Aneuploidy effects on human gene expression across three cell types.

Aneuploidy syndromes impact multiple organ systems but understanding of tissue-specific aneuploidy effects remains limited-especially for the comparison between peripheral tissues and relatively inaccessible tissues like brain. Here, we address this gap in knowledge by studying the transcriptomic effects of chromosome X, Y, and 21 aneuploidies in lymphoblastoid cell lines, fibroblasts and iPSC-derived neuronal cells (LCLs, FCL, and iNs, respectively). We root our analyses in sex chromosome aneuploidies, which offer a uniquely wide karyotype range for dosage effect analysis. We first harness a large LCL RNA-seq dataset from 197 individuals with one of 6 sex chromosome dosages (SCDs: XX, XXX, XY, XXY, XYY, and XXYY) to i) validate theoretical models of SCD sensitivity and ii) define an expanded set of 41 genes that show obligate dosage sensitivity to SCD and are all in cis (i.e., reside on the X or Y chromosome). We then use multiple complementary analyses to show that cis effects of SCD in LCLs are preserved in both FCLs (n = 32) and iNs (n = 24), whereas trans effects (i.e., those on autosomal gene expression) are mostly not preserved. Analysis of additional datasets confirms that the greater cross-cell type reproducibility of cis vs. trans effects is also seen in trisomy 21 cell lines. These findings i) expand our understanding of X, Y, and 21 chromosome dosage effects on human gene expression and ii) suggest that LCLs may provide a good model system for understanding cis effects of aneuploidy in harder-to-access cell types.

Paternal USP26 mutations raise Klinefelter syndrome risk in the offspring of mice and humans.

Current understanding holds that Klinefelter syndrome (KS) is not inherited, but arises randomly during meiosis. Whether there is any genetic basis for the origin of KS is unknown. Here, guided by our identification of some USP26 variations apparently associated with KS, we found that knockout of Usp26 in male mice resulted in the production of 41, XXY offspring. USP26 protein is localized at the XY body, and the disruption of Usp26 causes incomplete sex chromosome pairing by destabilizing TEX11. The unpaired sex chromosomes then result in XY aneuploid spermatozoa. Consistent with our mouse results, a clinical study shows that some USP26 variations increase the proportion of XY aneuploid spermatozoa in fertile men, and we identified two families with KS offspring wherein the father of the KS patient harbored a USP26-mutated haplotype, further supporting that paternal USP26 mutation can cause KS offspring production. Thus, some KS should originate from XY spermatozoa, and paternal USP26 mutations increase the risk of producing KS offspring.

Recent advances in mechanisms ensuring the pairing, synapsis and segregation of XY chromosomes in mice and humans.

Sex chromosome aneuploidies are among the most common variations in human whole chromosome copy numbers, with an estimated prevalence in the general population of 1:400 to 1:1400 live births. Unlike whole-chromosome aneuploidies of autosomes, those of sex chromosomes, such as the 47, XXY aneuploidy that causes Klinefelter Syndrome (KS), often originate from the paternal side, caused by a lack of crossover (CO) formation between the X and Y chromosomes. COs must form between all chromosome pairs to pass meiotic checkpoints and are the product of meiotic recombination that occurs between homologous sequences of parental chromosomes. Recombination between male sex chromosomes is more challenging compared to both autosomes and sex chromosomes in females, as it is restricted within a short region of homology between X and Y, called the pseudo-autosomal region (PAR). However, in normal individuals, CO formation occurs in PAR with a higher frequency than in any other region, indicating the presence of mechanisms that promote the initiation and processing of recombination in each meiotic division. In recent years, research has made great strides in identifying genes and mechanisms that facilitate CO formation in the PAR. Here, we outline the most recent and relevant findings in this field. XY chromosome aneuploidy in humans has broad-reaching effects, contributing significantly also to Turner syndrome, spontaneous abortions, oligospermia, and even infertility. Thus, in the years to come, the identification of genes and mechanisms beyond XY aneuploidy is expected to have an impact on the genetic counseling of a wide number of families and adults affected by these disorders.

