Cas9-directed long-read sequencing to resolve optical genome mapping findings in leukemia diagnostics.

Leukemias are genetically heterogeneous and diagnostics therefore includes various standard-of-care (SOC) techniques, including karyotyping, SNP-array and FISH. Optical genome mapping (OGM) may replace these as it detects different types of structural aberrations simultaneously and additionally detects much smaller aberrations (500 bp vs 5-10 Mb with karyotyping). However, its resolution may still be too low to define clinical relevance of aberrations when they are located between two OGM labels or when labels are not distinct enough. Here, we test the potential of Cas9-directed long-read sequencing (LRS) as an additional technique to resolve such potentially relevant new findings. From an internal Bionano implementation study we selected ten OGM calls that could not be validated with SOC methods. Per variant we designed crRNAs for Cas9 enrichment, prepared libraries and sequenced them on a MinION/GridION device. We could confirm all aberrations and, importantly, the actual breakpoints of the OGM calls were located between 0.2 and 5.5 kb of the OGM-estimated breakpoints, confirming the high reliability of OGM. Furthermore, we show examples of redefinition of aberrations between labels that enable judgment of clinical relevance. Our results suggest that Cas9-directed LRS can be a relevant and flexible secondary technique in diagnostic workflows including OGM.

Hyperkeratotic hand eczema: Eczema or not?

BACKGROUND: Hyperkeratotic hand eczema (HHE) is a typical clinical hand eczema subtype with a largely unknown pathophysiology. OBJECTIVE: To investigate histopathology, expression of keratins (K), epidermal barrier proteins, and adhesion molecules in HHE. METHODS: Palmar skin biopsies (lesional and perilesional) were obtained from seven HHE patients and two healthy controls. Moreover, 135 candidate genes associated with palmoplantar keratoderma were screened for mutations. RESULTS: Immunofluorescence staining showed a significant reduction of K9 and K14 in lesional skin. Upregulation was found for K5, K6, K16, and K17 in lesional skin compared with perilesional and healthy palmar skin. Further, upregulation of involucrin and alternating loricrin staining, both in an extracellular staining pattern, was found. Filaggrin expression was similar in lesional, perilesional, and control skin. No monogenetic mutations were found. CONCLUSION: Currently, the phenotype of HHE is included in the hand eczema classification system; however, it can be argued whether this is justified. The evident expression of filaggrin and involucrin in lesional skin does not support a pathogenesis of atopic eczema. The upregulation of K6, K16, and K17 and reduction of K9 and K14 might contribute to the underlying pathogenesis. Unfortunately, comparison with hand eczema studies is not possible yet, because similar protein expression studies are lacking.

Improving the diagnostic yield of exome- sequencing by predicting gene-phenotype associations using large-scale gene expression analysis.

The diagnostic yield of exome and genome sequencing remains low (8-70%), due to incomplete knowledge on the genes that cause disease. To improve this, we use RNA-seq data from 31,499 samples to predict which genes cause specific disease phenotypes, and develop GeneNetwork Assisted Diagnostic Optimization (GADO). We show that this unbiased method, which does not rely upon specific knowledge on individual genes, is effective in both identifying previously unknown disease gene associations, and flagging genes that have previously been incorrectly implicated in disease. GADO can be run on www.genenetwork.nl by supplying HPO-terms and a list of genes that contain candidate variants. Finally, applying GADO to a cohort of 61 patients for whom exome-sequencing analysis had not resulted in a genetic diagnosis, yields likely causative genes for ten cases.

Preantral follicular atresia occurs mainly through autophagy, while antral follicles degenerate mostly through apoptosis.

There is a general agreement that granulosa cell apoptosis is the cause of antral follicle attrition. Less clear is whether this pathway is also activated in case of preantral follicle degeneration, as several reports mention that the incidence of granulosa cell apoptosis in preantral follicles is negligible. Our objective is therefore to determine which cell-death pathways are involved in preantral and antral follicular degeneration.Atretic preantal and antral follicles were investigated using immunohistochemistry and laser-capture microdissection followed by quantitative real-time reverse transcription polymerase chain reaction. Microtubule-associated light-chain protein 3 (LC3), sequestosome 1 (SQSTM1/P62), Beclin1, autophagy-related protein 7 (ATG7), and cleaved caspase 3 (cCASP3) were used as markers for autophagy and apoptosis, respectively. P62 immunostaining was far less intense in granulosa cells of atretic compared to healthy preantral follicles, while no difference in LC3 and BECLIN1 immunostaining intensity was observed. This difference in P62 immunostaining was not observed in atretic antral follicles. mRNA levels of LC3 and P62 were not different between healthy and atretic (pre)antral follicles. ATG7 immunostaining was observed in granulosa cells of preantral atretic follicles, not in granulosa cells of degenerating antral follicles. The number of cCASP3-positive cells was negligible in preantral atretic follicles, while numerous in atretic antral follicles. Taken together, we conclude that preantral and antral follicular atresia is the result of activation of different cell-death pathways as antral follicular degeneration is initiated by massive granulosa cell apoptosis, while preantral follicular atresia occurs mainly via enhanced granulosa cell autophagy.

