Osteosclerotic bone dysplasia in siblings with a Fam20C mutation

Abstract

Fradin M, Stoetzel C, Muller J, Koob M, Christmann D, Debry C, Kohler M, Isnard M, Astruc D, Desprez P, Zorres C, Flori E, Dollfus H, Doray B. Osteosclerotic bone dysplasia in siblings with a Fam20C mutation.

Raine syndrome is an autosomal recessive disorder caused by mutations in the FAM20C gene. FAM20C codes for the human homolog of DMP4, a dentin matrix protein highly expressed in odontoblasts and moderately in bone. DMP4 is probably playing a role in the mineralization process. Since the first case reported in 1989 by Raine et al. 21 cases have been published delineating a phenotype which associates dysmorphic features, cerebral calcifications, choanal atresia or stenosis and thoracic/pulmonary hypoplasia. Kan and Kozlowski suggested the name of Raine syndrome to describe this new lethal osteosclerotic bone dysplasia. All the cases described were lethal during the neonatal period except for the last two reported patients aged 8 and 11 years who presented severe mental retardation. Here we describe two sisters, with an attenuated phenotype of Raine syndrome, who present an unexpectedly normal psychomotor development at ages 4 and 1, respectively. Identification of a homozygous mutation in the FAM20C gene confirmed the Raine syndrome diagnosis, thus contributing to the expansion of the Raine syndrome phenotype. This case report also prompted us to revisit the FAM20 gene classification and allowed us to highlight the ancestral status of Fam20C.

Conflicts of interest

The authors declare no conflict of interest.

Case report

Case report 1

We report herein on two sisters born from first cousin healthy parents of Algerian origin (Fig. 1). During the first pregnancy, the ultrasound examination at 21 weeks' gestation showed diffuse hyperechogenicity of the cerebral parenchyma and abnormal flat fetal facial profile suggestive of a Binder phenotype. The fetal karyotype was normal (46, XX) and infectious screening was negative. The fetal MRI examination at 30 gestational weeks showed increased diffuse perivascular signals.

Figure 1. Face of girl from observation 1, at 6 months (a) and 4 years (b, c) of age. Dysmorphic features include high forehead, hypertelorism with bilateral epicanthal folds and slightly downslanting palpebral fissures, nasal root hypoplasia and anteverted nares, dysplastic and posteriorly angulated ears with prominent lobule. (d, e) Face of girl from observation 2, at 1 year of age. The more severe dysmorphic features include brachycephaly, bilateral epicanthal folds, midface and nasal root hypoplasia with absence of nasal crest and micrognathia. (f) Fetal MRI from observation 2: signal abnormality within parieto-occipital and periventricular white matter. (g) CT scan from observation 2 at birth: cerebral calcifications within parieto-occipital and periventricular white matter. (h) Spine radiograph from observation 2 at the age of 3 months: increased density of vertebral bodies and calcifications of several intervertebral disks. (i) Femur and pelvis radiographs from observation 2 aged 3 months: absence of ossification of sacrum below S1 and presence of chain-like calcifications. (j, k) Pelvis and lower leg radiograph from observation 1 aged 3 years: reduction of osteosclerosis with satisfying cortico-medullary differentiation.

Normal vaginal delivery occurred at 37 weeks' gestation. The Apgar score was 10/10. The girl's birth weight was 3320 g, length was 49 cm and occipitofrontal circumference (OFC) was 34 cm. Craniofacial abnormalities included hypertelorism, epicanthus, hypoplastic and flat nose, associated with a nasal pyriform aperture and stenosis of the anterior segment of nasal fossae without choanal atresia. Blood analysis showed normal levels of circulating phosphate and calcium. Cardiac ultrasound showed a patent foramen ovale. Radiographs showed cerebral calcifications and revealed diffuse osteosclerosis including cranial base but normal cranial sutures. Cerebral ultrasound revealed periventricular white matter and basal ganglia hyperechogenicity suggestive of calcifications. The CT scan confirmed that nodular calcifications were located in the subependymal regions, the parieto-occipital subcortical white matter and the basal ganglia. Surgery for the nasal pyriform aperture stenosis was performed at 10 days of life. The girl was able to walk at 14 months. At the time of writing, she is 4 years old, has normal psychomotor development, especially for speech and pictural drawing and goes to regular school. Her height, weight and OFC are within the normal ranges. She has no sensorial anomalies and no history of respiratory distress. Craniofacial examination shows attenuation of the dysmorphic features (Fig. 1). Her palate is high and her teeth are small with enamel dysplasia. Radiographs show persistent bilateral cranial calcifications but no further osteosclerosis. Abdominal ultrasound examination reveals renal cortex calcifications. The brain CT scan reveals accentuation of calcifications located in the pallidum, and stabilization of the other calcifications. Blood circulating phosphate, calcium, parathormone, vitamin D are normal, as well as renal function and blood cell count.

Case report 2

Three years later, the second pregnancy of the couple showed a recurrence of the same ultrasound findings associated with polyhydramnios. The fetal female karyotype was normal and fetal MRI showed cerebral calcifications and the same profile suggestive of Binder phenotype.

