechanism drives non-Mendelian transmission in mice

Selfish genetic elements drive in meiosis to distort their transmission ratio and increase their representation in gametes, violating Mendel's law of segregation. The two established paradigms for meiotic drive, gamete killing and biased segregation, are fundamentally different. In gamete killing, typically observed with male meiosis, selfish elements sabotage gametes that do not contain them. By contrast, killing is predetermined in female meiosis, and selfish elements bias their segregation to the single surviving gamete (i.e., the egg in animal meiosis). Here, we show that a selfish element on mouse chromosome 2, Responder to drive 2 (R2d2), drives using a hybrid mechanism in female meiosis, incorporating elements of both killing and biased segregation. We propose that if R2d2 is destined for the polar body, it manipulates segregation to sabotage the egg by causing aneuploidy, which is subsequently lethal in the embryo, ensuring that surviving progeny preferentially contain R2d2. In heterozygous females, R2d2 orients randomly on the metaphase spindle but lags during anaphase and preferentially remains in the egg, regardless of its initial orientation. Thus, the egg genotype is either euploid with R2d2 or aneuploid with both homologs of chromosome 2, with only the former generating viable embryos. Consistent with this model, R2d2 heterozygous females produce eggs with increased aneuploidy for chromosome 2, increased embryonic lethality, and increased transmission of R2d2. In contrast to typical gamete killing of sisters produced as daughter cells in a single meiosis, R2d2 prevents production of any viable gametes from meiotic divisions in which it should have been excluded from theegg.

Exploring self-reported visual function and vision-related anxiety in patients with RPGR-associated retinal degeneration

Variants in the retinitis pigmentosa GTPase regulator (RPGR) gene are responsible for the majority of X-linked retinitis pigmentosa cases, which not only affects male patients but also some heterozygous females. Vision-related disability and anxiety of patients with RPGR-associated retinal degeneration have never been explored before. This study aimed to evaluate self-reported visual function and vision-related anxiety in a Portuguese cohort of male and female patients with RPGR-associated retinal degeneration using two validated patient-reported outcome measures. Cross-sectional data of thirty-two genetically-tested patients was examined, including scores of the Michigan retinal degeneration questionnaire (MRDQ) and Michigan vision-related anxiety questionnaire. Patients were classified according to retinal phenotypes in males (M), females with male phenotype (FM), and females with radial or focal pattern. Both M and FM revealed higher rod-function and cone-function anxiety scores (p < 0.017). Most MRDQ disability scores were higher in M and FM (p < 0.004). Overall, positive correlations (p < 0.004) were found between every MRDQ domain and both anxiety scores. In RPGR-associated retinal degeneration, males and females with male phenotype show similar levels of increased vision-related anxiety and disability. Every MRDQ visual function domain showed a strong correlation with anxiety scores.

Taf1 knockout is lethal in embryonic male mice and heterozygous females show weight and movement disorders.

The TATA box-binding protein-associated factor 1 (TAF1) is a ubiquitously expressed protein and the largest subunit of the basal transcription factor TFIID, which plays a key role in initiation of RNA polymerase II-dependent transcription. TAF1 missense variants in human males cause X-linked intellectual disability, a neurodevelopmental disorder, and TAF1 is dysregulated in X-linked dystonia-parkinsonism, a neurodegenerative disorder. However, this field has lacked a genetic mouse model of TAF1 disease to explore its mechanism in mammals and treatments. Here, we generated and validated a conditional cre-lox allele and the first ubiquitous Taf1 knockout mouse. We discovered that Taf1 deletion in male mice was embryonically lethal, which may explain why no null variants have been identified in humans. In the brains of Taf1 heterozygous female mice, no differences were found in gross structure, overall expression and protein localisation, suggesting extreme skewed X inactivation towards the non-mutant chromosome. Nevertheless, these female mice exhibited a significant increase in weight, weight with age, and reduced movement, suggesting that a small subset of neurons was negatively impacted by Taf1 loss. Finally, this new mouse model may be a future platform for the development of TAF1 disease therapeutics.

NGS-Based Identification of Two Novel PCDH19 Mutations in Female Patients with Early-Onset Epilepsy.

