Pia Hermanns

RMRP mutations in cartilage‐hair hypoplasia

Cartilage hair hypoplasia (CHH) or McKusick type metaphyseal chondrodysplasia (MCD) (OMIM # 250250) is due to either the homozygous or compound heterozygous mutations in the nuclear encoded, non‐coding RNA gene RMRP. Twenty‐seven CHH patients were referred for molecular evaluation of the clinical diagnosis. RMRP mutations were found in 22 patients. The phenotype in one of the five mutation‐negative patients was fully congruent with the adopted case definition of CHH. In a second of these patients, the diagnosis of Schmid type MCD (OMIM # 156500) was made and confirmed by the detection of a mutation in the COL10A1 gene. The remaining patients most likely represent one or more MCDs hitherto not yet delineated. The pattern of cumulative growth in infancy and early childhood in the latter four patients was the single feature with greatest negative predictive power for CHH. Fourteen mutations detected here, had not been reported previously. In this ethnically heterogeneous population, we performed a retrospective study to compare the prevalence of clinical features compared to previous reports based mostly on more ethnically homogenous groups.

Brachy-syndactyly caused by loss of Sfrp2 function

Wnt signaling pathways are regulated both at the intracellular and extracellular levels. During embryogenesis, the in vivo effects of the secreted frizzled‐related protein (Sfrp) family of Wnt inhibitors are poorly understood. Here, we show that inactivation of Sfrp2 results in subtle limb defects in mice with mesomelic shortening and consistent shortening of all autopodal elements that is clinically manifested as brachydactyly. In addition, there is soft‐tissue syndactyly of the hindlimb. The brachydactyly is caused by decreased chondrocyte proliferation and delayed differentiation in distal limb chondrogenic elements. These data suggest that Sfrp2 can regulate both chondrogenesis and regression of interdigital mesenchyme in distal limb. Sfrp2 can also repress canonical Wnt signaling by Wnt1, Wnt9a, and Wnt4 in vitro. Sfrp2−/− and TOPGAL/Sfrp2−/− mice have a mild increase in beta‐catenin and beta‐galactosidase staining, respectively, in some phalangeal elements. This however does not exclude a potential concurrent effect on non‐canonical Wnt signaling in the growth plate. In combination with what is known about BMP and Wnt signaling in human brachydactylies, our data establish a critical role for Sfrp2 in proper distal limb formation and suggest SFPR2 could be a novel candidate gene for human brachy–syndactyly defects. J. Cell. Physiol. 217: 127–137, 2008.

Expression profile of Papss2 (3′-phosphoadenosine 5′-phosphosulfate synthase 2) during cartilage formation and skeletal development in the mouse embryo

Sulfation of proteoglycans is a very important posttranslational modification in chondrocyte growth and development. The enzyme 3′‐phosphoadenosine 5′‐phosphosulfate synthase (PAPSS) catalyzes the biosynthesis of PAPS (3′‐phosphoadenosine 5′‐phosphosulfate), which serves as the universal sulfate donor compound for all sulfotransferase reactions (Schwartz and Domowicz [ 2002 ] Glycobiology 109:143–151). Two major isoenzymes, PAPS synthase 1 (PAPSS1) and PAPS synthase 2 (PAPSS2) were identified in higher organisms for the synthesis of PAPS. PAPSS1 is the more prominent isoform and is ubiquitously expressed in human adult tissues, including cartilage, while PAPSS2 shows a more restricted expression pattern and appears to be the major variant in growth plate cartilage (Fuda et al. [ 2002 ] Biochem J 365(Pt 2):497–504). Mutations within the murine and the human PAPSS2 genes are responsible for diseases affecting the skeletal system (Kurima et al. [ 1998 ] Proc Natl Acad Sci USA 95:8681–8685; ul Haque et al. [ 1998 ] Nat Genet 20:157–162), like the spondyloepimetaphyseal dysplasia (SEMD) Pakistani type. To further elucidate the function of Papss2 within the developing skeleton, we investigated the expression pattern of the murine gene at different developmental stages. We detected Papss2 mRNA starting from 11.5 days post coitum (dpc) at the sites of first chondrogenic condensations and the expression continued in all cartilaginous elements tested of 12.5 dpc, 13.5 dpc, 16.5 dpc embryos, and newborn mice. Papss2 transcripts were also observed in other tissues such as heart, tongue, kidney, and neuronal tissues. However, the most significant levels of Papss2 mRNA were found in condensing and proliferating chondrocytes, whereas hypertrophic chondrocytes show a dramatic down‐regulation of Papss2 mRNA expression, indicating an important role of the gene product for cartilage growth and development in mouse embryo. Developmental Dynamics 236:1313–1318, 2007.

