Karyotype Analysis Research Articles

Endosperm culture-based allotriploid hybrid production from an interspecific cross of Haemanthus spp.: new insights into polyploidization and hybridization

BackgroundAllopolyploid plants are valuable for plant breeding because they have the advantage of polyploidization and hybridization, such as increased vigor and adaptability. Although biparental triploid endosperms have the potential to be used to produce allotriploid plants, the approach remains largely unexplored. Therefore, this study aimed to produce allotriploid plants from the endosperms of interspecific crosses between Haemanthus pauculifolius and H. albiflos.ResultsPrecisely identified embryo and endosperm pairs were used. Embryos were grown on half-strength Murashige and Skoog (MS) medium, and endosperms from interspecific crossing were cultured to induce callus formation and shoot regeneration, which then developed into plantlets. MS medium supplemented with 4-amino-3,5,6-trichloropicolinic acid (picloram) and 6-benzylaminopurine (BAP), or 2,4-dichloro phenoxy acetic acid (2,4-D) and BAP were used for callus induction, and callus formation rates were measured. Flow cytometry, karyotyping, and Sanger sequencing of the nuclear internal transcribed spacer (ITS) region, chloroplast (trnL-trnF region, matK gene), and mitochondrial (nad1 gene) DNA were performed on plantlets derived from embryos and endosperms, along with their parental plants. In this study, a total of 18 pairs of diploid and triploid plantlets were obtained from the embryo and endosperm, respectively. Callus formation rates were significantly higher on media with picloram and BAP compared to 2,4-D and BAP. ITS sequencing and karyotype analyses detected that all the 16 pairs of plantlets analyzed were hybrids, indicating that most endosperm-derived plantlets were allotriploid with a parental chromosome ratio of 2:1 (maternal: paternal). In addition, chloroplast DNA sequencing revealed maternal inheritance in the endosperm-derived plantlets, consistent with embryo-derived plantlets.ConclusionsThis study is the first to demonstrate the production of allotriploid hybrid plants through endosperm culture using seeds from interspecific crosses, as supported by cellular and genetic analyses. Additionally, the study established a novel system for simultaneously producing diploid and allotriploid hybrids from a single seed, providing valuable materials to study the effects of polyploidization and hybridization in allopolyploid plants. These findings contribute to plant breeding strategies and advance our understanding of hybridization, polyploidization, and allopolyploid plant development.

Molecular characterization of a rare TP63 variant associated with split-hand/split-foot malformation 4 and incomplete penetrance: disruption of the p63-Dlx signaling pathway

BackgroundSplit-hand/foot malformation (SHFM) is a congenital disability characterized by the absence or hypoplasia of the central ray of the hands and/or feet. This study reports a causative variant in the TP63 gene in a Chinese family exhibiting limb anomalies associated with SHFM4.MethodsEnrolled in this study was a Chinese family with limb anomalies without any other clinical features. Karyotype analysis and chromosomal microarray analysis (CMA) were conducted to identify chromosomal abnormalities. Whole exome sequencing (WES) was utilized to investigate sequence variants, while RNA sequencing assessed differentially expressed genes, with findings confirmed through quantitative PCR (qPCR).ResultsKaryotype analysis and CMA revealed no chromosomal abnormalities in the family. Subsequently, WES identified a rare heterozygous variant of NM_003722.5: c.956G > A (p.Arg319His) in the TP63 gene in the proband, which was inherited from her father who also presented with limb deformities. However, both of the sister and grandfather of the proband had the same variant but exhibited normal limb morphology. RNA sequencing results demonstrated an increased expression level of TP63 and its downstream genes (PERP, CDH3, and DLX5) compared with the controls, indicating an enrichment of cell adhesion molecules the differentially expressed genes in the patient. However, significant differences were noted only for the CDH3 and DLX5 genes in qPCR analysis (p<0.05).ConclusionThis study identifies, for the first time, the TP63 gene variant c.956G > A (p.Arg319His) as a causative factor for SHFM4 in Chinese individuals with incomplete penetrance. In addition, we hypothesize that the p.Arg319His variant functions as a gain-of-function variant, leading to the upregulation of cell adhesion target genes. Such upregulation then disrupts the p63-Dlx signaling pathway and causes AER stratification failure.

Prenatal diagnosis and clinical pregnancy outcome of fetuses with conotruncal defects in a Chinese cohort.

