Kniest and Stickler dysplasia phenotypes caused by collagen type II gene (COL2A1) defect.

Kniest and Stickler dysplasia are two chondrodysplasias characterized by specific phenotypes. No basic defect has been found in patients with Kniest dysplasia, whereas Stickler dysplasia is one of four chondrodysplasias for which mutations of type II procollagen gene (COL2A1) have been identified. We studied a 2-year-old girl presenting with manifestations of Kniest dysplasia and her mother showing a Stickler phenotype. Analysing COL2A1 in both patients, we detected the same 28 basepair deletion spanning the 3'-exon/intron boundary of exon 12 in mother and daughter. We were able to prove a somatic mosaic status for this mutation in the mother which accounts for her milder Stickler-like phenotype.

Imprint switching on human chromosome 15 may involve alternative transcripts of the SNRPN gene.

Imprinting on human chromosome 15 is regulated by an imprinting centre, which has been mapped to a 100-kb region including exon 1 of SNRPN. From this region we have identified novel transcripts, which represent alternative transcripts of the SNRPN gene. The novel exons lack protein coding potential and are expressed from the paternal chromosome only. We have also identified intragenic deletions and a point mutation in patients who have Angelman or Prader-Willi syndrome due to a parental imprint switch failure. This suggests that imprint switching on human chromosome 15 may involve alternative SNRPN transcripts.

Mutations in LMX1B cause abnormal skeletal patterning and renal dysplasia in nail patella syndrome.

The LIM-homeodomain protein Lmx1b plays a central role in dorso-ventral patterning of the vertebrate limb. Targeted disruption of Lmx1b results in skeletal defects including hypoplastic nails, absent patellae and a unique form of renal dysplasia (see accompanying manuscript by H. Chen et al.; ref. 2). These features are reminiscent of the dominantly inherited skeletal malformation nail patella syndrome (NPS). We show that LMX1B maps to the NPS locus and that three independent NPS patients carry de novo heterozygous mutations in this gene. Functional studies show that one of these mutations disrupts sequence-specific DNA binding, while the other two mutations result in premature termination of translation. These data demonstrate a unique role for LMX1B in renal development and in patterning of the skeletal system, and suggest that alteration of Lmx1b/LMX1B function in mice and humans results in similar phenotypes. Furthermore, we provide evidence for the first described mutations in a LIM-homeodomain protein which account for an inherited form of abnormal skeletal patterning and renal failure.

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.

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.

One gene, two transcripts: isolation of an alternative transcript encoding for the autoantigen La/SS-B from a cDNA library of a patient with primary Sjögrens' syndrome.

A cDNA library was prepared from peripheral blood lymphocytes of an autoimmune patient with primary Sjögrens' syndrome. The cDNA library was screened with the patients own autoimmune serum being monospecific for the nuclear autoantigen La/SS-B. Thereby an alternative type of La mRNA was identified that differed from the known La mRNA due to an exchange of the exon 1. Sequencing of the genomic region between the exons 1 and 2 showed that the alternative 5'-end is a part of the intron. In addition, the presence of an alternative promoter site, which exists within the intron downstream of the exon 1, became evident. In consequence, the alternative La mRNA is the result of a promoter switching combined with an alternative splicing mechanism. In the intron, further transcription factor binding sites, including a NF-kappa B element, were identified leading to the suggestion that the expression of the gene encoding for the nuclear autoantigen La/SS-B alters in dependence on disease conditions.

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.

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.

Loss of heterozygosity studies and deletion mapping identify two putative chromosome 14q tumor suppressor loci in renal oncocytomas.

Renal oncocytoma is considered to be a benign tumor that shares some phenotypic features with chromophobe renal cell carcinoma (RCC). Recently, we described high frequencies of allelic loss at 1p, 2p, 6p, 10p, 13q, 14q, 17p, and 21q, which correlate significantly with the chromophobe subtype of RCC. To investigate the genetic relationship between these two entities, we examined 12 oncocytomas for loss of heterozygosity (LOH) at these regions. In addition, we included markers for 3p, 5q, 7q, 11p, and 22q. The only chromosomal region showing similarly high frequencies of allelic loss for both subtypes was 14q. Therefore, a genetic relationship between renal oncocytoma and chromophobe RCC seems questionable. Eight of 12 oncocytomas (67%) showed LOH at 14q, a frequency that was significantly higher (P < 0.001, chi(2) test) than the frequencies of LOH in all other regions. To define regions potentially harboring novel tumor suppressor genes, we performed multifluorescence microsatellite analysis with 13 markers spanning 14q. Interstitial deletions at different regions of 14q were detected, with the highest frequencies at D14S258 (14q23-24.3) and D14S292 (14q32.1-32.2). 14q LOH might be associated with advanced-stage RCCs or other tumors, but it does not seem to indicate progression in oncocytomas. Its role in pathogenesis of renal oncocytomas remains to be clarified. Here, we provide evidence for two distinct tumor suppressor gene loci at 14q in renal oncocytoma, which will be useful for further fine-mapping studies of these critical regions.