Developmental and epileptic encephalopathy-9 (DEE9) is characterized by seizure onset in infancy, mild to severe intellectual impairment, and psychiatric features and is caused by a mutation in the PCDH19 gene on chromosome Xq22. The rare, unusual X-linked type of disorder affects heterozygous females and mosaic males; transmitting males are unaffected. In our study, 165 patients with epilepsy were tested by Next Generation Sequencing (NGS)-based panel and exome sequencing using Illumina technology. PCDH19 screening identified three point mutations, one indel, and one 29 bp-long deletion in five unrelated female probands. Two novel mutations, c.1152_1180del (p.Gln385Serfs*6) and c.830_831delinsAA (p.Phe277*), were identified and found to be de novo pathogenic. Moreover, among the three inherited mutations, two originated from asymptomatic mothers and one from an affected father. The PCDH19 c.1682C>T and c.1711G>T mutations were present in the DNA samples of asymptomatic mothers. After targeted parental testing, X chromosome inactivation tests and Sanger sequencing were carried out for mosaicism examination on maternal saliva samples in the two asymptomatic PCDH19 mutation carrier subjects. Tissue mosaicism and X-inactivation tests were negative. Our results support the opportunity for reduced penetrance in DEE9 and contribute to expanding the genotype-phenotype spectrum of PCDH19-related epilepsy.

#2199 Kidney involvement aspects in women with Fabry disease from the Romanian cohort

Abstract Background and Aims Fabry disease (FD) is an X-linked rare lysosomal storage disease causing progressive nervous system, kidney and heart disorders. Progressive kidney impairment is common, and an important cause of morbidity and mortality. In FD females, treatment guidelines recommend therapy initiation when there is evidence of organ involvement, but determining the proper moment to start FD specific treatment could be a real challenge in the cases. The aim of our retrospective study is to investigate clinical and histological aspects of kidney involvement in female patients with genetically confirmed FD evaluated in our Expert Center for Rare Diseases of the Reno-Urinary System between 2015–2023, with focus on the clinical phenotype and initiation of FD specific treatment. Method All patients had comprehensive assessment for target organ manifestations of Fabry disease. Renal function was assessed using serum creatinine, albumin creatinine ratio and proteinuria. Also, 14 (40%) FD females underwent kidney biopsy (KB) in order to reveal the FD specific lesions and/or to establish the indication for FD pathogenic therapy. Kidney biopsy specimens were analyzed by light and electron microscopy. Results Our study included 35 heterozygous adult females with FD from 25 unrelated families. Most females (91.4%) were diagnosed through family screening, while 3 females (8.6%) were index cases. The mean age at diagnosis was 43.8 ± 15.3 years (range 17-73 years), although mean age of symptoms onset was 34.3 ± 13.5 years (range 14-61 years). Six women from our cohort were considered healthy carriers, three females presented only neurological manifestations, nine patients had combined renal and neurological involvement, one patient had cardiac and renal involvement and the others presented multiple organ involvement. Significant co-morbidities were: arterial hypertension in 13 patients, diabetes mellitus in 2 patients, and obesity in 9 patients. Overall baseline mean eGFR was 79.9 ± 28.2 (range 25-135) ml/min/1.73 m2. Eleven patients had no obvious kidney involvement, 10 patients presented chronic kidney disease (CKD) stage 2, 11 patients CKD stage 3, one CKD stage 4 and 1 was hemodialyzed. Seventeen patients presented normal UACR, thirteen patients showed microalbuminuria, and four patients presented macroalbuminuria. Overt proteinuria (&amp;gt; 0.3 g/24 h) was noticed in 8 patients. Thirteen females performed KB before the initiation of FD specific treatment, and one patient was already treated. At the moment of KB, five patients had normal biologic kidney assessment, three patients presented CKD stage 2 (microalbuminuria in 2 cases and proteinuria in 1 case), and five patients presented CKD stage 3 (normoalbuminuria in 1 case, microalbuminuria in 1 case and overt proteinuria in 3 cases). All KBs showed lysosomal accumulation in the podocytes, 92.8% showed inclusions in the parietal cells of the Bowman capsule, 85.7% in the tubules, while vascular inclusions were found in 78.5% of cases. Also, we observed segmental glomerular sclerosis in 5 cases, interstitial fibrosis and tubular atrophy in 3 cases, each. Only 2 patients were treated with pathogenic therapy before the first evaluation in our center. After multidisciplinary evaluation, 22 patients (62.8%) from our cohort received recommendation to initiate FD specific treatment, and 11 patients entered in our follow-up program. Evidence of specific FD lesions in KB was a strong reason to support the recommendation to initiate FD specific treatment. Conclusion Our results showed that kidney involvement in FD females can vary from asymptomatic or mildly symptomatic, to severely symptomatic, with end stage kidney disease. Thus, females should be carefully evaluated and monitored in a multidisciplinary team, in order to diagnosed FD affected organs. Also, we underline that a normal standard assessment of the kidney cannot rule out kidney involvement in female patients with FD, and strengthen the importance of KB for detection of early kidney involvement and for additional support for early initiation of FD specific therapy.