This study aimed to explore genetic etiologies of conotruncal defects (CTDs) in fetuses by analyzing the results of different genetic tests and to assess pregnancy outcomes of fetuses with CTD in a Chinese prenatal cohort. A total of 146 fetuses that underwent invasive prenatal genetic testing for CTD at the prenatal diagnosis center of the International Peace Maternity and Child Health Hospital between January 2018 and December 2022 were retrospectively analyzed. All of them underwent chromosomal microarray analysis (CMA) and karyotype analysis, but only 27 underwent whole-exome sequencing (WES). The results of these different genetic testing methods were collected and compared. Data on pregnancy outcomes and neonate prognosis were obtained from the electronic medical records and postpartum telephone follow-up. Among the 146 fetuses with CTD, the chromosome abnormality rate was 22.6% (33/146), and the detection rate of abnormal chromosomes in CMA and karyotype was 15.8% (23/146). Among the 27 pregnant women who accepted WES, five had pathogenic variants. The detection rate of abnormal chromosomes by CMA was higher in infants with an interrupted aortic arch (IAA) than in the other infant groups (60.0%, χ2 = 11.661, P = 0.045, c2-test or Fisher's exact test). Congenital heart disease is a complex congenital heart disease with an etiology closely related to genetic abnormalities. Therefore, invasive genetic testing and CMA are recommended as first-line tests for all fetuses with CTD detected by prenatal ultrasound, especially IAA. WES should be recommended when necessary.

Establishment of a human ovarian endometrioid carcinoma cell line by constitutive expression of cyclin-dependent kinase 4, cyclin D1 and telomerase reverse transcriptase

Only a few human ovarian endometrioid carcinoma cell lines are currently available, partly due to the difficulty of establishing cell lines from low-grade cancers. Here, using a cell immortalization strategy consisting of i) inactivation of the p16INK4a-pRb pathway by constitutive expression of mutant cyclin-dependent kinase 4 (R24C) (CDK4R24C) and cyclin D1, and ii) acquisition of telomerase reverse transcriptase (TERT) activity, we established a human ovarian endometrioid carcinoma cell line from a 46-year-old Japanese woman. That line, designated JFE-21, has proliferated continuously for over 6 months with a doubling time of ~ 55 h. JFE-21 cells exhibit polygonal shapes and proliferate without contact inhibition to form a monolayer in a jigsaw puzzle-like arrangement. Ultrastructurally, JFE-21 cells exhibit well-developed rough endoplasmic reticulum, mitochondria and lysosomes in the cytoplasm, with cells contacting each other via desmosomes. G-band karyotype analysis indicated that cells had a near-tetraploid karyotype. Immunofluorescence staining revealed that the expression profile of a series of ovarian carcinoma markers in JFE-21 cells was consistent with ovarian endometrioid carcinoma. Moreover, Sanger sequencing of DNA polymerase ε (POLE) gene and immunohistochemical analysis of mismatch repair (MMR) proteins revealed that JFE-21 cells were classified as the no specific molecular profile (NSMP) subtype. In addition, JFE-21 cells were sensitive to paclitaxel and carboplatin administered to the donor as therapy. These findings indicate that constitutive expression of CDK4R24C, cyclin D1 and TERT genes may be an option to establish cell lines from low-grade cancers, including ovarian endometrioid carcinoma.

A novel compound heterozygous mutation in the DYNC2H1 gene in a Chinese family with Jeune syndrome

BackgroundThe dynein cytoplasmic two heavy chain 1 (DYNC2H1) gene encodes a cytoplasmic dynein subunit. Cytoplasmic dyneins transport cargo towards the minus end of microtubules and are thus termed the “retrograde” cellular motor. Mutations in DYNC2H1 are the main causative mutations of short rib-thoracic dysplasia syndrome type III with or without polydactyly (SRTD3). Early diagnosis of SRTD3 prenatally by ultrasound alone is difficult. In this case, a couple who gave birth to three consecutive babies with SRTD3 requested fertility guidance to avoid having another baby with SRTD3.MethodsCytogenetic and molecular genetic analyses of amniotic fluid via whole-genome sequencing (WGS), routine G-banded karyotype analysis, fluorescent quantitative polymerase chain reaction, and whole-exome sequencing (WES) were performed at 19 weeks. Peripheral blood samples from the parents were also screened by Sanger sequencing for SRTD3-related mutations.ResultsTwo compound heterozygous mutations, c.10,594 C > T and c.7720G > A, in the DYNC2H1 gene were identified, which were inherited from the mother and father, respectively. The foetus’s mother is heterozygous for the c.10,594 C > T variant, and the foetus’s father is heterozygous for the c.7720G > A variant. The mutation c.10,594 C > T, which is a nonsense mutation believed to be pathogenic, has been previously reported. The mutation c.7720G > A, which is a missense mutation, has yet to be reported. Moreover, no chromosomal abnormalities or pathogenic copy number variations (CNVs) were detected in the foetus. The patient did not become pregnant after PGT-M and IVF-ET. This family subsequently accepted donated eggs; a successful pregnancy occurred, and a healthy girl was born.ConclusionThe compound heterogeneous mutations in DYNC2H1 ultimately accounts for the diversity of disease phenotypes reported in this study and can be used to guide future pregnancies. Our findings expand the mutation spectrum of DYNC2H1 in this rare disease and highlight the value of WES in the diagnosis of skeletal dysplasia with unclear prenatal indications.