Developmental expression of splicing variants of fibroblast growth factor receptor 3 (FGFR3) in mouse.

A characteristic feature of the fibroblast growth factor receptor (FGFR) family is the structural diversity generated by alternative splicing. The FGFR3 gene encodes two splice variants because of the mutually exclusive use of the exons IIIb and IIIc. In the present study we examined the expression of the two different splice forms IIIb and IIIc of FGFR3 in developing mouse embryos (12 days p.c., 14 days p.c., 20 days p.c.). The overall level of the IIIc exon splice product surpassed that of the IIIb exon form. The IIIc mRNA was detected in the developing brain and in the spinal cord. Outside the nervous system very strong expression was observed in the vertebra and in all other bony structures. In contrast, the IIIb splice form was restricted to epithelial structures with no expression detected in the central nervous system and bone.

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.

Kniest dysplasia: Dr. W. Kniest, his patient, the molecular defect.

Kniest dysplasia is a severe chondrodysplasia caused by the defective formation of type II collagen. We report about Dr. Kniest, who first described the condition in 1952, and his patient, who, at the age of 50 years is severely handicapped with short stature, restricted joint mobility, and blindness but is mentally alert and leads an active life. Molecular analysis of the patient's DNA showed a single base (G) deletion involving the GT dinucleotide at the start of intron 18 destroying a splice site of the COL2A1 gene. This is in accordance with molecular findings in other patients with Kniest dysplasia and confirms, in the original patient, that the disorder is caused by small inframe deletions often due to exon skipping as a result of COL2A1 splice site mutations.

A specific collagen type II gene (COL2A1) mutation presenting as spondyloperipheral dysplasia.

We report on a patient with a skeletal dysplasia characterized by short stature, spondylo-epiphyseal involvement, and brachydactyly E-like changes. This condition has been described as spondyloperipheral dysplasia and the few published cases suggest autosomal dominant inheritance with considerable clinical variability. We found our sporadic case to be due to a collagen type II defect resulting from a specific COL2A1 mutation. This mutation is the first to be located at the C-terminal outside the helical domain of COL2A1. A frameshift as consequence of a 5 bp duplication in exon 51 leads to a stop codon. The resulting truncated C-propeptide region seems to affect helix formation and produces changes of chondrocyte morphology, collagen type II fibril structure and cartilage matrix composition. Our case with its distinct phenotype adds another chondrodysplasia to the clinical spectrum of type II collagenopathies.

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.

Detection of hepatitis B virus DNA by polymerase chain reaction in serum and liver of children with chronic hepatitis B negative for hepatitis B virus DNA by conventional hybridization tests.

Hepatitis B virus (HBV) DNA was detected by polymerase chain reaction in the serum of 87 and liver tissue of 40 children with chronic hepatitis B, negative for HBV DNA by dot blot and Southern blot hybridization, respectively. In sera HBV DNA could be detected in 73 hepatitis B surface antigen carriers; 14 were hepatitis B e antigen (HBeAg), 56 were anti-HBe-seropositive and 3 had neither HBeAg nor positive anti-HBe. In 14 anti-HBe-positive patients no HBV DNA could be found. Viral sequences in liver tissue were present in 33 specimens; 20 were HBeAg and 13 were anti-HBe-seropositive. All of the 7 negative children had anti-HBe. Our results confirm polymerase chain reaction to be a more sensitive method to detect HBV DNA in the liver compared with conventional hybridization techniques. Every HBeAg-positive carrier as well as the majority of anti-HBe-positive patients show ongoing viral replication. This is of special clinical relevance, because these children must be considered infectious.

Ehlers-Danlos syndrome type VII: phenotype and genotype.

A patient suffering from a severe form of Ehlers-Danlos syndrome is presented (EDS type VII). The presence of bilateral congenital hip dislocation, generalized joint hypermobility and a soft hyperelastic skin with abnormal scarring suggested a specific collagen type I defect. SDS-PAGE analysis of collagens secreted into the medium of fibroblast cultures showed a retarded migration of more than half of the alpha 2(I) chains. CNBr peptide mapping of the HPLC-purified altered chain localized the mutant locus to the N-terminal region of the protein. cDNA analysis of the corresponding gene COL1A2 revealed, in addition to the expected collagen sequence, a transcript missing the entire exon 6. This exon encodes a major crosslinking site within collagen fibres as well as the N-propeptidase cleavage site. The skipping of exon 6 is caused by a splice site mutation substituting an A for a G at the first nucleotide of intron 6.

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.