Inefficient Sox9 upregulation and absence of Rspo1 repression lead to sex reversal in the B6.XYTIR mouse gonad†.

Sry on the Y-chromosome upregulates Sox9, which in turn upregulates a set of genes such as Fgf9 to initiate testicular differentiation in the XY gonad. In the absence of Sry expression, genes such as Rspo1, Foxl2, and Runx1 support ovarian differentiation in the XX gonad. These two pathways antagonize each other to ensure the development of only one gonadal sex in normal development. In the B6.YTIR mouse, carrying the YTIR-chromosome on the B6 genetic background, Sry is expressed in a comparable manner with that in the B6.XY mouse, yet, only ovaries or ovotestes develop. We asked how testicular and ovarian differentiation pathways interact to determine the gonadal sex in the B6.YTIR mouse. Our results showed that (1) transcript levels of Sox9 were much lower than in B6.XY gonads while those of Rspo1 and Runx1 were as high as B6.XX gonads at 11.5 and 12.5 days postcoitum. (2) FOXL2-positive cells appeared in mosaic with SOX9-positive cells at 12.5 days postcoitum. (3) SOX9-positive cells formed testis cords in the central area while those disappeared to leave only FOXL2-positive cells in the poles or the entire area at 13.5 days postcoitum. (4) No difference was found at transcript levels of all genes between the left and right gonads up to 12.5 days postcoitum, although ovotestes developed much more frequently on the left than the right at 13.5 days postcoitum. These results suggest that inefficient Sox9 upregulation and the absence of Rspo1 repression prevent testicular differentiation in the B6.YTIR gonad.

Visualizing histone H4K20me1 in knock-in mice expressing the mCherry-tagged modification-specific intracellular antibody.

During development and differentiation, histone modifications dynamically change locally and globally, associated with transcriptional regulation, DNA replication and repair, and chromosome condensation. The level of histone H4 Lys20 monomethylation (H4K20me1) increases during the G2 to M phases of the cell cycle and is enriched in facultative heterochromatin, such as inactive X chromosomes in cycling cells. To track the dynamic changes of H4K20me1 in living cells, we have developed a genetically encoded modification-specific intracellular antibody (mintbody) probe that specifically binds to the modification. Here, we report the generation of knock-in mice in which the coding sequence of the mCherry-tagged version of the H4K20me1-mintbody is inserted into the Rosa26 locus. The knock-in mice, which ubiquitously expressed the H4K20me1-mintbody, developed normally and were fertile, indicating that the expression of the probe does not disturb the cell growth, development, or differentiation. Various tissues isolated from the knock-in mice exhibited nuclear fluorescence without the need for fixation. The H4K20me1-mintbody was enriched in inactive X chromosomes in developing embryos and in XY bodies during spermatogenesis. The knock-in mice will be useful for the histochemical analysis of H4K20me1 in any cell types.

Incidence, prevalence, age at diagnosis, and mortality in individuals with 45,X/46,XY mosaicism: A population-based registry study.

PURPOSE: To assess the population-based incidence, prevalence, and age at diagnosis of individuals with 45,X/46,XY mosaicism (and associated variants) and describe the associated mortality pattern. In addition, a systematic literature review of papers providing prevalence data of 45,X/46,XY mosaicism was performed. METHODS: A population-based epidemiological study of all individuals diagnosed with 45,X/46,XY mosaicism between 1960 and 2019. Mortality was analyzed using data from the Danish Causes of Death Register. One-hundred randomly age- and sex-matched general population controls per case were identified for comparison. RESULTS: One-hundred-thirty-seven males and 46 females with 45,X/46,XY mosaicism were identified. The apparent prevalence was 5.6 per 100,000 liveborn males and 2.1 per 100,000 liveborn females. The incidence of males with 45,X/46,XY increased during the study (P > .0001) but was stable for females (P = .4). Males were significantly older than females when diagnosed (median age = 29.1, interquartile range: 3.4-41.3) years versus 13.3 (interquartile range: 2.1-19.1) years, P = .002). All-cause mortality was doubled in males with 45,X/46,XY (Hazard Ratio = 2.0, 95% confidence interval: 1.2-3.3) and quadrupled in females (Hazard Ratio = 4.0, confidence interval: 2.0-7.9). CONCLUSION: The apparent population-based prevalence of males and females with 45,X/46,XY is 5.6 and 2.1 per 100,000 liveborn males and females, respectively. Diagnosis of males with 45,X/46,XY males is increasing. 45,X/46,XY mosaicism is associated with an increased all-cause mortality.

