Novel KIAA0753 mutations extend the phenotype of skeletal ciliopathies.

The skeletal ciliopathies are a heterogeneous group of disorders with a significant clinical and genetic variability and the main clinical features are thoracic hypoplasia and short tubular bones. To date, 25 genes have been identified in association with skeletal ciliopathies. Mutations in the KIAA0753 gene have recently been associated with Joubert syndrome (JBTS) and orofaciodigital (OFD) syndrome. We report biallelic pathogenic variants in KIAA0753 in four patients with short-rib type skeletal dysplasia. The manifestations in our patients are variable and ranging from fetal lethal to viable and moderate skeletal dysplasia with narrow thorax and abnormal metaphyses. We demonstrate that KIAA0753 is expressed in normal fetal human growth plate and show that the affected fetus, with a compound heterozygous frameshift and a nonsense mutation in KIAA0753, has an abnormal proliferative zone and a broad hypertrophic zone. The importance of KIAA0753 for normal skeletal development is further confirmed by our findings that zebrafish embryos homozygous for a nonsense mutation in kiaa0753 display altered cartilage patterning.

Tooth agenesis in osteogenesis imperfecta related to mutations in the collagen type I genes.

BACKGROUND: Osteogenesis imperfecta (OI) is a heterogeneous group of disorders of connective tissue, mainly caused by mutations in the collagen type I genes (COL1A1 and COL1A2). Tooth agenesis is a common feature of OI. We investigated the association between tooth agenesis and collagen type I mutations in individuals with OI. SUBJECTS AND METHODS: In this cohort study, 128 unrelated individuals with OI were included. Panoramic radiographs were analyzed regarding dentinogenesis imperfecta (DGI) and congenitally missing teeth. The collagen I genes were sequenced in all individuals, and in 25, multiplex ligation-dependent probe amplification was performed. RESULTS: Mutations in the COL1A1 and COL1A2 genes were found in 104 of 128 individuals. Tooth agenesis was diagnosed in 17% (hypodontia 11%, oligodontia 6%) and was more frequent in those with DGI (P = 0.016), and in those with OI type III, 47%, compared to those with OI types I, 12% (P = 0.003), and IV, 13% (P = 0.017). Seventy-five percent of the individuals with oligodontia (≥6 missing teeth) had qualitative mutations, but there was no association with OI type, gender, or presence of DGI. CONCLUSION: The prevalence of tooth agenesis is high (17%) in individuals with OI, and OI caused by a qualitative collagen I mutation is associated with oligodontia.

Decreased fracture rate, pharmacogenetics and BMD response in 79 Swedish children with osteogenesis imperfecta types I, III and IV treated with Pamidronate.

BACKGROUND: Osteogenesis imperfecta (OI) is an inherited heterogeneous bone fragility disorder, usually caused by collagen I mutations. It is well established that bisphosphonate treatment increases lumbar spine (LS) bone mineral density (BMD), as well as improves vertebral geometry in severe OI; however, fracture reduction has been difficult to prove, pharmacogenetic studies are scarce, and it is not known at which age, or severity of disease, treatment should be initiated. MATERIALS AND METHODS: COL1A1 and COL1A2 were analyzed in 79 children with OI (type I n=33, type III n=25 and type IV n=21) treated with Pamidronate. Data on LS BMD, height, and radiologically confirmed non-vertebral and vertebral fractures were collected prior to, and at several time points during treatment. RESULTS: An increase in LS BMD Z-score was observed for all types of OI, and a negative correlation to Δ LS BMD was observed for both age and LS BMD Z-score at treatment initiation. Supine height Z-scores were not affected by Pamidronate treatment, The fracture rate was reduced for all OI types at all time points during treatment (overall p<0.0003, <0.0001 and 0.0003 for all OI types I, III and IV respectively). The reduced fracture rate was maintained for types I and IV, while an additional decrease was observed over time for type III. The fracture rate was reduced also in individuals with continued low BMD after >4yrs Pamidronate. Twice as many boys as girls with OI type I were treated with Pamidronate, and the fracture rate the year prior treatment was 2.2 times higher for boys (p=0.0236). Greater Δ LS BMD, but smaller Δ fracture numbers were observed on Pamidronate for helical glycine mutations in COL1A1 vs. COL1A2. Vertebral compression fractures did not progress in any individual during treatment; however, they did not improve in 9%, and these individuals were all >11years of age at treatment initiation (p<0.0001). CONCLUSION: Pamidronate treatment in children with all types of OI increased LS BMD, decreased fracture rate, and improved vertebral compression fractures. Fracture reduction was prompt and maintained during treatment, irrespective of age at treatment initiation and collagen I mutation type.

