Characterization of germ cell differentiation in the male mouse through single-cell RNA sequencing.

Spermatogenesis in the mouse has been extensively studied for decades. Previous methods, such as histological staining or bulk transcriptome analysis, either lacked resolution at the single-cell level or were focused on a very narrowly defined set of factors. Here, we present the first comprehensive, unbiased single-cell transcriptomic view of mouse spermatogenesis. Our single-cell RNA-seq (scRNA-seq) data on over 2,500 cells from the mouse testis improves upon stage marker detection and validation, capturing the continuity of differentiation rather than artificially chosen stages. scRNA-seq also enables the analysis of rare cell populations masked in bulk sequencing data and reveals new insights into the regulation of sex chromosomes during spermatogenesis. Our data provide the basis for further studies in the field, for the first time providing a high-resolution reference of transcriptional processes during mouse spermatogenesis.

Functional null mutations in the gonosomal homologue gene TBL1Y are associated with non-syndromic coarctation of the aorta.

In patients with congenital heart defects, chromosomal anomalies are 100 times more frequent than in control subjects. Coarctation of the aorta can be detected in 15-20% of patients with Ullrich-Turner syndrome. By extensively reviewing literature involving breakpoint analysis of gonosomal deletions in Ullrich- Turner syndrome patients with and without coarctation of the aorta, we identified several gonosomal homolgous gene pairs of interest. Four of these homologous gene pairs were investigated by standard DNA sequencing in a cohort of 83 patients with non-syndromic coarctation of the aorta. Subsequently stability of mutant RNA and protein was analyzed to verify functional relevance of detected mutations. We identified two unreported missense mutations in Exon 8 (p.D69H) and 9 (p.R176W) of TBL1Y. Bioinformatic analysis and 3D modelling predicted that both mutations lead to TBL1Y loss of function. In RT-PCR and Western blot analyses of HEK293 cells transfected with a vector carrying the full-length TBL1Y (wild-type and mutant), we documented the predicted protein instability by showing protein decay for both mutant proteins. TBL1Y is similar to its gonosomal homologue, TBL1X, and its autosomal homologue, TBLR1, on chromosome 3. Both genes are part of co-repressor machineries and required for transcriptional activation by transcription factors that involve CtBP1/2, which contributes to Notch signaling. Several studies have shown that Notch signalling is important for proper development of the left ventricular outflow tract. Our findings suggest that TBL1Y is involved in the genesis of non-syndromic coarctation of the aorta.

Primary erythromelalgia in a 12-year-old boy: positive response to sodium channel blockers despite negative SCN9A mutations.

Erythromelalgia is a rare disorder characterized by recurrent pain attacks, swelling and redness in the distal extremities. The primary forms of the disorder are caused by mutations in voltage-gated sodium channels. Treatment is difficult and controlled therapeutic studies offer little to no guidance. We report on a 12-year-old boy and his first occurrence of primary erythromelalgia. Genetic findings for mutations in the SCN9A gene, which encodes for the α-subunit of sodium channel NaV1.7, were negative. Although initial treatment with sodium nitroprusside was ineffective, subsequent medication with lidocaine and mexiletine, in combination with gabapentin, was successful. Despite negative findings for mutations in the sodium channels, the use of sodium channel blockers should be considered in these patients.

Balanced translocation in a patient with craniosynostosis disrupts the SOX6 gene and an evolutionarily conserved non-transcribed region.