P-06 WHEN IS GENDER SELECTION IN CONGENITAL ADRENAL HYPERPLASIA?

Abstract Introduction Congenital adrenal hyperplasia (CAH) is one of the most common (classical type; nonclassical type) genetic diseases and is inherited in an autosomal recessive manner. This disease can lead to adrenal insufficiency, fluid-electrolyte disorder, and ambiguous genitalia as a result of axis dysfunction in cortisol biosynthesis. The optimal time for gender identity formation is between the ages of 18 and 36 months. The gender assignment of patients with CAH should be done as soon as possible before people around them know about it. Clinical Case A 23-year-old patient with known CAH due to 21-hydroxylase deficiency was referred to our clinic by a psychiatrist 1 year ago with the diagnosis of gender dysphoria. In his history, the patient was admitted first to the hospital with complaints of nausea and vomiting only one month after birth by his family. During examination at that time, BP was 70/40mmHg, and genital examination revealed ambiguous genitalia. In laboratory tests, Na:128mEq/l, K:5.1 mEq/l, DHEA-SO4: 4mcg/dl, basal cortisol: 1.18 mcg/dl and 17-OH progesterone: 6.8 ng/ml were found, and Karyotype analysis was reported as 46 XX. According to these results, the patient was diagnosed with CAH. Genetic analysis revealed CYP21A2 IVS2-13 C&amp;gt;G homozygous mutation. The patient who has classical type CAH was accepted as a phenotypically and genetically female child by her family and had undergone 2 correction operations since the age of 1 due to ambiguous genitalia. Between 2003 and 2013, he continued his follow-up in the pediatric endocrinology department and used Hydrocortisone + Fludrocortisone treatment in varying doses. He expressed that he did not go for regular check-ups and did not use his medications until 2018. Due to being untreated for a long time, his testosterone levels were high as male normal ranges, and his phenotype was male. The Ferriman-Gallwey score was determined as 22, and reconstruction scars and narrow vaginal opening were observed in the genital examination. In the laboratory test without any treatment were 17-OH-progesterone &amp;gt; 20ng/ml, basal cortisol: 7.8 mcg/dl, ACTH: 570 pg/ml, total testosterone: 524 ng/L, estradiol: 42 ng/L, DHEAS:252 mcg/dl, FSH:4.5 IU/L, LH: 0.76 IU/L, Na:136 mEq/L, K:4.3 mEq/L. The patient, who had simple virilizing CAH and gender dysphoria together, was first planned to start steroid replacement therapy in terms of CAH treatment. According to his clinical course, a decision was made to re-evaluate him in terms of identity change and testosterone replacement therapy. Conclusion The majority of CAH patients were raised as chromosomally female-identifying and living as female despite masculinized gender behaviors. Only 5.2% of these patients experienced gender dissatisfaction. Recent studies have shown that rather than intrauterine hormone exposure, regular treatment during the growth period and gender orientation during follow-up are more accurate and important regarding psychosocial satisfaction.Figure 1Possesses a masculine phenotype without using any testosterone