A case report of a normal fertile woman with 46,XX/46,XY somatic chimerism reveals a critical role for germ cells in sex determination.

Individuals with 46,XX/XY chimerism can display a wide range of characteristics, varying from hermaphroditism to complete male or female, and can display sex chromosome chimerism in multiple tissues, including the gonads. The gonadal tissues of females contain both granulosa and germ cells. However, the specific sex chromosome composition of the granulosa and germ cells in 46,XX/XY chimeric female is currently unknown. Here, we reported a 30-year-old woman with secondary infertility who displayed a 46,XX/46,XY chimerism in the peripheral blood. FISH testing revealed varying degrees of XX/XY chimerism in multiple tissues of the female patient. Subsequently, the patient underwent preimplantation genetic testing (PGT) treatment, and 26 oocytes were retrieved. From the twenty-four biopsied mature oocytes, a total of 23 first polar bodies (PBs) and 10 second PBs were obtained. These PBs and two immature metaphase I (MI) oocytes only displayed X chromosome signals with no presence of the Y, suggesting that all oocytes in this chimeric female were of XX germ cell origin. On the other hand, granulosa cells obtained from individual follicles exhibited varied proportions of XX/XY cell types, and six follicles possessed 100% XX or XY granulosa cells. A total of 24 oocytes were successfully fertilized, and 12 developed into blastocysts, where 5 being XY and 5 were XX. Two blastocysts were transferred with one originating from an oocyte aspirated from a follicle containing 100% XY granulosa cells. This resulted in a twin pregnancy. Subsequent prenatal diagnosis confirmed normal male and female karyotypes. Ultimately, healthy boy-girl twins were delivered at full term. In summary, this 46,XX/XY chimerism with XX germ cells presented complete female, suggesting that germ cells may exert a significant influence on the sexual determination of an individual, which provide valuable insights into the intricate processes associated with sexual development and reproduction.

Genetic sex determination in three closely related hydrothermal vent gastropods, including one species with intersex individuals.

Molluscs have undergone many transitions between separate sexes and hermaphroditism, which is of interest in studying the evolution of sex determination and differentiation. Here, we combined multi-locus genotypes obtained from restriction site-associated DNA (RAD) sequencing with anatomical observations of the gonads of three deep-sea hydrothermal vent gastropods of the genus Alviniconcha living in the southwest Pacific. We found that all three species (Alviniconcha boucheti, Alviniconcha strummeri, and Alviniconcha kojimai) share the same male-heterogametic XY sex-determination system but that the gonads of XX A. kojimai individuals are invaded by a variable proportion of male reproductive tissue. The identification of Y-specific RAD loci (found only in A. boucheti) and the phylogenetic analysis of three sex-linked loci shared by all species suggested that X-Y recombination has evolved differently within each species. This situation of three species showing variation in gonadal development around a common sex-determination system provides new insights into the reproductive mode of poorly known deep-sea species and opens up an opportunity to study the evolution of recombination suppression on sex chromosomes and its association with mixed or transitory sexual systems.

Unusual sex chromosomal DSD in a domestic Shorthair cat with a 37,X/38,XY mosaic karyotype.