Craniosynostosis is a congenital developmental disorder involving premature fusion of cranial sutures, which results in an abnormal shape of the skull. Significant progress in understanding the molecular basis of this phenotype has been made for a small number of syndromic craniosynostosis forms. Nevertheless, in the majority of the approximately 100 craniosynostosis syndromes and in non-syndromic craniosynostosis the underlying gene defects and pathomechanisms are unknown. Here we report on a male infant presenting at birth with brachycephaly, proptosis, midfacial hypoplasia, and low set ears. Three dimensional cranial computer tomography showed fusion of the lambdoid sutures and distal part of the sagittal suture with a gaping anterior fontanelle. Mutations in the genes for FGFR2 and FGFR3 were excluded. Standard chromosome analysis revealed a de novo balanced translocation t(9;11)(q33;p15). The breakpoint on chromosome 11p15 disrupts the SOX6 gene, known to be involved in skeletal growth and differentiation processes. SOX6 mutation screening of another 104 craniosynostosis patients revealed one missense mutation leading to the exchange of a highly conserved amino acid (p.D68N) in a single patient and his reportedly healthy mother. The breakpoint on chromosome 9 is located in a region without any known or predicted genes but, interestingly, disrupts patches of evolutionarily highly conserved non-genic sequences and may thus led to dysregulation of flanking genes on chromosome 9 or 11 involved in skull vault development. The present case is one of the very rare reports of an apparently balanced translocation in a patient with syndromic craniosynostosis, and reveals novel candidate genes for craniosynostoses and cranial suture formation.

Comparative architectural aspects of regions of conserved synteny on human chromosome 11p15.3 and mouse chromosome 7 (including genes WEE1 and LMO1).

Human chromosome 11p15.3 is associated with chromosome aberrations in the Beckwith Wiedemann Syndrome and implicated in the pathogenesis of different tumor types including lung cancer and leukemias. To date, only single tumor-relevant genes with linkage to this region (e.g. LMO1) have been found suggesting that this region may harbor additional potential disease associated genes. Although this genomic area has been studied for years, the exact order of genes/chromosome markers between D11S572 and the WEE1 gene locus remained unclear. Using the FISH technique and PAC clones of the flanking markers we determined the order of the genomic markers. Based on these clones we established a PAC contig of the respective region. To analyse the chromosome area in detail the synteny of the orthologous region on distal mouse chromosome 7 was determined and a corresponding mouse clone contig established, proving the conserved order of the genes and markers in both species: "cen-WEE1-D11S2043-ZNF143-RANBP7-CEGF1- ST5-D11S932-LMO1-D11S572-TUB-tel", with inverted order of the murine genes with respect to the telomere/centromere orientation. The region covered by these contigs comprises roughly 1.6 MB in human as well as in mouse. The genomic sequence of the two subregions (around WEE1 and LMO1) in both species was determined using a shotgun sequencing strategy. Comparative sequence analysis techniques demonstrate that the content of repetitive elements seems to decline from centromere to telomere (52.6% to 34.5%) in human and in the corresponding murine region from telomere to centromere (41.87% to 27.82%). Genomic organisation of the regions around WEE1 and LMO1 was conserved, although the length of gene regions varied between the species in an unpredictable ratio. CpG islands were found conserved in putative promoter regions of the known genes but also in regions which so far have not been described as harboring expressed sequences.

Comparative genomic sequencing reveals a strikingly similar architecture of a conserved syntenic region on human chromosome 11p15.3 (including gene ST5) and mouse chromosome 7.

Comparative genomics is a superior way to identify phylogenetically conserved features like genes or regions involved in gene regulation. The comparison of extended orthologous chromosomal regions should also reveal other characteristic traits essential for chromosome or gene function. In the present study we have sequenced and compared a region of conserved synteny from human chromosome 11p15.3 and mouse chromosome 7. In human, this region is known to contain several genes involved in the development of various disorders like Beckwith-Wiedemann overgrowth syndrome and other tumor diseases. Furthermore, in the neighboring chromosome region 11p15.5 extensive imprinting of genes has been reported which might extend to region 11p15.3. The analysis of approximately 730 kb in human and 620 kb in mouse led to the identification of eleven genes. All putative genes found in the mouse DNA were also present in the same order and orientation in the human chromosome. However, in the human DNA one putative gene of unknown function could be identified which is not present in the orthologous position of the mouse chromosome. The sequence similarity between human and mouse is higher in transcribed and exon regions than in non-transcribed segments. Dot plot analysis, however, reveals a surprisingly well-conserved sequence similarity over the entire analyzed region. In particular, the positions of CpG islands, short regions of very high GC content in the 5' region of putative genes, are similar in human and mouse. With respect to base composition, two distinct segments of significantly different GC content exist as well in human as in the mouse. With a GC content of 45% the one segment would correspond to "isochore H1" and the other segment (39% GC in human, 40% GC in mouse) to "isochore L1/L2". The gene density (one gene per 66 kb) is slightly higher than the average calculated for the complete human genome (one gene per 90 kb). The comparison of the number and distribution of repetitive elements shows that the proportion of human DNA made up by interspersed repeats (43.8%) is significantly higher than in the corresponding mouse DNA (30.1%). This partly explains why the human DNA is longer between the landmark genes used to define the orthologous positions in human and mouse.