Birth occurred at 38 weeks of gestation after a normal delivery. Birth length was short (43 cm) whereas weight (3144 g) and OFC (35.5 cm) were normal. The Apgar score was 9/9 because of a slight respiratory failure. Clinical examination of this female newborn showed brachycephaly, bilateral epicanthus, a Binder phenotype appearance with midface hypoplasia and microretrognatism, brachydactyly and slight thoracic narrowness. Blood phosphate and calcium were normal. Abdominal ultrasound was normal. Cardiac ultrasound revealed a patent foramen ovale of 2.5 mm. A CT scan showed a nasal pyriform aperture stenosis, without choanal atresia and osteosclerosis of the basilar region and absence of ossification of C3 to C5 vertebral bodies (M. Koob et al., submitted). Calcification of the parieto-occipital, frontal periventricular and basal ganglia were noted. Radiographs also showed the absence of ossification of the sacrum below S1 contrasting with the presence of sacral chain-like calcifications, calcifications of several intervertebral disks, and generalized osteosclerosis.

The child had surgical treatment of her pyriform aperture stenosis at 4 days of life, and underwent three other surgical procedures, including glossopexia, before leaving hospital at 4 months of life.

At 1 year old, the girl has a normal psychomotor development. She sat at 7 months and almost walks. Her height is still at −2 DS, but her weight and OFC are of medium range. She has no history of respiratory distress but frequent episodes of nasal obstruction. She has bilateral epicanthus, hypoplasia of nasal root, a narrow palate, and brachydactyly. The abdominal ultrasound reveals the presence of kidney calcifications. Radiographs show decreasing osteosclerosis and vertebral segmentation defects whereas the CT scan shows accentuation of frontal white matter and pallidum calcifications. Blood cell count is normal as blood calcium level. Circulating phosphate is slightly low at 1.32 mmol/l (normal ranges: 1.5–2.3 mmol/l), while parathormone and vitamin D levels are normal. There is moderately elevated alkaline phosphatases (378 U/l, normal ranges: 123–283 U/l) which could reflect bone remodeling.

Materials and methods

Informed consent was obtained from patients and their parents.

DNA sequencing and mutation screening

Genomic DNA was extracted from blood samples according to standard protocols using the Flexigene DNA Kit from Qiagen (Qiagen, Hilden, Germany).

Coding exons and flanking intronic regions of Fam20C were PCR amplified from 50 ng of genomic DNA. Based on the sequences retrieved from the NCBI map viewer (1) and the Ensembl website (2), nine sets of primers were designed using the primer3 (v.04.0) software (3). Bidirectional sequencing of the purified PCR products was performed using the ABI Big Dye Terminator 3.1 Sequencing Kit on an ABI3130 automated capillary sequencer (Applied Biosystems, Foster City, CA). Sequences were aligned and compared with reference sequences using the SeqScape software (v.2.5) (Applied Biosystems).

DNA analysis with microsatellite markers

The highly informative microsatellite markers D7S2477 was chosen to study the Fam20C locus. The sequences were obtained from the UCSC Genome Browser (4) and markers were performed on a CEQ8800 genetic analysis system (Beckman Coulter Inc., USA). Experimental conditions are available on request.

Bioinformatics

FAM20 sequences were collected using the eggNOG database (5) covering 55 eukaryotes ranging from Arabidopsis thaliana to Homo sapiens. The FAM20C, FAM20A and FAM20B proteins sequences were extracted from the KOG 3829 orthologous group. A high quality multiple sequence alignment (MSA) of all proteins from this dataset was then computed using AQUA (6). The MSA was manually refined, taking into account secondary structures, resulting in a final MSA including 102 protein sequences distributed as following: 27 FAM20A, 33 FAM20B and 42 FAM20C. Notably, the reference human FAM20C protein in Ensembl (2) is truncated from 570 to 274 amino acids. Therefore, we added the corresponding sequence (ID: DMP4_HUMAN and ACC: Q8IXL6) from the UniProt (7). The identified mutation matches the proline at position 314 in this reference sequence. The complete MSA is available upon request.

Based on this MSA, a phylogenetic tree was computed using the PhyML program (8). The parameters were set to compute 100 bootstrap replicates and to optimize topology, branch lengths and rate parameters. The results were visualized using the iTOL software (9).

Mutation analysis

The SIFT (10) and PolyPhen (11) programs were used to predict whether the missense mutation might impact the protein structure and function. The FAM20C MSA was used as input for the SIFT programs.

Results

Microsatellites informative analysis at the FAM20C locus showed that the two sisters are homozygous at this locus. Subsequent sequencing of FAM20C identified a homozygous variation c.940C>T (p.P314S) for the two affected girls, each parent being heterozygous for the variation (Fig. 2a). This variation was not found in 172 control chromosomes.

