Mutations in RELT cause autosomal recessive amelogenesis imperfecta

Amelogenesis imperfecta (AI) is a collection of isolated (non-syndromic) inherited diseases affecting dental enamel formation or a clinical phenotype in syndromic conditions. We characterized three consanguineous AI families with generalized irregular hypoplastic enamel with rapid attrition that perfectly segregated with homozygous defects in a novel gene: RELT that is a member of the tumor necrosis factor receptor superfamily (TNFRSF). RNAscope in situ hybridization of wild-type mouse molars and incisors showed specific Relt mRNA expression by secretory stage ameloblasts and by odontoblasts. Relt^−/− mice generated by CRISPR/Cas9 exhibited incisor and molar enamel malformations. Relt^−/− enamel had a rough surface and underwent rapid attrition. Normally unmineralized spaces in the deep enamel near the dentino-enamel junction (DEJ) were as highly mineralized as the adjacent enamel, which likely altered the mechanical properties of the DEJ. Phylogenetic analyses showed the existence of selective pressure on RELT gene outside of tooth development, indicating that the human condition may be syndromic, which possibly explains the history of small stature and severe childhood infections in two of the probands. Knowing a TNFRSF member is critical during the secretory stage of enamel formation advances our understanding of amelogenesis and improves our ability to diagnose human conditions featuring enamel malformations.     

A new form of skeletal dysplasia with amelogenesis imperfecta and platyspondyly

We report two patients, born of consanguineous parents, affected by a disorder resulting in mild growth retardation. Hallmarks are amelogenesis imperfecta (absence of the enamel cap) associated with brachyolmia-like anomalies: platyspondyly with short pedicles, narrow intervertebral and interpedicular distances, rectangular-shaped vertebrae with posterior scalloping and herniation of the nuclei, and broad femoral necks. Inheritance appears to be autosomal recessive.     

Characterisation of molecular defects in X-linked amelogenesis imperfecta (AIH1)

Amelogenins are an heterogenous family of proteins produced by ameloblasts of the enamel organ during tooth development. Disturbances of enamel formation occur in amelogenesis imperfecta, a clinically heterogenous group of inherited disorders characterised by defective enamel biomineralisa-tion. An amelogenin gene, AMGX, has been mapped to the short of the X chromosome (Xp22.1—p22.3) and has been implicated in the molecular pathology of X-linked amelogenesis imperfecta (AIH1). We have identified three families exhibiting AIH1 and screened the AMGX gene for mutations using single-strand conformational polymorphism analysis and DNA sequencing. Three novel mutations were identified: a C-T substitution in exon 5, and a G-T substitution and single cytosine deletion in exon 6, confirming the existence of extensive allelic heterogeneity in this condition. The identification of family-specific mutations will enable early identification of affected individuals and correlation of clinical phenotype with genotype will facilitate an objective system of disease classification. © 1995 Wiley-Liss, Inc.     

Whole-exome sequencing,without prior linkage,identifies a mutation in LAMB3 as a cause of dominant hypoplastic amelogenesis imperfecta

The conventional approach to identifying the defective gene in a family with an inherited disease is to find the disease locus through family studies. However, the rapid development and decreasing cost of next generation sequencing facilitates a more direct approach. Here, we report the identification of a frameshift mutation in LAMB3 as a cause of dominant hypoplastic amelogenesis imperfecta (AI). Whole-exome sequencing of three affected family members and subsequent filtering of shared variants, without prior genetic linkage, sufficed to identify the pathogenic variant. Simultaneous analysis of multiple family members confirms segregation, enhancing the power to filter the genetic variation found and leading to rapid identification of the pathogenic variant. LAMB3 encodes a subunit of Laminin-5, one of a family of basement membrane proteins with essential functions in cell growth, movement and adhesion. Homozygous LAMB3 mutations cause junctional epidermolysis bullosa (JEB) and enamel defects are seen in JEB cases. However, to our knowledge, this is the first report of dominant AI due to a LAMB3 mutation in the absence of JEB.     