Regulation of glomerular basement membrane collagen expression by LMX1B contributes to renal disease in nail patella syndrome.

Basement membrane (BM) morphogenesis is critical for normal kidney function. Heterotrimeric type IV collagen, composed of different combinations of six alpha-chains (1-6), is a major matrix component of all BMs (ref. 2). Unlike in other BMs, glomerular BM (GBM) contains primarily the alpha 3(IV) and alpha 4(IV) chains, together with the alpha 5(IV) chain. A poorly understood, coordinated temporal and spatial switch in gene expression from ubiquitously expressed alpha 1(IV) and alpha 2(IV) collagen to the alpha 3(IV), alpha 4(IV) and alpha 5(IV) chains occurs during normal embryogenesis of GBM (ref. 4). Structural abnormalities of type IV collagen have been associated with diverse biological processes including defects in molecular filtration in Alport syndrome, cell differentiation in hereditary leiomyomatosis, and autoimmunity in Goodpasture syndrome; however, the transcriptional and developmental regulation of type IV collagen expression is unknown. Nail patella syndrome (NPS) is caused by mutations in LMX1B, encoding a LIM homeodomain transcription factor. Some patients have nephrosis-associated renal disease characterized by typical ultrastructural abnormalities of GBM (refs. 8,9). In Lmx1b(-/-) mice, expression of both alpha(3)IV and alpha(4)IV collagen is strongly diminished in GBM, whereas that of alpha1, alpha2 and alpha5(IV) collagen is unchanged. Moreover, LMX1B binds specifically to a putative enhancer sequence in intron 1 of both mouse and human COL4A4 and upregulates reporter constructs containing this enhancer-like sequence. These data indicate that LMX1B directly regulates the coordinated expression of alpha 3(IV) and alpha 4(IV) collagen required for normal GBM morphogenesis and that its dysregulation in GBM contributes to the renal pathology and nephrosis in NPS.

A novel mutation in FGFR-3 disrupts a putative N-glycosylation site and results in hypochondroplasia.

Fibroblast growth factor receptor 3 (FGFR3) is a glycoprotein that belongs to the family of tyrosine kinase receptors. Specific mutations in the FGFR3 gene are associated with autosomal dominant human skeletal disorders such as hypochondroplasia, achondroplasia, and thanatophoric dysplasia. Hypochondroplasia (HCH), the mildest form of this group of short-limbed dwarfism disorders, results in approximately 60% of cases from a mutation in the intracellular FGFR3-tyrosine kinase domain. The remaining cases may either be caused by defects in other FGFR gene regions or other yet unidentified genes. We describe a novel HCH mutation, the first found outside the common mutation hot spot of this condition. This point mutation, an N328I exchange in the extracellular Ig domain III of the receptor, seems to be unique as it affects a putative N-glycosylation site that is conserved between different FGFRs and species. The amino acid exchange itself most probably has no impact on the three-dimensional structure of the receptor domain, suggesting that the phenotype is the result of altered receptor glycosylation and its pathophysiological consequences.

Mtr1, a novel biallelically expressed gene in the center of the mouse distal chromosome 7 imprinting cluster, is a member of the Trp gene family.