BACKGROUND: Sex chromosome abnormalities associated with disorders of sexual development (DSD) are rarely described in cats, mainly due to the lack of chromossome studies that precisely reveal the condition. Genetic approaches are therefore required in order to detect sex chromossomes abnormalities as variations in the number and structure of chromosomes, or the presence of a second cell line as mosaicim or chimerism. CASE PRESENTATION: A male Shorthair cryptorchid cat was presented with clinical signs of anorexia, tenesmus and hyperthermia. Ultrasonography revealed a fluid-filled structure, with approximately 1 cm in diameter, adjacent to the descending colon. Computed tomography evidenced a tubular structure, ventral to the descending colon and caudal to the bladder, which extended cranially, through two branches. Histopathological evaluation confirmed the presence of two atrophic uterine horns and one hypoplastic testicle with epididymis at the end of one of the uterine horns. The end of the other uterine horn was attached to a structure composed by a mass of adipocytes. Cytogenetic analysis revealed a mosaic 37,X/38,XY karyotype. The two cell lines were found in 15% and 85% of the lymphocytes, respectively. Genetic analysis confirmed the presence of SRY and ZFY genes in blood and hair bulbs, and revealed a marked reduction in SRY expression in the testicle. Additionally, this case presented exceptionally rare features, such as a Leydig' cell tumour and a chronic endometritis in both uterine horns. CONCLUSIONS: Complete imaging workup, cytogenetic analysis and SRY gene expression should be systematically realized, in order to properly classify disorders of sexual development (DSD) in cats.

The RNA-binding protein FUS/TLS interacts with SPO11 and PRDM9 and localize at meiotic recombination hotspots.

In mammals, meiotic recombination is initiated by the introduction of DNA double strand breaks (DSBs) into narrow segments of the genome, defined as hotspots, which is carried out by the SPO11/TOPOVIBL complex. A major player in the specification of hotspots is PRDM9, a histone methyltransferase that, following sequence-specific DNA binding, generates trimethylation on lysine 4 (H3K4me3) and lysine 36 (H3K36me3) of histone H3, thus defining the hotspots. PRDM9 activity is key to successful meiosis, since in its absence DSBs are redirected to functional sites and synapsis between homologous chromosomes fails. One protein factor recently implicated in guiding PRDM9 activity at hotspots is EWS, a member of the FET family of proteins that also includes TAF15 and FUS/TLS. Here, we demonstrate that FUS/TLS partially colocalizes with PRDM9 on the meiotic chromosome axes, marked by the synaptonemal complex component SYCP3, and physically interacts with PRDM9. Furthermore, we show that FUS/TLS also interacts with REC114, one of the axis-bound SPO11-auxiliary factors essential for DSB formation. This finding suggests that FUS/TLS is a component of the protein complex that promotes the initiation of meiotic recombination. Accordingly, we document that FUS/TLS coimmunoprecipitates with SPO11 in vitro and in vivo. The interaction occurs with both SPO11ß and SPO11α splice isoforms, which are believed to play distinct functions in the formation of DSBs in autosomes and male sex chromosomes, respectively. Finally, using chromatin immunoprecipitation experiments, we show that FUS/TLS is localized at H3K4me3-marked hotspots in autosomes and in the pseudo-autosomal region, the site of genetic exchange between the XY chromosomes.

Optimization of the synthesis of BET BD2 selective inhibitor XY153.

XY153 is a promising BET BD2 inhibitor with an IC50 value of 0.79 nM against BRD4 BD2. It shows 354-fold selectivity over BRD4-BD1 and 6-fold selectivity over other BET BD2 domains. However, the reported synthesis route of XY153 and its derivatives are extremely poor-yielding. After the synthesis of three key fragments, XY153 can only be obtained with a yield of 1.3 % in the original four-step reaction. In this study, we reported a three-step alternative route in the synthesis process of XY153. The reaction conditions for this route were thoroughly investigated and optimized, resulting in a significantly improved yield of 61.5 %. This efficient synthesis route establishes a robust chemical foundation for the rapid synthesis of XY153 derivatives as BET BD2 inhibitors in the near future.

Monte Carlo Based Techniques for Quantum Magnets with Long-Range Interactions.