P-33 PRIMARY OVARIAN INSUFFICIENCY AS A COMPONENT OF AUTOIMMUNE POLYGLANDULAR SYNDROME

Abstract Introduction Primary ovarian insufficiency (POI) is characterized by amenorrhea secondary to ovarian dysfunction accompanied by high gonadotropin levels in women before the age of 40. Autoimmunity is estimated to account for 5-17% of POI etiologies. Although it varies according to its subtype, the prevalence of POI in patients with autoimmune polyglandular syndrome (APS) is reported to be between 16% and 40%. It may take several years to recognize the syndrome. Generally, a multidisciplinary approach is needed to diagnose and treat the cases accurately. The present case report described the diagnostic process of such a patient. Clinical Case A 19-year-old female patient applied to our outpatient clinic with the complaint of disruption of menstrual cycles for nine months. She was diagnosed with type 1 diabetes mellitus and chronic autoimmune thyroiditis for nine years and was receiving insulin glargine, insulin aspart, and levothyroxine treatment. She reported that her menarche age was 12 years, and she had regular menstrual cycles until the previous year; it was learned that she first experienced oligomenorrhea and then amenorrhea for nine months. Her body mass index was within normal limits on examination, and secondary sex characteristics were fully developed. Laboratory tests revealed fasting plasma glucose 180 mg/dL, HbA1c: 9.5%, TSH: 2.2 mIU/L, free T4: 1.4 ng/dL, Anti-TPO: 120 IU/mL, (&amp;lt;34), prolactin: 18 mcg/L, basal serum cortisol: 15 mcg/dL. β-hCG was negative. Karyotype analysis was determined as 46, XX without structural chromosomal abnormality. Following the progesterone challenge test, the patient had no bleeding, and serum FSH level, which was checked twice, was high, serum estradiol was low, and AMH was also low at 0.1 ng/mL. The patient was diagnosed as POI based on this clinic and laboratory features. Bone density was found to be within normal limits in bone densitometry. The patient, who currently does not seek fertility and does not consider oocyte freezing, was started on hormone replacement therapy with transdermal estradiol and oral micronized progesterone. Conclusion POI is rarely seen in women before the age of 30, but patients with APS should be screened and closely followed up to identify other organ dysfunctions that may be targets of the immune system. When POI is diagnosed at an early age, attention should be given to other accompanying autoimmune diseases and polyglandular syndrome.

P-05 17Β-HYDROXYSTEROID DEHYDROGENASE 3 DEFICIENCY: TWO CASE REPORTS

Abstract Introduction 17-β-hydroxysteroid dehydrogenase type3 (17β-HSD3) is an enzyme that produces testosterone from androstenedione, encoded by the HSD17B3 gene. Its deficiency causes 46 XY disorder of sex development (DSD) with autosomal recessive inheritance. 46 XY individuals with this enzyme deficiency can exhibit a wide range of phenotypes, ranging from normal female external genitalia to ambiguous genitalia. In early childhood, the diagnosis might be clinically indistinguishable from other causes of 46 XY DSD such as partial androgen insensitivity syndrome (AIS) and 5α-reductase type 2 deficiency. Laboratory diagnosis is based on a low testosterone/androstenedione ratio. The diagnosis is confirmed by molecular genetic testing. If the diagnosis is missed in early infancy, patients present with severe virilization symptoms and amenorrhea during puberty. We present two siblings followed and managed with a presumed diagnosis of AIS until the genetic testing was performed. Clinical Case 1 A 4-month-old patient with female external genitalia was operated on with suspicion of bilateral inguinal hernia, and pathologic investigation revealed immature testicular tissue. No uterus and ovaries could be detected by pelvic ultrasound. The karyotype analysis was consistent with 46 XY, and the patient was followed up with the diagnosis of AIS. The patient’s parents were first cousins. The patient was started on estradiol replacement when she was 13 years old. At the age of 21, she underwent vaginoplasty due to a 2 cm long vaginal canal. A genetic analysis was available when the patient was 21 years old. Next generation sequencing revealed a previously reported homozygous c.277+5g&amp;gt;A mutation in exon 3 of HSD17B3 gene. The diagnosis was established as 17β-HSD3 deficiency. Clinical Case 2 After the index case was diagnosed with AIS karyotype analyses were performed on three other siblings at the time. The patient’s 7 years old sibling, who had a complete female-appearing genitalia, had a karyotype of 46XY. As the presumed diagnosis was AIS, bilateral orchiectomy was performed after counselling. Estradiol replacement was started at the age of 14 and complete breast development was achieved. Vaginal length was reported as 8 cm. Further genetic analysis was performed at the age of 27 and it revealed that the two siblings shared the same genetic mutation. Both preferred to retain the female gender identity during adulthood. Two other female siblings with a karyotype of 46XX and both parents had a heterozygous c.277+5g&amp;gt;A mutation in the HSD17B3 gene. Conclusion Although clinical evaluation and laboratory test results might point out to 17-β-HSD3 deficiency, the definite diagnosis is established with genetic testing. As these patients might develop male gender identity, awareness of the disease and early genetic testing is of importance for the prevention of misdiagnoses, counselling for gender identity, deciding on surgical interventions, and management of these patients.