Nuclease-free precise genome editing corrects MECP2 mutations associated with Rett syndrome.

Rett syndrome is an acquired progressive neurodevelopmental disorder caused by de novo mutations in the X-linked MECP2 gene which encodes a pleiotropic protein that functions as a global transcriptional regulator and a chromatin modifier. Rett syndrome predominantly affects heterozygous females while affected male hemizygotes rarely survive. Gene therapy of Rett syndrome has proven challenging due to a requirement for stringent regulation of expression with either over- or under-expression being toxic. Ectopic expression of MECP2 in conjunction with regulatory miRNA target sequences has achieved some success, but the durability of this approach remains unknown. Here we evaluated a nuclease-free homologous recombination (HR)-based genome editing strategy to correct mutations in the MECP2 gene. The stem cell-derived AAVHSCs have previously been shown to mediate seamless and precise HR-based genome editing. We tested the ability of HR-based genome editing to correct pathogenic mutations in Exons 3 and 4 of the MECP2 gene and restore the wild type sequence while preserving all native genomic regulatory elements associated with MECP2 expression, thus potentially addressing a significant issue in gene therapy for Rett syndrome. Moreover, since the mutations are edited directly at the level of the genome, the corrections are expected to be durable with progeny cells inheriting the edited gene. The AAVHSC MECP2 editing vector was designed to be fully homologous to the target MECP2 region and to insert a promoterless Venus reporter at the end of Exon 4. Evaluation of AAVHSC editing in a panel of Rett cell lines bearing mutations in Exons 3 and 4 demonstrated successful correction and rescue of expression of the edited MECP2 gene. Sequence analysis of edited Rett cells revealed successful and accurate correction of mutations in both Exons 3 and 4 and permitted mapping of HR crossover events. Successful correction was observed only when the mutations were flanked at both the 5' and 3' ends by crossover events, but not when both crossovers occurred either exclusively upstream or downstream of the mutation. Importantly, we concluded that pathogenic mutations were successfully corrected in every Rett line analyzed, demonstrating the therapeutic potential of HR-based genome editing.

An egg sabotaging mechanism drives non-Mendelian transmission in mice.

During meiosis, homologous chromosomes segregate so that alleles are transmitted equally to haploid gametes, following Mendel's Law of Segregation. However, some selfish genetic elements drive in meiosis to distort the transmission ratio and increase their representation in gametes. The established paradigms for drive are fundamentally different for female vs male meiosis. In male meiosis, selfish elements typically kill gametes that do not contain them. In female meiosis, killing is predetermined, and selfish elements bias their segregation to the single surviving gamete (i.e., the egg in animal meiosis). Here we show that a selfish element on mouse chromosome 2, R2d2, drives using a hybrid mechanism in female meiosis, incorporating elements of both male and female drivers. If R2d2 is destined for the polar body, it manipulates segregation to sabotage the egg by causing aneuploidy that is subsequently lethal in the embryo, so that surviving progeny preferentially contain R2d2. In heterozygous females, R2d2 orients randomly on the metaphase spindle but lags during anaphase and preferentially remains in the egg, regardless of its initial orientation. Thus, the egg genotype is either euploid with R2d2 or aneuploid with both homologs of chromosome 2, with only the former generating viable embryos. Consistent with this model, R2d2 heterozygous females produce eggs with increased aneuploidy for chromosome 2, increased embryonic lethality, and increased transmission of R2d2. In contrast to a male meiotic driver, which kills its sister gametes produced as daughter cells in the same meiosis, R2d2 eliminates "cousins" produced from meioses in which it should have been excluded from the egg.

Sex differences in physiological response to increased neuronal excitability in a knockin mouse model of pediatric epilepsy.