Short Stature in KBG Syndrome: First Responses to Growth Hormone Treatment.

BACKGROUND: KBG syndrome is a rare disorder characterized by intellectual disability and associated with macrodontia of the upper central incisors, specific craniofacial findings, short stature and skeletal anomalies. Genetic corroboration of a clinical diagnosis has been possible since 2011, upon identification of heterozygous mutations in or a deletion of the ANKRD11 gene. METHODS: We summarized the height data of 14 adults and 18 children (age range 2-16 years) with a genetically confirmed diagnosis of KBG syndrome. Two of these children were treated with growth hormones. RESULTS: Stature below the 3rd centile or -1.88 standard deviation score (SDS) was observed in 72% of KBG children and in 57% of KBG adults. Height below -2.50 SDS was observed in 62% of KBG children and in 36% of KBG adults. The mean SDS of height in KBG children was -2.56 and in KBG adults -2.17. Two KBG children on growth hormone therapy increased their height by 0.6 and 1 SDS within 1 year, respectively. The former also received a gonadotropin-releasing hormone agonist due to medical necessity. CONCLUSION: Short stature is prevalent in KBG syndrome, and spontaneous catch-up growth beyond childhood appears limited. Growth hormone intervention in short KBG children is perceived as promising.

SLC26A2 disease spectrum in Sweden - high frequency of recessive multiple epiphyseal dysplasia (rMED).

Diastrophic dysplasia (DTD) is an autosomal recessive skeletal dysplasia caused by SLC26A2 mutations. Clinical features include short stature, joint contractures, spinal deformities, and cleft palate. SLC26A2 mutations also result in other skeletal dysplasias, including the milder recessive multiple epiphyseal dysplasia (rMED). DTD is overrepresented in Finland and we speculated that this may have influenced the prevalence and spectrum of SLC26A2-related skeletal conditions also in Sweden. We reviewed the patient registry at Department of Clinical Genetics, Karolinska University Hospital, Stockholm to identify subjects with SLC26A2 mutations. Seven patients from six families were identified; clinical data were available for six patients. All but one patient had one or two copies of the Finnish SLC26A2 founder mutation IVS1+2T>C. Arg279Trp mutation was present in compound heterozygous form in five patients with phenotypes consistent with rMED. Their heights ranged from -2.6 to -1.4 standard deviation units below normal mean and radiographic features included generalised epiphyseal dysplasia and double-layered patellae. Two rMED patients had hypoplastic C2 and cervical kyphosis, a severe manifestation previously described only in DTD. Our study confirms a high prevalence of rMED in Sweden and expands the phenotypic manifestations of rMED.

Novel and recurrent TRPV4 mutations and their association with distinct phenotypes within the TRPV4 dysplasia family.

BACKGROUND: Mutations in TRPV4, a gene that encodes a Ca(2+) permeable non-selective cation channel, have recently been found in a spectrum of skeletal dysplasias that includes brachyolmia, spondylometaphyseal dysplasia, Kozlowski type (SMDK) and metatropic dysplasia (MD). Only a total of seven missense mutations were detected, however. The full spectrum of TRPV4 mutations and their phenotypes remained unclear. OBJECTIVES AND METHODS: To examine TRPV4 mutation spectrum and phenotype-genotype association, we searched for TRPV4 mutations by PCR-direct sequencing from genomic DNA in 22 MD and 20 SMDK probands. RESULTS: TRPV4 mutations were found in all but one MD subject. In total, 19 different heterozygous mutations were identified in 41 subjects; two were recurrent and 17 were novel. In MD, a recurrent P799L mutation was identified in nine subjects, as well as 10 novel mutations including F471del, the first deletion mutation of TRPV4. In SMDK, a recurrent R594H mutation was identified in 12 subjects and seven novel mutations. An association between the position of mutations and the disease phenotype was also observed. Thus, P799 in exon 15 is a hot codon for MD mutations, as four different amino acid substitutions have been observed at this codon; while R594 in exon 11 is a hotspot for SMDK mutations. CONCLUSION: The TRPV4 mutation spectrum in MD and SMDK, which showed genotype-phenotype correlation and potential functional significance of mutations that are non-randomly distributed over the gene, was presented in this study. The results would help diagnostic laboratories establish efficient screening strategies for genetic diagnosis of the TRPV4 dysplasia family diseases.

Metacarpophalangeal pattern profile analysis in Leri-Weill dyschondrosteosis.