P-34 XX MALE SYNDROME DIAGNOSED AT AGE 52

Abstract Introduction XX male syndrome (de la Chapelle syndrome) is a rare disorder of sex development linked to a 46,XX karyotype. Depending on the presence of the SRY gene, two variants of this disease are distinguished: SRY-positive and SRY-negative ones. The SRY (sex-determining region Y) gene is involved in the differentiation of Sertoli cells and development of the tests. Typically, de la Chapelle syndrome is diagnosed in childhood or adolescence. We present a case where XX male syndrome was diagnosed at age 52. Clinical Case A 52-year-old man came to our clinic complaining of excess weight, decreased libido, and fatigue with little physical activity. Physical examination discovered full masculinization, microorchidism, gynecomastia, obesity. At the time of his visit, he was undergoing chemotherapy for β-cell lymphoma. It is known that due to severe pain in the spine, he had been injecting himself with dexamethasone every day for six months, which led to secondary adrenal insufficiency. When he came, we stopped dexamethasone and initiated hydrocortisone. We reduced the dosage until it was completely discontinued over 4 months. The function of the adrenal glands was completely restored, which was shown by laboratory tests. On the first visit patient said that over the past 9-10 years he had been periodically receiving testosterone therapy due to low testosterone level. The decrease in testosterone was explained by obesity. About 10 years ago, investigation was performed due to infertility. Investigation showed a low testosterone level, a high FSH and LH level, azoospermia, a small testicular volume in the ultrasound were detected. Hypergonadotropic hypogonadism was diagnosed. Karyotyping analysis revealed a chromosomal complement of 46,XX. Molecular genetic testing confirmed the SRY-positive variant of de la Chapelle syndrome. Thus, we understand the reason for patient's low testosterone, and replacement therapy with testosterone was prescribed. And we recommended assisted reproductive technologies using donor sperm. Conclusion It is important to remember that in men of any age with hypogonadism and infertility, chromosomal abnormalities must be excluded (and not only in children and adolescents).Figure 1:Karyogram of the patient, 46,XX

Automated karyogram analysis for early detection of genetic and neurodegenerative disorders: a hybrid machine learning approach.

Anomalous chromosomes are the cause of genetic diseases such as cancer, Alzheimer's, Parkinson's, epilepsy, and autism. Karyotype analysis is the standard procedure for diagnosing genetic disorders. Identifying anomalies is often costly, time-consuming, heavily reliant on expert interpretation, and requires considerable manual effort. Efforts are being made to automate karyogram analysis. However, the unavailability of large datasets, particularly those including samples with chromosomal abnormalities, presents a significant challenge. The development of automated models requires extensive labeled and incredibly abnormal data to accurately identify and analyze abnormalities, which are difficult to obtain in sufficient quantities. Although the deep learning-based architecture has yielded state-of-the-art performance in medical image anomaly detection, it cannot be generalized well because of the lack of anomalous datasets. This study introduces a novel hybrid approach that combines unsupervised and supervised learning techniques to overcome the challenges of limited labeled data and scalability in chromosomal analysis. An Autoencoder-based system is initially trained with unlabeled data to identify chromosome patterns. It is fine-tuned on labeled data, followed by a classification step using a Convolutional Neural Network (CNN). A unique dataset of 234,259 chromosome images, including the training, validation, and test sets, was used. Marking a significant achievement in the scale of chromosomal analysis. The proposed hybrid system accurately detects structural anomalies in individual chromosome images, achieving 99.3% accuracy in classifying normal and abnormal chromosomes. We also used a structural similarity index measure and template matching to identify the part of the abnormal chromosome that differed from the normal one. This automated model has the potential to significantly contribute to the early detection and diagnosis of chromosome-related disorders that affect both genetic health and neurological behavior.

Assessment of Long-Term Cultivated In Vitro Willow Clones according to Chromosomal Analysis.