Long-range interactions are relevant for a large variety of quantum systems in quantum optics and condensed matter physics. In particular, the control of quantum-optical platforms promises to gain deep insights into quantum-critical properties induced by the long-range nature of interactions. From a theoretical perspective, long-range interactions are notoriously complicated to treat. Here, we give an overview of recent advancements to investigate quantum magnets with long-range interactions focusing on two techniques based on Monte Carlo integration. First, the method of perturbative continuous unitary transformations where classical Monte Carlo integration is applied within the embedding scheme of white graphs. This linked-cluster expansion allows extracting high-order series expansions of energies and observables in the thermodynamic limit. Second, stochastic series expansion quantum Monte Carlo integration enables calculations on large finite systems. Finite-size scaling can then be used to determine the physical properties of the infinite system. In recent years, both techniques have been applied successfully to one- and two-dimensional quantum magnets involving long-range Ising, XY, and Heisenberg interactions on various bipartite and non-bipartite lattices. Here, we summarise the obtained quantum-critical properties including critical exponents for all these systems in a coherent way. Further, we review how long-range interactions are used to study quantum phase transitions above the upper critical dimension and the scaling techniques to extract these quantum critical properties from the numerical calculations.

Evolutionary dynamics of sex-biased gene expression in a young XY system: insights from the brown alga genus Fucus.

Sex-biased gene expression is considered to be an underlying cause of sexually dimorphic traits. Although the nature and degree of sex-biased expression have been well documented in several animal and plant systems, far less is known about the evolution of sex-biased genes in more distant eukaryotic groups. Here, we investigate sex-biased gene expression in two brown algal dioecious species, Fucus serratus and Fucus vesiculosus, where male heterogamety (XX/XY) has recently emerged. We find that in contrast to evolutionary distant plant and animal lineages, male-biased genes do not experience high turnover rates, but instead reveal remarkable conservation of bias and expression levels between the two species, suggesting their importance in sexual differentiation. Genes with consistent male bias were enriched in functions related to gamete production, along with sperm competition and include three flagellar proteins under positive selection. We present one of the first reports, outside of the animal kingdom, showing that male-biased genes display accelerated rates of coding sequence evolution compared with female-biased or unbiased genes. Our results imply that evolutionary forces affect male and female sex-biased genes differently on structural and regulatory levels, resulting in unique properties of differentially expressed transcripts during reproductive development in Fucus algae.

Molecular parallelism in the evolution of a master sex-determining role for the anti-Mullerian hormone receptor 2 gene (amhr2) in Midas cichlids.

The evolution of sex chromosomes and their differentiation from autosomes is a major event during genome evolution that happened many times in several lineages. The repeated evolution and lability of sex-determination mechanisms in fishes makes this a well-suited system to test for general patterns in evolution. According to current theory, differentiation is triggered by the suppression of recombination following the evolution of a new master sex-determining gene. However, the molecular mechanisms that establish recombination suppression are known from few examples, owing to the intrinsic difficulties of assembling sex-determining regions (SDRs). The development of forward-genetics and long-read sequencing have generated a wealth of data questioning central aspects of the current theory. Here, we demonstrate that sex in Midas cichlids is determined by an XY system, and identify and assemble the SDR by combining forward-genetics, long-read sequencing and optical mapping. We show how long-reads aid in the detection of artefacts in genotype-phenotype mapping that arise from incomplete genome assemblies. The male-specific region is restricted to a 100-kb segment on chromosome 4 that harbours transposable elements and a Y-specific duplicate of the anti-Mullerian receptor 2 gene, which has evolved master sex-determining functions repeatedly. Our data suggest that amhr2Y originated by an interchromosomal translocation from chromosome 20 to 4 pre-dating the split of Midas and Flier cichlids. In the latter, it is pseudogenized and translocated to another chromosome. Duplication of anti-Mullerian genes is a common route to establishing new sex determiners, highlighting the role of molecular parallelism in the evolution of sex determination.

Undetectable anti-Mullerian hormone and inhibin B do not preclude the presence of germ cell tumours in 45,X/46,XY or 46,XY gonadal dysgenesis.