We recently described a novel putative Ca(2+) channel gene, MTR1, which shows a high level of homology to the human TRPC7 gene and the melastatin 1 (MLSN1) gene, another Trp (transient receptor potential protein)-related gene whose transcript was found to be downregulated in metastatic melanomas. It maps to human chromosome band 11p15.5, which is associated with the Beckwith-Wiedemann syndrome and predisposition to a variety of neoplasias. Here we report the isolation and characterization of the murine orthologue Mtr1. The chromosomal localization on distal chromosome 7 places it in a cluster of imprinted genes, flanked by the previously described Tapa1 and Kcnq1 genes. The Mtr1 gene encodes a 4.4-kb transcript, present in a variety of fetal and adult tissues. The putative open reading frame consists of 24 exons, encoding 1158 amino acids. Transmembrane prediction algorithms indicate the presence of six membrane-spanning domains in the proposed protein. Imprinting analysis, using RT-PCR on RNA from reciprocal mouse crosses harboring a sequence polymorphism, revealed biallelic expression of Mtr1 transcripts at all stages and tissues examined.

LMX1B transactivation and expression in nail-patella syndrome.

Lmx1b, a member of the LIM homeodomain protein family, is essential for the specification of dorsal limb fates at the zeugopodal and autopodal level in vertebrates. We and others have shown that a skeletal dysplasia, nail-patella syndrome (NPS), results from mutations in LMX1B. While it is a unique mesenchymal determinant of dorsal limb patterning during vertebrate development, the mechanism by which LMX1B mutations generate the NPS phenotype has not been addressed at a transcriptional level or correlated with its spatial pattern of gene expression. In this study, in situ hybridizations of Lmx1b on murine limb sections reveal strong expression in dorsal mesenchymal tissues (precursors of muscle, tendons, joints and patella) and, interestingly, also in anterior structures of the limb, explaining the anterior to posterior gradient of joint and nail dysplasia observed in NPS patients. Transfection studies showed that both the LIM domain-interacting protein, LDB1, and the helix-loop-helix protein, E47/shPan1, can regulate LMX1B action. While co--transfections of E47/shPan1 with LMX1B result in a synergistic effect on reporter activity, LDB1 down-regulated LMX1B-mediated transactivation irrespective of E47/shPan1. Mutant LMX1B proteins containing human mutations affecting each of the helices or the N-terminal arm of the homeodomain abolished transactivation, while LIM B and truncation mutations retained residual activity. These mutations fail to act in a dominant-negative manner on wild-type LMX1B in mixing studies, thereby supporting haploinsufficiency as the mechanism underlying NPS pathogenesis.

Identification and characterization of MTR1, a novel gene with homology to melastatin (MLSN1) and the trp gene family located in the BWS-WT2 critical region on chromosome 11p15.5 and showing allele-specific expression.

Alterations within human chromosomal region 11p15.5 are associated with the Beckwith-Wiedemann syndrome (BWS) and predisposition to a variety of neoplasias, including Wilms' tumors (WTs), rhabdoid tumors and rhabdomyosarcomas. To identify candidate genes for 11p15. 5-related diseases we compared human genomic sequence with expressed sequence tag and protein databases from different organisms to discover evolutionarily conserved sequences. Herein we describe the identification and characterization of a novel human transcript related to a putative Caenorhabditis elegans protein and the trp (transient receptor potential) gene. The highest homologies are observed with the human TRPC7 and with melastatin 1 ( MLSN1 ), whose transcript is downregulated in metastatic melanomas. Other genes related to and interacting with the trp family include the Grc gene, which codes for a growth factor-regulated channel protein, and PKD1/PKD2, involved in polycystic kidney disease. The novel gene presented here (named MTR1 for MLSN1 - and TRP -related gene 1) resides between TSSC4 and KvLQT1. MTR1 is expressed as a 4.5 kb transcript in a variety of fetal and adult tissues. The putative open reading frame is encoded in 24 exons, one of which is alternatively spliced leading to two possible proteins of 872 or 1165 amino acids with several predicted membrane-spanning domains in both versions. MTR1 transcripts are present in a large proportion of WTs and rhabdomyosarcomas. RT-PCR analysis of somatic cell hybrids harboring a single human chromosome 11 demonstrated exclusive expression of MTR1 in cell lines carrying a paternal chromosome 11, indicating allele-specific inactivation of the maternal copy by genomic imprinting.