Creation and long-term in vitro maintenance of valuable genotype collection is one of the modern approach to conservation of valuable gene pool of woody plants. However, during prolonged cultivation, genetic variability of cells and tissues may accumulate and lead to the loss of valuable characteristics of parental plants. It is therefore important to assess the genetic (including cytogenetic) stability of collection clones. The purpose of this work was to study the karyotype features (number, ploidy level and chromosome size) of various willow clones (S. dasyclados Wimm., S. viminalis L., S. purpurea L., S. caspica Pall., х S. palustrisHost.) under conditions of long-term in vitro storage. No such research has been conducted on willow so far. Nevertheless, there is evidence of significant influence of ploidy level on growth, productivity, and composition of wood for willow plants. Our study was based on the plants of five micropropagated willow clones rated as promising for plantation forestry. The plants used in the study were maintained in vitro for a long period (14 years) by rare subculturing (once in 5 months) on a hormone-free1/2 WPM nutrient medium under standard cultivation conditions (25± 2°C, 16 h light/8 h dark, 2.0 klx). Throughout the entire in vitro cultivation period, the plants showed proper growth and development, high regeneration activity, and no visible signs of somaclonal variation. Willow is one of the rather difficult objects for karyotype analysis. The authors improved the method of preparation and analysis of specimens. During the long-term cultivation, the clones showed cytogenetic stability, maintaining the ploidy (2n = 2х = 38 or 2n= 4х = 76) and the mixoploid nature of the original plants. Data obtained gives an update on the sizes of chromosomes of clones with different ploidy. The absolute chromosome length for diploid clones varies from 0.8 to 2.1 μm; tetraploid, from 0.9 to 2.5 μm. There were no statistically significant differences in the average chromosome length between diploid and tetraploid clones. All studied willow clones during long-term (for 14years) in vitro cultivation on a hormone-free nutrient media retain the karyotype of the respective species.

Karyotype variation patterns and phenotypic responses of hybrid progenies of triploid loquat (Eriobotrya japonica) provide new insight into aneuploid germplasm innovation

Abstract The sexual reproduction of triploids induces chromosomal karyotype variations, which are significant for germplasm resource innovation. Most triploid plants are with low fertility. Therefore, triploid offspring karyotypes’ variation pattern and phenotypic response remain poorly understood. Here, we employed three diploids with diverse genetic distances as male parents to cross-pollinate the female fertile triploid loquat Q24 to construct three experimental populations. The chromosome numbers of 93.82% of hybrid plants were 34~46 in three hybrid populations. All 168 aneuploids with 160 karyotypes and a small percentage of euploids were detected among 178 hybrids by the improved molecular karyotype analysis method. Further analysis revealed that when being transmitted to offspring, chromosome 5 of Q24 as disomy was with the highest frequency (&amp;gt; 50%), while chromosome 12 was with the lowest frequency (≤ 30%). The frequency of Q24’s chromosomes being transmitted to offspring as disomy was influenced by the gene function on the chromosomes and the number of inter-chromosome collinear gene links. Whole genome resequencing showed that the Q24 alleles exhibited segregation distortions in the offspring aneuploid population. Transgenic experiments demonstrated that the EjRUN1 gene, which was on one segregation distortion region of Q24, promoted the seed viability of triploid Arabidopsis. Furthermore, chromosome number, dosage, and male parent genotype affected the aneuploid phenotype. These findings advance the understanding of genome genetic characteristics of triploid loquat, and provide a reference for germplasm innovation of loquat rapidly through triploid sexual reproduction.

Evaluating Chromosomal Mosaicism in Prenatal Diagnosis: The Complementary Roles of Chromosomal Microarray Analysis and Karyotyping.

To explore the impact of invitro cell subculture on prenatal diagnostic sample results and compare the efficacy of conventional karyotyping and chromosomal microarray analysis (CMA) in detecting chromosome mosaicism. We conducted a retrospective analysis of G-banding karyotyping and CMA data from 2007 amniocentesis cases to investigate chromosome mosaicism. Chromosome mosaicism was detected in 1.49% of cases (30/2007). Sex chromosome mosaicism was the most common form of mosaicism. Among the 30 mosaicisms, 18 results were consistent between the two methods. In four cases, CMA indicated mosaicism but the karyotypes were normal. In eight cases, CMA was normal while the karyotypes suggested mosaicism. CMA and karyotyping complement each other in prenatal genetic diagnosis. Combining both methods enhances detection accuracy, particularly in cases of chromosomal mosaicism, which may be missed after the subculture of adherent cells in karyotype analysis.

Clinical and genetic diagnosis of first cohort of differences of sexual development in the Iranian population.

Differences of sex development (DSD) refer to various congenital conditions affecting the urogenital and hormonal systems. Accurate diagnosis and personalized management are crucial for supporting patients through complex decisions, such as those related to gender identity. This study represents the first comprehensive investigation into DSD inIran, analyzing patient's clinical and genetic data between 1991 and 2020. Karyotype analysis was performed on 69 patients without a molecular diagnosis, with sex chromosome DSD excluded. Presence of SRY gene evaluated in all sex reversal patients. Whole exome sequencing (WES) was used for 26 undiagnosed patients, revealing pathogenic variants in WT1, NR5A1, DHX37, AR, CYP17A1, and LHCGR genes. The most common diagnosis was testicular TDSD, identified in 42 patients (60.86 %), with the SRY gene being the primary cause in 36 of these patients. The study highlights the importance of genetic analysis in identifying novel and rare gene variants, particularly within the steroid hormone and gonad differentiation pathways, for both 46, XY and 46, XX DSD. These findings emphasize the need for genetic analysis in providing personalized patient care and tailored counseling to help individuals navigate complex decisions, including those involving gender identity.