Epilepsy is a common neurological disease; however, few if any of the currently marketed antiseizure medications prevent or cure epilepsy. Discovery of pathological processes in the early stages of epileptogenesis has been challenging given the common use of preclinical models that induce seizures in physiologically normal animals. Moreover, despite known sex dimorphism in neurological diseases, females are rarely included in preclinical epilepsy models. We characterized sex differences in mice carrying a pathogenic knockin variant (p.N1768D) in the Scn8a gene that causes spontaneous tonic-clonic seizures (TCs) at ∼3 months of age and found that heterozygous females are more resilient than males in mortality and morbidity. To investigate the cellular mechanisms that underlie female resilience, we utilized blood-brain barrier (BBB) and hippocampal transcriptomic analyses in heterozygous mice before seizure onset (pre-TC) and in mice that experienced ∼20 TCs (post-TC). In the pre-TC latent phase, both sexes exhibited leaky BBB; however, patterns of gene expression were sexually dimorphic. Females exhibited enhanced oxidative phosphorylation and protein biogenesis, while males activated gliosis and CREB signaling. After seizure onset (chronic phase), females exhibited a metabolic switch to lipid metabolism, while males exhibited increased gliosis and BBB dysfunction and a strong activation of neuroinflammatory pathways. The results underscore the central role of oxidative stress and BBB permeability in the early stages of epileptogenesis, as well as sex dimorphism in response to increasing neuronal hyperexcitability. Our results also highlight the need to include both sexes in preclinical studies to effectively translate results of drug efficacy studies.

Effect of PCDH19 missense mutations on cell-to-cell proximity and neuronal development under heterotypic conditions.

The mutation of the X-linked protocadherin (PCDH) 19 gene in heterozygous females causes epilepsy. However, because of the erosion of X-chromosome inactivation (XCI) in female human pluripotent stem cells, precise disease modeling often leads to failure. In this study, using a mathematical approach and induced pluripotent stem cells retaining XCI derived from patients with PCDH19 missense mutations, we found that heterotypic conditions, which are composed of wild-type and missense PCDH19, led to significant cell-to-cell proximity and impaired neuronal differentiation, accompanied by the aberrant accumulation of doublecortin, a microtubule-associated protein. Our findings suggest that ease of adhesion between cells expressing either wild-type or missense PCDH19 might lead to aberrant cell aggregation in early embryonic phases, causing poor neuronal development.

PICH deficiency limits the progression of MYC-induced B-cell lymphoma

Plk1-interacting checkpoint helicase (PICH) is a DNA translocase involved in resolving ultrafine anaphase DNA bridges and, therefore, is important to safeguard chromosome segregation and stability. PICH is overexpressed in various human cancers, particularly in lymphomas such as Burkitt lymphoma, which is caused by MYC translocations. To investigate the relevance of PICH in cancer development and progression, we have combined novel PICH-deficient mouse models with the Eμ-Myc transgenic mouse model, which recapitulates B-cell lymphoma development. We have observed that PICH deficiency delays the onset of MYC-induced lymphomas in Pich heterozygous females. Moreover, using a Pich conditional knockout mouse model, we have found that Pich deletion in adult mice improves the survival of Eμ-Myc transgenic mice. Notably, we show that Pich deletion in healthy adult mice is well tolerated, supporting PICH as a suitable target for anticancer therapies. Finally, we have corroborated these findings in two human Burkitt lymphoma cell lines and we have found that the death of cancer cells was accompanied by chromosomal instability. Based on these findings, we propose PICH as a potential therapeutic target for Burkitt lymphoma and for other cancers where PICH is overexpressed.

Targeted long-read sequencing identified a causal structural variant in X-linked nephrogenic diabetes insipidus.

X-linked nephrogenic diabetes insipidus (NDI) is a rare genetic renal disease caused by pathogenic variants in the AVPR2 gene. Single nucleotide variants and small insertions/deletions in AVPR2 are reliably detected by routine clinical sequencing. Nevertheless, structural variants involving AVPR2 are challenging to identify accurately by conventional genetic testing. Here, we report a novel deletion of AVPR2 in a Czech family identified for the first time by targeted long-read sequencing (T-LRS). A male proband with X-linked NDI underwent clinical sequencing of the AVPR2 gene that failed and thus indicated possible whole-gene deletion. Therefore, PCR mapping and subsequent targeted long-read sequencing (T-LRS) using a Pacific Biosciences sequencer were applied to search for the suspected deletion. To validate the deletion breakpoints and prove variant segregation in the family with X-linked NDI, Sanger sequencing of the deletion junction was performed. Quantitative real-time PCR was further carried out to confirm the carrier status of heterozygous females. By T-LRS, a novel 7.5 kb deletion of AVPR2 causing X-linked NDI in the proband was precisely identified. Sanger sequencing of the deletion junction confirmed the variant breakpoints and detected the deletion in the probands´ mother, maternal aunt, and maternal cousin with X-linked NDI. The carrier status in heterozygous females was further validated by quantitative real-time PCR. Identifying the 7.5 kb deletion gave a precise molecular diagnosis for the proband, enabled genetic counselling and genetic testing for the family, and further expanded the spectrum of structural variants causing X-linked NDI. Our results also show that T-LRS has significant potential for accurately identifying putative structural variants.