Taurodontism of the mandibular first permanent molar distinguishes between the tricho-dento-osseous (TDO) syndrome and amelogenesis imperfecta

Seow WK. Taurodontism of the mandibular first permanent molar distinguishes between the tricho-dento-osseous (TDO) syndrome and amelogenesis imperfecta.
Clin Genet 1993: 43: 240–246. © Munksgaard, 1993
The diagnosis of tricho-dento-osseous (TDO) syndrome is often confused with that of a variant of amelogenesis imperfecta (AI) which shows similar dental features of hypomaturation-hypoplastic enamel defects and putative taurodontism. In this controlled study, an objective, biometric measurement technique was used to determine the prevalence and severity of taurodontism of the mandibular first permanent molar in 23 patients with AI and one patient with TDO syndrome compared with age- and sex-matched controls. The published radiographs of previous cases of TDO and hypomaturation-hypoplastic AI were also reviewed with regard to the presence and severity of taurodontism. The results indicate that in all cases of TDO, taurodontism of the molars including mandibular first permanent molars was consistently present and in a severe form. By contrast, the taurodontic defects present in all cases of AI, including the hypomaturation-hypoplastic variant were not significantly different from matched, healthy controls. Of significance is the fact that in all the AI cases, none of the taurodontic defects were present on the mandibular first permanent molars. The results indicate that true taurodontism as indicated by a change in the mandibular first permanent molars occurs only in the TDO syndrome. This feature may be used to differentiate clearly between TDO and AI.     

Novel synonymous and missense variants in FGFR1 causing Hartsfield syndrome

Hartsfield syndrome is a rare clinical entity characterized by holoprosencephaly and ectrodactyly with the variable feature of cleft lip/palate. In addition to these symptoms patients with Hartsfield syndrome can show developmental delay of variable severity, isolated hypogonadotropic hypogonadism, central diabetes insipidus, vertebral anomalies, eye anomalies, and cardiac malformations. Pathogenic variants in FGFR1 have been described to cause phenotypically different FGFR1‐related disorders such as Hartsfield syndrome, hypogonadotropic hypogonadism with or without anosmia, Jackson–Weiss syndrome, osteoglophonic dysplasia, Pfeiffer syndrome, and trigonocephaly Type 1. Here, we report three patients with Hartsfield syndrome from two unrelated families. Exome sequencing revealed two siblings harboring a novel de novo heterozygous synonymous variant c.1029G>A, p.Ala343Ala causing a cryptic splice donor site in exon 8 of FGFR1 likely due to gonadal mosaicism in one parent. The third case was a sporadic patient with a novel de novo heterozygous missense variant c.1868A>G, p.(Asp623Gly).     

Novel missense loss-of-function mutations of WNT1 in an autosomal recessive Osteogenesis imperfecta patient

Osteogenesis imperfecta (OI) is a heritable skeletal disorder characterized by bone fragility and low bone mass. Recently, loss-of-function mutations of WNT1 have been reported to be causative in OI or osteoporosis. We report an OI patient with novel compound heterozygous WNT1 missense mutations, p.Glu123Asp and p.Cys153Gly. Both mutations are found in the exon 3, and the p.Glu123Asp is the most proximal N-terminus missense mutation among the reported WNT1 missense mutations in OI patients. In vitro functional analysis reveals that while expression of wildtype WNT1 stimulates canonical WNT1-mediated β-catenin signaling, that of individual WNT1 mutant fails to do so, indicative of the pathogenic nature of the WNT1 variants. Although the pathogenic mechanism of WNT1 defects in OI has yet to be uncovered, these findings further contribute to the implications and importance of functional relevance of WNT1 in skeletal disorders.     

Ultrastructural Study of Tooth Enamel with Amelogenesis Imperfecta in Al-Nephrocalcinosis Syndrome

This paper describes the ultrastructure of the affected enamel and the clinical features in two siblings with the syndrome of nephrocalcinosis and amelogenesis imperfecta. Nephrocalcinosis was diagnosed by intravenous pyelography, and confirmed by ultrasonography and CT scan. Amelogenesis imperfecta AI was diagnosed clinically and histologically.

Light microscopy showed that the affected enamel surfaces were rough and the enamel was hypoplastic and mainly positively birefringent. Scanning electron microscopy revealed a rough and extensively cracked enamel surface covered with oval shaped blister-like protrusions. TEM snowed porous enamel consisting of loosely packed and randomly oriented thin ribbon-like crystals with little or no prismatic structure.