PURPOSE: To analyze the metacarpophalangeal profile (MCPP) in individuals with Leri-Weill dyschondrosteosis (LWD) and to assess its value as a possible contributor to early diagnosis. MATERIAL AND METHODS: Hand profiles of 39 individuals with a diagnosis of LWD were calculated and analyzed. Discriminant analysis was applied to differentiate between LWD and normal individuals. RESULTS: There was a distinct pattern profile in LWD. Mean pattern profile showed two bone-shortening gradients, with increasing shortening from distal to proximal and from medial to lateral. Distal phalanx 2 was disproportionately long and second metacarpal was disproportionately short. Discriminant analysis yielded correct classification in 72% of analyzed cases. CONCLUSION: MCPP is not age-related and the analysis can be applied at any age, facilitating early diagnosis of LWD. In view of its availability, low costs, and diagnostic value, MCPP analysis should be considered as a routine method in the patients of short stature where LWD is suspected.

Analysis of short stature homeobox-containing gene ( SHOX) and auxological phenotype in dyschondrosteosis and isolated Madelung deformity.

Dyschondrosteosis (DCO; also called Léri-Weill syndrome) is a skeletal dysplasia characterised by disproportionate short stature because of mesomelic shortening of the limbs. Madelung deformity is a feature of DCO that is distinctive, variable in expressivity and frequently observed. Mutations of the SHOX (short stature homeobox-containing) gene have been previously described as causative in DCO. Isolated Madelung deformity (IMD) without the clinical characteristics of DCO has also been described in sporadic and a few familial cases but the genetic defect underlying IMD is unknown. In this study, we have examined 28 probands with DCO and seven probands with IMD for mutations in the SHOX gene by using polymorphic CA-repeat analysis, fluorescence in situ hybridisation (FISH), Southern blotting, direct sequencing and fibre-FISH analyses. This was combined with auxological examination of the probands and their family members. Evaluation of the auxological data showed a wide intra- and interfamilial phenotype variability in DCO. Out of 28 DCO probands, 22 (79%) were shown to have mutations in the SHOX gene. Sixteen unrelated DCO families had SHOX gene deletions. Four novel DCO-associated mutations were found in different families. In two additional DCO families, the previously described nonsense mutation (Arg195Stop) was detected. We conclude that mutations in the SHOX gene are the major factor in the pathogenesis of DCO. In a female proband with severe IMD and her unaffected sister, we detected an intrachromosomal duplication of the SHOX gene.

Asn540Lys mutation in fibroblast growth factor receptor 3 and phenotype in hypochondroplasia.

UNLABELLED: Hypochondroplasia is characterized by a disproportionate short stature with rhizomelic shortening of the limbs. Amino acid substitutions Asn540Lys, Asn540Thr and Ile538Val in the fibroblast growth factor receptor 3 (FGFR3) are considered to cause hypochondroplasia. In this study we examined the FGFR3 gene for the previously described hypochondroplasia mutations and the phenotype of 23 probands with clinically and radiologically diagnosed hypochondroplasia. For the phenotype comparison, the patients were divided into two groups: Group 1: hypochondroplasia with Asn540Lys substitution; Group 2: hypochondroplasia with no mutations identified so far. A three-generation family negative for the known hypochondroplasia mutations was examined with polymorphic markers flanking the FGFR1, FGFR2 and FGFR3 genes. Nine (39%) of 23 probands were found to be heterozygous for the Asn540Lys substitution. The individuals positive for the Asn540Lys substitution were significantly more disproportionate than the individuals without this mutation. In this respect, a genotype-phenotype correlation was found in our patients. However, some individuals belonging to the group without mutations identified so far showed similarly abnormal proportions. Genotyping/haplotyping in the three-generation family with hypochondroplasia showed that FGFR1, FGFR2 and FGFR3 genes were not linked to the hypochondroplasia phenotype in this family, thus further confirming the genetic heterogeneity of hypochondroplasia. CONCLUSION: Individuals with hypochondroplasia heterozygous for the Asn540Lys substitution are significantly more disproportionate than individuals without this mutation. Our study further confirms the clinical and genetic heterogeneity of hypochondroplasia.

Mutations in short stature homeobox containing gene (SHOX) in dyschondrosteosis but not in hypochondroplasia.