OBJECTIVE: Individuals with 45,X/46,XY or 46,XY gonadal dysgenesis are at increased risk of germ cell malignancies. Therefore, prophylactic bilateral gonadectomy is advised in girls and considered in boys with atypical genitalia for undescended, macroscopically abnormal gonads. However, severely dysgenetic gonads may not contain germ cells rendering gonadectomy unnecessary. Therefore, we investigate if undetectable preoperative serum anti-Müllerian hormone (AMH) and inhibin B can predict the absence of germ cells, (pre)malignant or otherwise. DESIGN, PATIENTS AND MEASUREMENTS: Individuals who had undergone bilateral gonadal biopsy and/or gonadectomy because of suspected gonadal dysgenesis in 1999-2019 were included in this retrospective study if preoperative AMH and/or inhibin B were available. Histological material was reviewed by an experienced pathologist. Haematoxylin and eosin and immunohistochemical stainings for SOX9, OCT4, TSPY and SCF (KITL) were used. RESULTS: Thirteen males and 16 females were included, 20 with 46,XY and 9 with 45,X/46,XY DSD. Three females had dysgerminoma alongside gonadoblastoma; two gonadoblastoma, one germ cell neoplasia in situ (GCNIS) and three males had pre-GCNIS and/or pre-gonadoblastoma. Gonadoblastoma and/or dysgerminoma were present in 3/11 individuals with undetectable AMH and inhibin B, one of whom also had non-(pre)malignant germ cells. Of the other 18, in whom AMH and/or inhibin B were detectable, only one had no germ cells. CONCLUSIONS: Undetectable serum AMH and inhibin B cannot reliably predict the absence of germ cells and germ cell tumours in individuals with 45,X/46,XY or 46,XY gonadal dysgenesis. This information should help in counselling about prophylactic gonadectomy, taking into account both the germ cell cancer risk and potential for gonadal function.

Comparison of Antiaromatic Properties in a Series of Structurally Isomeric Naphthothiophene-Fused s-Indacenes.

Fusion of aromatic subunits to stabilize an antiaromatic core allows the isolation and study of otherwise unstable paratropic systems. A complete study of a series of six naphthothiophene-fused s-indacene isomers is herein described. Additionally, the structural modifications resulted in increased π-π overlap in the solid state, which was further explored through changing the sterically blocking mesityl group to (triisopropylsilyl)ethynyl in three derivatives. The computed antiaromaticity of the six isomers is compared to the observed physical properties, such as NMR chemical shift, UV-vis, and CV data. We find that the calculations predict the most antiaromatic isomer and give a general estimation of the relative degree of paratropicity for the remaining isomers, when compared to the experimental results.

Additional evidence for the role of chromosomal imbalances and SOX8, ZNRF3 and HHAT gene variants in early human testis development.

BACKGROUND: Forty-six ,XY Differences/Disorders of Sex Development (DSD) are characterized by a broad phenotypic spectrum ranging from typical female to male with undervirilized external genitalia, or more rarely testicular regression with a typical male phenotype. Despite progress in the genetic diagnosis of DSD, most 46,XY DSD cases remain idiopathic. METHODS: To determine the genetic causes of 46,XY DSD, we studied 165 patients of Tunisian ancestry, who presented a wide range of DSD phenotypes. Karyotyping, candidate gene sequencing, and whole-exome sequencing (WES) were performed. RESULTS: Cytogenetic abnormalities, including a high frequency of sex chromosomal anomalies (85.4%), explained the phenotype in 30.9% (51/165) of the cohort. Sanger sequencing of candidate genes identified a novel pathogenic variant in the SRY gene in a patient with 46,XY gonadal dysgenesis. An exome screen of a sub-group of 44 patients with 46,XY DSD revealed pathogenic or likely pathogenic variants in 38.6% (17/44) of patients. CONCLUSION: Rare or novel pathogenic variants were identified in the AR, SRD5A2, ZNRF3, SOX8, SOX9 and HHAT genes. Overall our data indicate a genetic diagnosis rate of 41.2% (68/165) in the group of 46,XY DSD.

MRNIP interacts with sex body chromatin to support meiotic progression, spermatogenesis, and male fertility in mice.