Refinement of the 12q14 microdeletion syndrome: primordial dwarfism and developmental delay with or without osteopoikilosis

In their studies on the molecular basis of osteopoikilosis, Menten et al have identified three individuals with microdeletions on chromosome 12q14.4, which removed several genes including LEMD3, the osteopoikilosis gene. In addition to osteopoikilosis, affected individuals had growth retardation and developmental delay. We now report a smaller 12q14.4 microdeletion in a boy with severe pre and postnatal growth failure, and mild developmental delay; the patient was small at birth and presented with poor feeding and failure to thrive during the first 2u2009years of life, similar to the phenotype of primordial dwarfism or severe Silver-Russell syndrome (SRS). The 12q14 deletion did not include LEMD3, and no signs of osteopoikilosis were observed on skeletal radiographs. Among the deleted genes, HMGA2 is of particular interest in relationship to the aberrant somatic growth in our patient, as HMGA2 variants have been linked to stature variations in the general population and loss of function of Hmga2 in the mouse results in the pygmy phenotype that combines pre and postnatal growth failure, with resistance to the adipogenic effect of overfeeding. Sequencing of the remaining HMGA2 allele in our patient showed a normal sequence, suggesting that HMGA2 haploinsufficiency may be sufficient to produce the aberrant growth phenotype. We conclude that the 12q14.4 microdeletion syndrome can occur with or without deletion of LEMD3 gene; in LEMD3-intact cases, the phenotype includes primordial short stature and failure to thrive with moderate developmental delay, but osteopoikilosis is absent. Such cases will likely be diagnosed as Silver-Russell-like or as primordial dwarfism.

The natural history of severe anemia in cartilage-hair hypoplasia.

Anemia is seen in over 80% of patients with cartilage‐hair hypoplasia (CHH). While this is usually mild and self‐limited, some patients demonstrate a severe, persistent anemia resembling that seen in Diamond‐Blackfan anemia (DBA). This paper examines the natural history of 12 patients with CHH and severe anemia. Phenotypic features and mutation data (where available) were reviewed, but no significant differences were found that predicted severe anemia. Severe anemia is estimated to occur in approximately 6% of CHH patients and is permanent in more than half of these patients. Thrombocytosis, though not previously reported in CHH, was noted in five patients, similar to that seen in DBA. The role of possible gene–gene and gene–environment interactions is discussed.

Characterization of a New Syndrome That Associates Craniosynostosis, Delayed Fontanel Closure, Parietal Foramina, Imperforate Anus, and Skin Eruption: CDAGS

We describe the clinical characterization, molecular analyses, and genetic mapping of a distinct genetic condition characterized by craniosynostosis, delayed closure of the fontanel, cranial defects, clavicular hypoplasia, anal and genitourinary malformations, and skin eruption. We have identified seven patients with this phenotype in four families from different geographic regions and ethnic backgrounds. This is an autosomal recessive condition that brings together apparently opposing pathophysiologic and developmental processes, including accelerated suture closure and delayed ossification. Selected candidate genes--including RUNX2, CBFB, MSX2, ALX4, TWIST1, and RECQL4--were screened for mutations, by direct sequencing of their coding regions, and for microdeletions, by fluorescent in situ hybridization. No mutations or microdeletions were detected in any of the genes analyzed. A genomewide screen yielded the maximum estimated LOD score of +2.38 for markers D22S283 and D22S274 on chromosome 22q12-q13. We hypothesize that the gene defect in this condition causes novel context-dependent dysregulation of multiple signaling pathways, including RUNX2, during osteoblast differentiation and craniofacial morphogenesis.

Analysis of RPS19 in patients with cartilage-hair hypoplasia and severe anemia: Preliminary results