The value of increasing sequencing depth for noninvasive prenatal screening for whole chromosomal aneuploidy

To evaluate the value of increasing sequencing depths of non-invasive prenatal testing (NIPT) for fetal chromosomal aneuploidies based on the semiconductor sequencing platform. This study recruited a cohort of 59,800 singleton pregnancies from Guangdong Women and Children Hospital between January 2015 and December 2020, including 48,018 cases of NIPT and 11,782 cases of expanded NIPT. Cell-free DNA from plasma samples was sequenced at a sequencing depth of 0.15X for NIPT and 0.4X for expanded NIPT. Patients with positive NIPT results were offered fetal karyotyping or microarray analysis for confirmatory testing, and all pregnant women were followed up. A total of 892 cases were predicted as positive for chromosomal aneuploidies, including 682 cases of NIPT and 210 cases of expanded NIPT, with a positive rate of 1.42% and 1.78% (p = 0.0037), respectively. The positive predictive values (PPV) for trisomy 21/18/13 and other autosomal aneuploidies detected by NIPT were 84.80%, 69.23%, 25.00%, and 4.55%, respectively. For expanded NIPT, the positive predictive rates were 86.96%, 80.00%, 35.00%, and 8.77%, respectively. Both NIPT and expanded NIPT have the same PPV for detecting sex chromosome aneuploidies (approximately 50%). Additionally, one false-negative case was identified during the follow-up period. This was a trisomy 18 confirmed by prenatal diagnosis due to multiple ultrasound abnormalities during pregnancy. The study shows that increasing sequencing depth can significantly improve the PPV performance of aneuploidies, with a statistically significant difference, particularly in trisomy 18. The results may provide valuable guidance for clinical doctors during consultations.

Identification of a cryptic unbalanced translocation Der(22)t(12;22)(q24.33;q13.33) in a large Chinese family with Phelan-McDermid syndrome by nanopore sequencing

To explore the genetic cause of a four-generation severe intellectual disability in a Chinese family using nanopore sequencing and to provide genetic counseling and reproductive guidance for family members. Multiple genetic analyses of the proband and family members were performed, including chromosome karyotype analysis, whole exome sequencing, nanopore sequencing, PCR amplification, and Sanger sequencing. The results of G-binding karyotyping, CGG repeats for FMR1, GGC repeats for NOTCH2NCL, and trio-whole-exome sequencing were negative for the proband and his parents. Nanopore sequencing showed that the proband carried 12q24.33 microduplication (3.26 Mb) and 22q13.33 microdeletion (1.5 Mb). According to the guidelines of the American Society for Medical Genetics and Genomics (ACMG), the 22q13.33 microdeletion was classified as pathogenic, whereas the 12q24.33 microduplication was classified as a variant of uncertain significance (VUS). The precise karyotype and location of chromosomal breakpoints in the patient and family members were determined through PCR. According to the results of Sanger sequencing, a cryptic balanced translocation was detected in the proband’s father. Additionally, informative SNPs were identified near the breakpoints for preimplantation genetic testing for structure rearrangement (PGT-SR) treatment by nanopore sequencing. We identified a cryptic unbalanced translocation in a large Chinese family with Phelan-McDermid syndrome (22q13.33 deletion syndrome) by nanopore sequencing. Nanopore sequencing can be a powerful tool for the genetic diagnosis of unexplained intellectual disability and the detection of precise breakpoints of chromosomal rearrangement in PGT-SR treatment.

Chromosome-scale genome assembly of three-spotted seahorse (Hippocampus trimaculatus) with a unique karyotype

Three-spotted seahorse (Hippocampi trimaculata) is a unique fish with important economic and medicinal values, and its total chromosome number is potentially quite different from other seahorse species. Herein, we constructed a chromosome-level genome assembly for this special seahorse by integration of MGI short-read, PacBio HiFi long-read and Hi-C sequencing techniques. A 416.57-Mb haplotypic genome assembly was obtained. Subsequently, 99.38% of its scaffold sequences were anchored onto 18 chromosomes, with identification of 29.1% repeat sequences in the assembled genome. Additional karyotype analysis validated the diploid chromosomes of 2n = 36, which are remarkably different from other seahorses’ 2n = 42 or 44. The genome completeness (BUSCO score: 96.5%, CEGMA score: 97.87%) confirmed that this chromosome-scale assembly is indeed of high quality. Moreover, a total of 18,712 protein-coding genes were annotated, of which 96.36% could be predicted with functions. Based on construction of a phylogenetic tree, we estimated that Hippocampus and Syngnathoides diverged approximately 50.1 million years ago (Mya). Taken together, our genome data presented in this study provide a valuable genetic resource for numerical chromosome changes and in-depth evolutionary and functional investigations, as well as conservation and molecular breeding of this endangered teleost.