Distinct prophase arrest mechanisms in human male meiosis.

To prevent chromosomal aberrations being transmitted to the offspring, strict meiotic checkpoints are in place to remove aberrant spermatocytes. However, in about 1% of males these checkpoints cause complete meiotic arrest leading to azoospermia and subsequent infertility. Here, we unravel two clearly distinct meiotic arrest mechanisms that occur during prophase of human male meiosis. Type I arrested spermatocytes display severe asynapsis of the homologous chromosomes, disturbed XY-body formation and increased expression of the Y chromosome-encoded gene ZFY and seem to activate a DNA damage pathway leading to induction of p63, possibly causing spermatocyte apoptosis. Type II arrested spermatocytes display normal chromosome synapsis, normal XY-body morphology and meiotic crossover formation but have a lowered expression of several cell cycle regulating genes and fail to silence the X chromosome-encoded gene ZFX Discovery and understanding of these meiotic arrest mechanisms increases our knowledge of how genomic stability is guarded during human germ cell development.

Unraveling transcriptome dynamics in human spermatogenesis.

Spermatogenesis is a dynamic developmental process that includes stem cell proliferation and differentiation, meiotic cell divisions and extreme chromatin condensation. Although studied in mice, the molecular control of human spermatogenesis is largely unknown. Here, we developed a protocol that enables next-generation sequencing of RNA obtained from pools of 500 individually laser-capture microdissected cells of specific germ cell subtypes from fixed human testis samples. Transcriptomic analyses of these successive germ cell subtypes reveals dynamic transcription of over 4000 genes during human spermatogenesis. At the same time, many of the genes encoding for well-established meiotic and post-meiotic proteins are already present in the pre-meiotic phase. Furthermore, we found significant cell type-specific expression of post-transcriptional regulators, including expression of 110 RNA-binding proteins and 137 long non-coding RNAs, most of them previously not linked to spermatogenesis. Together, these data suggest that the transcriptome of precursor cells already contains the genes necessary for cellular differentiation and that timely translation controlled by post-transcriptional regulators is crucial for normal development. These established transcriptomes provide a reference catalog for further detailed studies on human spermatogenesis and spermatogenic failure.

Spermatogonial stem cell autotransplantation and germline genomic editing: a future cure for spermatogenic failure and prevention of transmission of genomic diseases.

BACKGROUND: Subfertility affects approximately 15% of all couples, and a severe male factor is identified in 17% of these couples. While the etiology of a severe male factor remains largely unknown, prior gonadotoxic treatment and genomic aberrations have been associated with this type of subfertility. Couples with a severe male factor can resort to ICSI, with either ejaculated spermatozoa (in case of oligozoospermia) or surgically retrieved testicular spermatozoa (in case of azoospermia) to generate their own biological children. Currently there is no direct treatment for azoospermia or oligozoospermia. Spermatogonial stem cell (SSC) autotransplantation (SSCT) is a promising novel clinical application currently under development to restore fertility in sterile childhood cancer survivors. Meanwhile, recent advances in genomic editing, especially the clustered regulatory interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) system, are likely to enable genomic rectification of human SSCs in the near future. OBJECTIVE AND RATIONALE: The objective of this review is to provide insights into the prospects of the potential clinical application of SSCT with or without genomic editing to cure spermatogenic failure and to prevent transmission of genetic diseases. SEARCH METHODS: We performed a narrative review using the literature available on PubMed not restricted to any publishing year on topics of subfertility, fertility treatments, (molecular regulation of) spermatogenesis and SSCT, inherited (genetic) disorders, prenatal screening methods, genomic editing and germline editing. For germline editing, we focussed on the novel CRISPR-Cas9 system. We included papers written in English only. OUTCOMES: Current techniques allow propagation of human SSCs in vitro, which is indispensable to successful transplantation. This technique is currently being developed in a preclinical setting for childhood cancer survivors who have stored a testis biopsy prior to cancer treatment. Similarly, SSCT could be used to restore fertility in sterile adult cancer survivors. In vitro propagation of SSCs might also be employed to enhance spermatogenesis in oligozoospermic men and in azoospermic men who still have functional SSCs albeit in insufficient numbers. The combination of SSCT with genomic editing techniques could potentially rectify defects in spermatogenesis caused by genomic mutations or, more broadly, prevent transmission of genomic diseases to the offspring. In spite of the promising prospects, SSCT and germline genomic editing are not yet clinically applicable and both techniques require optimization at various levels. WIDER IMPLICATIONS: SSCT with or without genomic editing could potentially be used to restore fertility in cancer survivors to treat couples with a severe male factor and to prevent the paternal transmission of diseases. This will potentially allow these couples to have their own biological children. Technical development is progressing rapidly, and ethical reflection and societal debate on the use of SSCT with or without genomic editing is pressing.