Dyschondrosteosis (DCO) and hypochondroplasia (HCH) are common skeletal dysplasias characterized by disproportionate short stature. The diagnosis of these conditions might be difficult to establish especially in early childhood. Point mutations and deletions of the short stature homeobox containing gene (SHOX) are detected in DCO and idiopathic short stature with some rhizomelic body disproportion, whereas mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are found in 40-70% of HCH cases. In this study, we performed mutational analysis of the coding region of the SHOX gene in five DCO and 18 HCH patients, all of whom tested negative for the known HCH-associated FGFR3 mutations. The polymorphic CA-repeat analysis, direct sequencing and Southern blotting were used for detection of deletions and point mutations. The auxological and radiological phenotype of these patients was carefully determined. Three novel mutations in DCO patients were found: (1) a deletion of one base (de1272G) (according to GenBank accession nos. Y11536, Y11535), resulting in a premature stop codon at position 75 of the amino acid sequence; (2) the transversion C485G resulting in the substitution Leu132Val; and (3) the transversion G549T causing an Arg153Leu substitution. These substitutions segregate with the DCO phenotype and affect evolutionarily conserved homeodomain residues, based on a comparison of homeobox containing proteins in 13 species. Moreover, these changes were not found in 80 unrelated, unaffected individuals. This strongly suggests that these mutations are pathogenic. The phenotype of our patients with DCO and HCH varied from mild to severe shortness and body disproportion. These results further support clinical and genetic heterogeneity of dyschondrosteosis and hypochondroplasia.

Demonstration of estrogen receptor-beta immunoreactivity in human growth plate cartilage.

Estrogens affect longitudinal bone growth through their action on endochondral bone formation. Two estrogen receptors are known, the classical estrogen receptor-alpha (ER alpha), newly demonstrated in human growth plate cartilage, and a recently cloned estrogen receptor-beta (ER beta). The present study aimed to localize a possible expression of ER beta protein in human growth plates. Tissue samples were obtained from tibial and femoral growth plates in four female pubertal patients undergoing epiphyseal surgery. Immunohistochemistry, using two different ER beta-specific antibodies, demonstrated positive staining for ER beta in hypertrophic epiphyseal chondrocytes from all patients. No staining was noted in resting or proliferative chondrocytes. These data suggest that in addition to ER alpha, human epiphyseal chondrocytes also express ER beta. The physiological role of ER beta in the regulation of longitudinal bone growth in humans remains to be elucidated.

A novel missense mutation Ile538Val in the fibroblast growth factor receptor 3 in hypochondroplasia. Mutations in brief no. 122. Online.

Hypochondroplasia and achondroplasia are skeletal dysplasias, characterized by autosomal dominant inheritance and disproportionate short stature, which occurs mainly due to growth failure of the extremities. Both dysplasias have been mapped to fibroblast growth factor receptor 3 (FGFR3) gene. For hypochondroplasia, two point mutations, both responsible for the Asn540Lys substitution in the region coding the tyrosine kinase domain have been reported. Here we report an A to G transition at position 1651, predicting an Ile538Val substitution in the FGFR3, in hypochondroplasia. The substitution is found in a swedish family with three affected members. The criteria for hypochondroplasia were disproportionate short stature and radiological evidence of shortened long bones and decrease or absence of normal increase in interpedicular distances of the lumbar column. The mutation was detected by direct sequencing and restriction enzyme Tai I digestion. The base change was not found in the FGFR3 genes of unaffected members of the family nor in seventy-five unrelated unaffected individuals, suggesting that it was not a polymorphism. The Ile538Val substitution is a conservative amino acid change (a hydrophobic amino acid incorporated for another hydrophobic amino acid). Nevertheless, it is located in the stretch of nine amino acids, which is highly conserved among all the human fibroblast growth factor receptors. Considering the location of this substitution and the segregation with the phenotype in this family, we propose that it is a causative mutation of hypochondroplasia. It is difficult to establish whether the Ile538Val substitution is rare in hypochondroplasia patients or whether the individuals, who have a moderate degree of short stature, rarely seek medical help for the short stature and consequently are rarely diagnosed as affected by hypochondroplasia.

Cerebrospinal fluid of newborn infants contains a deglycosylated form of the intermediate filament nestin.

Nestin is an intermediate filament protein found in CNS progenitor cells. Nestin reappears in CNS tumor cells and reactive astrocytes after CNS injury. In this study we investigated whether nestin could be detected in the cerebrospinal fluid (CSF) of newborn infants and whether expression levels change with gestational age (GA) and/or brain injury. Using Western blot analysis, we examined the expression of nestin in the CSF of newborn infants (GA 25-42 wk) with asphyxia (n = 14), periventricular leukomalacia and peri(intra)ventricular hemorrhage (n = 7), and in a control group (n = 11). Protein extract from the periventricular brain tissue of a 1-wk-old infant was also analyzed. Nestin was detected in all the CSF samples and in the protein extract from the periventricular brain tissue. Although the CSF levels of nestin expression did not change with increasing GA, the asphyxia group had significantly lower levels of nestin in the CSF. An unexpected finding was that brain-derived nestin had an apparent molecular mass of approximately 240 kD, whereas all analyzed CSF samples contained two nestin-immunoreactive proteins at 200 and 220 kD. Experimental deglycosylation of the 240-kD form reduced the molecular mass to 220 kD, indicating that nestin undergoes a specific deglycosylation upon release into the CSF.