In this issue of the journal, Williams et al. present 12 patients with cartilage-hair hypoplasia (CHH) and severe anemia. The macrocytic anemia seen in CHH is indistinguishable from the anemia seen in Diamond-Blackfan anemia (DBA). Mutational heterogeneity in RMRP does not explain the variability of the anemia phenotype in CHH, as most patients are homozygous for the 70 A>G transversion. Additionally, siblings of these patients are discordant for the severe anemia phenotype. The authors note that the gene responsible for CHH (RMRP) [Ridanpää et al., 2001] and the gene responsible for at least 25% of DBA (RPS19) [Draptchinskaia et al., 1999] are both involved in ribosomal biogenesis, and that both localize to the nucleolus [Clayton, 1994; Da Costa et al., 2003]. RPS19 is one of the 79 ribosomal proteins and located in the small 40S subunit of the ribosome. RMRP is the RNA component of the RNase Mitochondrial RNA Processing endonuclease (RNase MRP) [Chang and Clayton, 1987] that is involved in maturation of the 5.8S rRNA [Clayton, 2001], which is located in the 60S subunit of the ribosome. Additionally, there is evidence that both RNase MRP and RPS19 might interact with or affect levels of elongation factor 2 (eIF-2). RPS19 is known to be a component of the small subunit of the cytoplasmic ribosome where it serves a structural role and binds eIF-2 [Bommer et al., 1988]. Studies using anti 5.8S rRNA oligonucleotides demonstrated a significant and specific inhibition of protein synthesis in vitro [Walker et al., 1990]. Additional studies using mutant 5.8S rRNA provide in vivo evidence for a role in protein elongation or termination probably mediated by reduced levels of elongation factors 1 and 2, which are necessary for translocation of the tRNA-mRNA complex on the ribosome [Abou Elela et al., 1994; Abou Elela and Nazar, 1997]. While there are no studies that directly demonstrate that mutations in RMRP lead to decreased levels of eIF-2, this conclusion might be plausible based on what is currently known about processing and function of 5.8S rRNA. These observations suggest that RPS19 and RNase MRP might either interact directly, or might play a role in a common pathway involving eIF-2. This leads to the hypothesis that mutations in RPS19 could produce the severe anemia phenotype in patients with CHH. We present the results of RPS19 mutation analysis in three patients with CHH and severe anemia. RPS19 analysis was performed in one Amish (Patient #1) and two Finnish (Patient #2 and #3) patients with CHH and severe anemia who were previously enrolled in the CHH study at Baylor. The patient numbers correspond to those in the report by Williams et al. (in press), and clinical descriptions are provided in that report. All three of these patients were homozygous for the 70A>G transversion in RMRP. Analysis was also performed in five Caucasian controls not of Finnish or Amish ancestry. Genomic DNA was extracted from whole blood using the Genomic DNA Purification Kit (Gentra, Minneapolis, MN). Exons of RPS19 were amplified using the primers: ex1-F3: 50CCCTGTCACAGTTCCGCCC-30, ex1-R3: 50-CAGGCACGCGCTCTGAGG-30, ex2-F: 50-GGGAAAAGCACGATTCAGTC-30, ex3-R: 50-AGGGTTTCTCTCCCTCTTGG-30, ex4-F: 50-GGAGATGGTCACAGCTAGGC-30, ex4-R: 50-TGGGCTAGTCAGCCAGAGAG-30, ex5-F: 50-ATGCTTGCACAAACAACACC-30, ex5-R: 50-AGCAGCACCAACAGGAAAAG-30, ex6-F: 50-AGACCCAGTTTCCACGTCTG-30, ex6-R: 50-CAGCCATCCTTTCGTTTCTC-30 under standard PCR conditions at an annealing temperature of 588C in 50 mM KCl, 10 mM Tris/HCl (pH 8.3), 0.001% gelatin, 1.5 mM MgCl2. PCR products were purified using the UltraClean PCR Clean-up Kit (Mo BIO Laboratories, Solana Beach, CA) and sequenced with Big Dye Terminators (Applied Biosystem, Foster City, CA) and run on an ABI 3700 (Applied Biosystems) according to the manufacturer’s manual. ENSG00000105372 was used as a reference sequence. The research project was approved by the Institutional Review Board of the Baylor College of Medicine. Sequencing results are given in Table I. No mutations were found in RPS19. Several polymorphisms were identified, including three seen in all three patients (exon 2 þ89G>C; exon 4 þ14G>A; exon 5 þ70 C>T). The combination of polymorphisms was not seen in the control population (data not included in Table I).

RMRP (RNA component of mitochondrial RNA processing endoribonuclease)

Review on RMRP (RNA component of mitochondrial RNA processing endoribonuclease), with data on DNA, on the protein encoded, and where the gene is implicated.

Cartilage-hair hypoplasia (CHH)

Note: CHH was first described in the Amish, an isolated religious group in the USA by Victor McKusick in 1965. It is a multi-systemic disorder characterized by short stature, blond fine sparse hair, but this may be quite variable, and defective cellular immunity predominantly affecting T-cell mediated responses. Patients may have severe combined immunodeficiency, requiring bone marrow transplantation or they may be asymptomatic. Gastrointestinal dysfunctions are frequently observed such as mal-absorption or Hirschsprung1s disease. The incidence of CHH in the Amish is 1.5 in 1,000 births, whereas in Finland it is 1 in 18,000 to 23,000 live births.