Immune Gene Expression Profiling in Individuals with Turner Syndrome, Graves' Disease, and a Healthy Female by Single-Cell RNA Sequencing: A Comparative Study.

Turner syndrome (TS) can be determined by karyotype analysis, marked by the loss of one X chromosome in females. However, the genes involved in autoimmunity in TS patients remain unclear. In this study, we aimed to analyze differences in immune gene expression between a patient with TS, a healthy female, and a female patient with Graves' disease using single-cell RNA sequencing (scRNA-seq) analysis of antigen-specific CD4(+) T cells. We identified 43 differentially expressed genes in the TS patient compared with the healthy female and the female patient with Graves' disease. Many of these genes have previously been suggested to play a role in immune system regulation. This study provides valuable insights into the differences in immune-related gene expression between TS patients, healthy individuals, and those with autoimmune diseases.

Maternal X chromosome pericentric inversion resulting in the genetic analysis of offspring pedigrees with deletions at Xp22.33 and Xp22.33p11.3, and duplications at Xq27.3q28: Case report.

This study investigates the genetic cause of primary infertility and short stature in a woman, focusing on maternal X chromosome pericentric inversion and its impact on offspring genetic outcomes, including deletions at Xp22.33 and Xp22.33p11.3, and duplications spanning Xq27.3 to the distal end of the X chromosome's long arm. The proband presented with primary infertility, menstrual irregularities, and ultrasound findings indicating a small uterus. Peripheral blood G-banded karyotype analysis and single nucleotide polymorphism array analysis revealed a 46,X,rec(X)dup(Xq)inv(X)(p11.3q27)dmat karyotype in the proband, inherited from her mother. Genetic testing identified pathogenic deletions at Xp22.33 and Xp22.33p11.3, and a pathogenic duplication at Xq27.3q28. Genetic counseling and pedigree analysis were conducted to trace the maternal origin of the pericentric inversion and assess recurrence risks. The study confirmed the maternal X chromosome pericentric inversion caused the observed genetic abnormalities, with a 50% recurrence risk for X-linked inheritance. Maternal X chromosome pericentric inversion significantly affects offspring genetic outcomes. Assisted reproductive technologies, including in vitro fertilization with preimplantation genetic testing, are recommended to reduce recurrence risks in future pregnancies. Prenatal genetic testing is advised for natural conception to ensure fetal genetic health.

An investigation of a hemophilia A female with heterozygous intron 22 inversion and skewed X chromosome inactivation.

Hemophilia A (HA) is an X-linked recessive inherited bleeding disorder that typically affects men. Women are usually asymptomatic carriers, and rarely presenting with severe or moderately severe phenotype. This study aims to describe a case of a 17-year-old girl with moderate HA, investigating the mechanisms of her condition and the genetic basis within her family. We conducted coagulation tests and bleeding assessments to evaluate her bleeding phenotype. Molecular genetic examinations, karyotype analysis, X-chromosome inactivation testing, and targeted bioinformatic analysis were used to identify potential genetic etiologies. The proband exhibited a severe bleeding phenotype and was found to be a heterozygous carrier of an intron 22 inversion (Inv22) with a normal chromosomal karyotype. No other hemostatic defects were identified through whole exome sequencing. The proband's mother and monozygotic twin sister are also Inv22 carriers, yet remain asymptomatic with normal FVIII activity. X-chromosome inactivation experiments revealed unbalanced inactivation in the proband, leading to the silencing of the healthy X copy. Notably, several novel X-linked gene mutations (SHROOM2, RPGR, VCX3B, GAGE, GCNA, ZNF280C, CT45A, and XK) were identified in the proband compared to her monozygotic twin sister, though their impact on X-chromosome inactivation remains unclear. Our findings suggest that the proband's bleeding phenotype results from unbalanced X-chromosome inactivation. This research marks the first analysis of X chromosome-related gene mutations among monozygotic twins who are carriers of hemophilia A, laying the groundwork for further investigations into the disorder's pathogenesis in women and highlighting the complexities in genetic counseling.