Observations showed that hypoplasia together with hypocalcification and/or hypomaturation defects were present in the same tooth, indicating the possibility of an abnormality in interstitial matrix, leading to dystrophic calcification in the kidney and abnormal tooth enamel formation, or alternatively an involvement of two separate but closely linked genes.     

Circulating levels of sTNFR and discrepancy between cytotoxicity and immunoreactivity of TNF-α in patients with visceral leishmaniasis

Objectives To study the influence of soluble tumour necrosis factor (TNF) receptors (sTNFR) on bioactivity and immunoreactivity of TNF-α in patients with visceral leishmaniasis (Kala-azar) and to examine the association between circulating levels of sTNFR type I and type II with clinical manifestations of the disease.
Methods   Ten patients with Kala-azar were enrolled. Plasma samples for TNF-α and sTNFR were obtained on days 0, 7 and 21–28 of antimonial therapy. Bioactivity of TNF-α was measured by cytotoxicity to L-929 cells and immunoreactivity by enzyme-linked immunosorbent assay (ELISA). sTNFR-I and sTNFR-II were measured by ELISA.
Results   Measured by ELISA, TNF-α was detected at baseline in all patients (range from 22.3 to 163 pg/mL) and showed a linear decline over time on therapy ( r  = −0.49, P  = 0.007). In contrast, when measured by cytotoxicity assay, TNF-α was detected in only one patient at baseline (193 pg/mL) and in four patients at the end of therapy (38.7, 95, 133 and 232 pg/mL) and there was no linear association between TNF-α and duration of therapy ( r  = −0.18, P  = 0.45). sTNFR-I and sTNFR-II were detected in all patients before therapy. There was a strong positive correlation between plasma concentrations of sTNFR-I and sTNFR-II ( r  = 0.8, P  = 0.006). Levels of sTNFR-I and sTNFR-II declined exponentially with time on therapy.
Conclusions   We concluded that sTNFR-I and sTNFR-II are related to disease activity in patients with Kala-azar and that these circulating receptors may interfere with the biological activity of TNF-α in patients with Kala-azar.     

Higher serum levels of tumour necrosis factor and its soluble receptors are associated with ovarian tumours

Introduction

Tumour necrosis factor (TNF) and its soluble receptors type 1 (sTNF-R1) and type 2 (sTNF-R2) have been suggested as key mediators between apoptosis and cancer cell progression. The aim was to examine concentrations of the parameters in the serum of women with ovarian tumour and in the fluid from ovarian cysts of women with serous cystadenoma.

Material and methods

The study included 125 women with ovarian tumours. As a control, sera were obtained from 70 healthy female volunteers. Concentrations of TNF, sTNF-R1 and sTNF-R2 were measured by enzyme-linked immunosorbent assay (ELISA).

Results

Significant increases of TNF, sTNF-R1 and sTNF-R2 were found in the serum of women with ovarian tumour in comparison to the control (p < 0.0001). The highest levels of all studied parameters were observed in women with ovarian cancer. In the ovarian cyst fluid the concentrations of the evaluated parameters increased significantly as compared to the serum (p < 0.0001).

Conclusions

Our data showed changes in regulatory mechanisms of apoptosis in women with ovarian tumours which are associated with increased concentrations of all studied factors. Serum estimated TNF and especially sTNF-R may be used as complementary diagnostic markers in patients with ovarian tumours.     

Mutant forms of tumour necrosis factor receptor I that occur in TNF-receptor-associated periodic syndrome retain signalling functions but show abnormal behaviour