Defining the sequence requirements for the positioning of base J in DNA using SMRT sequencing.

Base J (ß-D-glucosyl-hydroxymethyluracil) replaces 1% of T in the Leishmania genome and is only found in telomeric repeats (99%) and in regions where transcription starts and stops. This highly restricted distribution must be co-determined by the thymidine hydroxylases (JBP1 and JBP2) that catalyze the initial step in J synthesis. To determine the DNA sequences recognized by JBP1/2, we used SMRT sequencing of DNA segments inserted into plasmids grown in Leishmania tarentolae. We show that SMRT sequencing recognizes base J in DNA. Leishmania DNA segments that normally contain J also picked up J when present in the plasmid, whereas control sequences did not. Even a segment of only 10 telomeric (GGGTTA) repeats was modified in the plasmid. We show that J modification usually occurs at pairs of Ts on opposite DNA strands, separated by 12 nucleotides. Modifications occur near G-rich sequences capable of forming G-quadruplexes and JBP2 is needed, as it does not occur in JBP2-null cells. We propose a model whereby de novo J insertion is mediated by JBP2. JBP1 then binds to J and hydroxylates another T 13 bp downstream (but not upstream) on the complementary strand, allowing JBP1 to maintain existing J following DNA replication.

Glucosylated hydroxymethyluracil, DNA base J, prevents transcriptional readthrough in Leishmania.

Some Ts in nuclear DNA of trypanosomes and Leishmania are hydroxylated and glucosylated to yield base J (ß-D-glucosyl-hydroxymethyluracil). In Leishmania, about 99% of J is located in telomeric repeats. We show here that most of the remaining J is located at chromosome-internal RNA polymerase II termination sites. This internal J and telomeric J can be reduced by a knockout of J-binding protein 2 (JBP2), an enzyme involved in the first step of J biosynthesis. J levels are further reduced by growing Leishmania JBP2 knockout cells in BrdU-containing medium, resulting in cell death. The loss of internal J in JBP2 knockout cells is accompanied by massive readthrough at RNA polymerase II termination sites. The readthrough varies between transcription units but may extend over 100 kb. We conclude that J is required for proper transcription termination and infer that the absence of internal J kills Leishmania by massive readthrough of transcriptional stops.

Molecular control of rodent spermatogenesis.

Spermatogenesis is a complex developmental process that ultimately generates mature spermatozoa. This process involves a phase of proliferative expansion, meiosis, and cytodifferentiation. Mouse models have been widely used to study spermatogenesis and have revealed many genes and molecular mechanisms that are crucial in this process. Although meiosis is generally considered as the most crucial phase of spermatogenesis, mouse models have shown that pre-meiotic and post-meiotic phases are equally important. Using knowledge generated from mouse models and in vitro studies, the current review provides an overview of the molecular control of rodent spermatogenesis. Finally, we briefly relate this knowledge to fertility problems in humans and discuss implications for future research. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.

The structural basis for recognition of base J containing DNA by a novel DNA binding domain in JBP1.

The J-binding protein 1 (JBP1) is essential for biosynthesis and maintenance of DNA base-J (ß-d-glucosyl-hydroxymethyluracil). Base-J and JBP1 are confined to some pathogenic protozoa and are absent from higher eukaryotes, prokaryotes and viruses. We show that JBP1 recognizes J-containing DNA (J-DNA) through a 160-residue domain, DB-JBP1, with 10 000-fold preference over normal DNA. The crystal structure of DB-JBP1 revealed a helix-turn-helix variant fold, a 'helical bouquet' with a 'ribbon' helix encompassing the amino acids responsible for DNA binding. Mutation of a single residue (Asp525) in the ribbon helix abrogates specificity toward J-DNA. The same mutation renders JBP1 unable to rescue the targeted deletion of endogenous JBP1 genes in Leishmania and changes its distribution in the nucleus. Based on mutational analysis and hydrogen/deuterium-exchange mass-spectrometry data, a model of JBP1 bound to J-DNA was constructed and validated by small-angle X-ray scattering data. Our results open new possibilities for targeted prevention of J-DNA recognition as a therapeutic intervention for parasitic diseases.