Meiosis has a principal role in sexual reproduction to generate haploid gametes in both sexes. During meiosis, the cell nucleus hosts a dynamic environment where some genes are transcriptionally activated, and some are inactivated at the same time. This becomes possible through subnuclear compartmentalization. The sex body, sequestering X and Y chromosomes during male meiosis and creating an environment for the meiotic sex chromosome inactivation (MSCI) is one of the best known and studied subnuclear compartments. Herein, we show that MRNIP forms droplet-like accumulations that fuse together to create a distinct subnuclear compartment that partially overlaps with the sex body chromatin during diplotene. We demonstrate that Mrnip-/- spermatocytes have impaired DNA double-strand break (DSB) repair, they display reduced sex body formation and defective MSCI. We show that Mrnip-/- undergoes critical meiocyte loss at the diplotene stage. Furthermore, we determine that DNA DSBs (induced by SPO11) and synapsis initiation (facilitated by SYCP1) precede Mrnip expression in testes. Altogether, our findings indicate that in addition to an emerging role in DNA DSB repair, MRNIP has an essential function in spermatogenesis during meiosis I by forming drop-like accumulations interacting with the sex body.

New observations on minifascicular neuropathy with sex-dependent gonadal dysgenesis: a case series with nerve ultrasound assessment.

BACKGROUND: Gonadal dysgenesis with minifascicular neuropathy (GDMN) is a rare autosomal recessive condition associated with biallelic DHH pathogenic variants. In 46, XY individuals, this disorder is characterized by an association of minifascicular neuropathy (MFN) and gonadal dysgenesis, while in 46, XX subjects only the neuropathic phenotype is present. Very few patients with GDMN have been reported so far. We describe four patients with MFN due to a novel DHH likely pathogenic homozygous variant and the results of nerve ultrasound assessment. METHODS: This retrospective observational study included 4 individuals from 2 unrelated Brazilian families evaluated for severe peripheral neuropathy. Genetic diagnosis was performed with a peripheral neuropathy next-generation sequencing (NGS) panel based on whole exome sequencing focused analysis that included a control SRY probe to confirm genetic sex. Clinical characterization, nerve conduction velocity studies, and high-resolution ultrasound nerve evaluation were performed in all subjects. RESULTS: Molecular analysis disclosed in all subjects the homozygous DHH variant p.(Leu335Pro). Patients had a striking phenotype, with marked trophic changes of extremities, sensory ataxia, and distal anesthesia due to a sensory-motor demyelinating polyneuropathy. One 46, XY phenotypically female individual had gonadal dysgenesis. High-resolution nerve ultrasound showed typical minifascicular formation and increased nerve area in at least one of the nerves assessed in all patients. CONCLUSION: Gonadal dysgenesis with minifascicular neuropathy is a severe autosomal recessive neuropathy characterized by trophic alterations in limbs, sensory ataxia, and distal anesthesia. Nerve ultrasound studies are very suggestive of this condition and may help to avoid invasive nerve biopsies.

Biochemical Features of X or Y Chromosome-Bearing Spermatozoa for Sperm Sexing.

This review presents information on biochemical features of spermatozoa bearing X or Y chromosome, enabling production of a sperm fraction with pre-defined sex chromosome. The almost only technology currently used for such separation (called sexing) is based on the fluorescence-activated cell sorting of sperm depending on DNA content. In addition to the applied aspects, this technology made it possible to analyze properties of the isolated populations of spermatozoa bearing X or Y chromosome. In recent years, existence of the differences between these populations at the transcriptome and proteome level have been reported in a number of studies. It is noteworthy that these differences are primarily related to the energy metabolism and flagellar structural proteins. New methods of sperm enrichment with X or Y chromosome cells are based on the differences in motility between the spermatozoa with different sex chromosomes. Sperm sexing is a part of the widespread protocol of artificial insemination of cows with cryopreserved semen, it allows to increase proportion of the offspring with the required sex. In addition, advances in the separation of X and Y spermatozoa may allow this approach to be applied in clinical practice to avoid sex-linked diseases.