Tumour necrosis factor (TNF)-receptor-associated periodic syndrome (TRAPS) is a hereditary autoinflammatory disorder involving autosomal-dominant missense mutations in TNF receptor superfamily 1A (TNFRSF1A) ectodomains. To elucidate the molecular effects of TRAPS-related mutations, we transfected HEK-293 cells to produce lines stably expressing high levels of either wild-type (WT) or single mutant recombinant forms of TNFRSF1A. Mutants with single amino acid substitutions in the first cysteine-rich domain (CRD1) were produced both as full-length receptor proteins and as truncated forms lacking the cytoplasmic signalling domain (deltasig). High-level expression of either WT or mutant full-length TNFRSF1A spontaneously induced apoptosis and interleukin-8 production, indicating that the mutations in CRD1 did not abrogate signalling. Consistent with this, WT and mutant full-length TNFRSF1A formed cytoplasmic aggregates that co-localized with ubiquitin and chaperones, and with the signal transducer TRADD, but not with the inhibitor, silencer of death domain (SODD). Furthermore, as expected, WT and mutant deltasig forms of TNFRSF1A did not induce apoptosis or interleukin-8 production. However, whereas the WT full-length TNFRSF1A was expressed both in the cytoplasm and on the cell surface, the mutant receptors showed strong cytoplasmic expression but reduced cell-surface expression. The WT and mutant deltasig forms of TNFRSF1A were all expressed at the cell surface, but a proportion of the mutant receptors were also retained in the cytoplasm and co-localized with BiP. Furthermore, the mutant forms of surface-expressed deltasig TNFRSF1A were defective in binding TNF-alpha. We conclude that TRAPS-related CRD1 mutants of TNFRSF1A possess signalling properties associated with the cytoplasmic death domain, but other behavioural features of the mutant receptors are abnormal, including intracellular trafficking and TNF binding.     

Biallelic variants in four genes underlying recessive osteogenesis imperfecta

Osteogenesis imperfecta (OI) is an inherited heterogeneous rare skeletal disorder characterized by increased bone fragility and low bone mass. The disorder mostly segregates in an autosomal dominant manner. However, several rare autosomal recessive and X-linked forms, caused by mutations in 18 different genes, have also been described in the literature.Here, we present five consanguineous families segregating OI in an autosomal recessive pattern. Affected individuals in the five families presented severe forms of skeletal deformities. It included frequent bone fractures with abnormal healing, short stature, facial dysmorphism, osteopenia, joint laxity, and severe scoliosis. In order to search for the causative variants, DNA of at least one affected individual in three families (A-C) were subjected to whole exome sequencing (WES). In two other families (D-E), linkage analysis using highly polymorphic microsatellite markers was followed by Sanger sequencing. Sequence analysis revealed two novels and three previously reported disease-causing variants. The two novel homozygous variants including [c.824G > A; p.(Cys275Tyr)] in the SP7 gene and [c.397C > T, p.(Gln133*)] in the SERPINF1 gene were identified in families A and B, respectively. The three previously reported homozygous variants including [c.497G > A; p.(Arg166His)] in the SPARC gene, (c.359-3C > G; intron 2) and [c.677C > T; p.(Ser226Leu)] in the WNT1 gene were identified in family C, D, and E.In conclusion, our findings provided additional evidence of involvement of homozygous sequence variants in the SP7, SERPINF1, SPARC and WNT1 genes causing severe OI. It also highlights the importance of extensive genetic investigations to search for the culprit gene in each case of skeletal deformity.     

Increased expression and occupancy of receptors for tumour necrosis factor on blood monocytes from tuberculosis patients.

Blood monocytes from tuberculosis patients release high amounts of tumour necrosis factor-alpha (TNF-alpha). Because the biological efficiency of TNF-alpha would depend on the expression of TNF-alpha receptors on target cells, we thought to analyse the capacity of blood monocytes from a group of patients with pulmonary tuberculosis to bind 125I-TNF-alpha. We report a slight but not significant enhancement in specific binding of 125I-TNF-alpha on monocytes of 15 consecutively studied patients compared with 10 controls. Per cent cell surface bound and internalized 125I-TNF-alpha was identical in the two groups. To evaluate the receptor occupancy by endogenously generated TNF-alpha, similar experiments were performed after cell exposure to low-pH glycine buffer. Under these conditions, specific binding of 125I-TNF-alpha was significantly higher on tuberculosis monocytes compared with control monocytes. Moreover, the occupancy of TNF-alpha receptors by endogenously generated TNF-alpha that was found to be significantly higher on tuberculosis monocytes than on control monocytes, was directly related to the enhanced capacity of mononuclear cells to generate TNF-alpha in vitro. It normalized after 3 months of antituberculous therapy. Scatchard analysis of the binding data revealed that tuberculosis infection caused a significant increase in high affinity 125I-TNF-alpha binding to monocytes without any significant change in the dissociation constant. Collectively, these results indicate an up-regulation of TNF-alpha generation and binding to blood monocytes in patients with pulmonary tuberculosis. They provide support to the hypothesis that TNF-alpha is of critical importance in the pathogenesis of this infection.