Isolation, characterization, and mapping of a zinc finger gene, ZFP95, containing both a SCAN box and an alternatively spliced KRAB A domain.

A new zinc finger gene of the Krüppel family was identified by screening a human fetal cartilage cDNA library with degenerate oligonucleotides. Sequence analysis indicates that ZFP95 contains 12 highly conserved zinc finger motifs at the C-terminus and a SCAN box as well as a KRAB A domain at the N-terminus of the protein. ZFP95 represents a member of a new subclass of Krüppel zinc finger proteins containing both a SCAN box and a KRAB domain. Sequence comparison revealed that ZFP95 is the human ortholog of murine Zfp95, which is differentially expressed during spermatogenesis. We demonstrate that ZFP95 is ubiquitously expressed in adult and fetal tissues with the strongest expression in testis. Two transcripts, 4. 2 and 4.6 kb, were detected in all tissues tested. In testis, a third transcript of 3.8 kb was present. RT-PCR analysis confirmed alternative splicing for the KRAB A domain and an upstream exon leading to three transcripts of ZFP95 with and without this transcriptional repressor domain. Finally, we show that ZFP95 maps on human chromosome 7q22 between the markers D7S651 and WI-5853.

LETM1, a novel gene encoding a putative EF-hand Ca(2+)-binding protein, flanks the Wolf-Hirschhorn syndrome (WHS) critical region and is deleted in most WHS patients.

Deletions within human chromosome 4p16.3 cause Wolf-Hirschhorn syndrome (WHS), which is characterized by severe mental and developmental defects. It is thought that haploinsufficiency of more than one gene contributes to the complex phenotype. We have cloned and characterized a novel gene (LETM1) that is deleted in nearly all WHS patients. LETM1 encodes a putative member of the EF-hand family of Ca(2+)-binding proteins. The protein contains two EF-hands, a transmembrane domain, a leucine zipper, and several coiled-coil domains. On the basis of its possible Ca(2+)-binding property and involvement in Ca(2+) signaling and/or homeostasis, we propose that haploinsufficiency of LETM1 may contribute to the neuromuscular features of WHS patients.

Mutation analysis of LMX1B gene in nail-patella syndrome patients.

Nail-patella syndrome (NPS), a pleiotropic disorder exhibiting autosomal dominant inheritance, has been studied for >100 years. Recent evidence shows that NPS is the result of mutations in the LIM-homeodomain gene LMX1B. To determine whether specific LMX1B mutations are associated with different aspects of the NPS phenotype, we screened a cohort of 41 NPS families for LMX1B mutations. A total of 25 mutations were identified in 37 families. The nature of the mutations supports the hypothesis that NPS is the result of haploinsufficiency for LMX1B. There was no evidence of correlation between aspects of the NPS phenotype and specific mutations.

Heterozygous glycine substitution in the COL11A2 gene in the original patient with the Weissenbacher-Zweymüller syndrome demonstrates its identity with heterozygous OSMED (nonocular Stickler syndrome).

The original patient with the Weissenbacher-Zweymüller syndrome was analyzed for mutations in two candidate genes expressed in cartilage (COL2A1 and COL11A2). No mutations were found in the COL2A1 gene but the COL11A2 gene contained a single-base mutation that converted a codon for an obligate glycine to a codon for glutamate at position alpha 2-955 (G955E). The results here and those published previously indicate that the Weissenbacher-Zweymüller syndrome (heterozygous OSMED), nonocular Stickler syndrome, and homozygous OSMED are all caused by mutations in the COL11A2 gene.