Figure 2. (a) Familial segregation of the c.940C>T (p.P314S) mutation with the two affected girls being homozygous and each parent being heterozygous. (b) Multiple sequence alignment of FAM20 family. The displayed region corresponds to the surrounding amino acids of the P314S mutation (highlighted by the red rectangle). When available representative sequences from the following species (covering major distinct metazoan clades) Homo sapiens, Mus musculus, Canis lupus, Ornithorhynchus anatinus, Takifugu rubripes, Ciona intestinalis, Drosophila melanogaster and Caenorhabditis elegans have been shown. The different FAM20 subfamilies are highlighted by different colors, green, orange and gray, respectively, for FAM20A, FAM20B and FAM20C. The amino acid coloring scheme is the one used by ClustalX. The figure has been prepared using Jalview (12). (c) Phylogenetic tree of FAM20 family. The different FAM20 subfamilies are highlighted by different colors, green, orange and gray, respectively, for FAM20A, FAM20B and FAM20C. Bootstrap support is indicated when superior to 70.

To assess the pathogenicity, we tested this missense variation using two bioinformatic prediction programs, PolyPhen (11) and SIFT (10). The predictions obtained were ‘probably damaging’ (score 2.651) and could ‘affect protein function’ (score 0.00), respectively. Sequence conservation analysis shows that the P314 is strictly conserved in all sequences of FAM20B proteins, covering the major metazoan species (Fig. 2b). Taken together these data suggest that the missense P314S in the FAM20C protein sequence is probably to be the causative mutation of the observed phenotype.

FAM20C is a member of a protein family encompassing three related subfamilies, namely FAM20A, FAM20B and FAM20C. So far the FAM20 family has been characterized only in metazoa and the FAM20B subfamily is thought to be the ancestor of the other two subfamilies (13).

Our analysis confirms previous results observed for the whole FAM20 family, with the N-terminus being the most variable part of the protein and the C-terminus the most conserved part. This statement is also true within each subfamily. On average, the FAM20B subfamily is more conserved within the vertebrates (mean percent identity ∼90.9%), compared to FAM20A (∼81.3%) and FAM20C (∼86.9%). Comparison of the human FAM20 subfamilies showed that FAM20A and FAM20C are more closely related (with 44.6% residue identity) with respect to FAM20B, which shares only 35% and 36.8% identity with FAM20A and FAM20C, respectively. Based on our MSA of all the FAM20 proteins currently available in complete genomes, we refined the presence and absence profiles of each subfamily (Fig. S1, supporting information) and clearly assign the only Caenorhabditis elegans sequence to the FAM20C subfamily and not to FAM20B as indicated previously (13). Furthermore, the phylogenetic tree (Fig. 2c and Fig. S2, supporting information for a reduced version with very high support) allows us to conclude that one ancestral FAM20 (a FAM20C-like) appeared in C. elegans, that gave rise to the FAM20C subfamily. Two duplication events then occurred: one within the arthropods leading to the FAM20B subfamily and one within the vertebrates leading to FAM20A. FAM20C is thus the ancestral subfamily.

Discussion

The family study reported here expands the phenotypic description of Raine syndrome from an extremely severe phenotype to milder non-lethal manifestations. Bioinformatics analysis based on the reported novel mutation and the literature allowed us to revisit the FAM20C gene history.

Among the 22 cases of Raine syndrome reported in the literature, (14,15,16–28) only the two most recently described cases survived after the neonatal period (28). The boys aged 8 and 11 years, respectively, at the time of the report, presented severe mental retardation in striking contrast with the two sisters reported herein, who have normal psychomotor development.

Since the recent identification of the FAM20C gene for Raine syndrome, (14,29) only nine patients have been studied using molecular analysis, resulting in the description of one case of homozygous deletion and eight cases of point mutations. These mutations involved splice site changes or missense mutations. Until now, it could be considered somewhat premature to suggest genotype/phenotype correlations, given the small number of patients and the fact that every case screened so far had unique mutations, all located in the evolutionary conserved C-terminal domain (CCD) (14). The missense mutation P314S identified in these two mildly affected sisters has not been previously documented. Nevertheless the segregation of the mutation in the family, the absence of the mutation in normal controls, the coherent prediction of PolyPhen and SIFT, and the high conservation of this residue located in the CCD indicates the pathogenicity of the mutation. The MSA of the complete sequence of the three FAM20 subfamilies shed light on the relationships within and between the subfamilies leading to a more precise view of the evolutionary history of the FAM20 family. In the light of our findings, we propose that the ancestral FAM20 protein is closer to the FAM20C subfamily than to FAM20B, as was previously hypothesized (13). Our new evolutionary scenario is more parsimonious, since only two duplications are necessary to generate the three FAM20 subfamilies. In order to further complete and validate the proposed scenario, more FAM20 sequences from additional genomes are required. The assignment of sequences to their correct subfamily is essential while predicting the pathogenicity of a new variant. Indeed, among other criteria, SIFT and PolyPhen use the sequence conservation as a proxy for discriminating an SNP from a mutation.

The two observations described here show that the clinical spectrum associated with mutations in FAM20C is much wider than the classical neonatal lethal presentation and the two severely affected siblings reported previously. The two sisters show good psychomotor development after adequate management during the neonatal period. The elder sister has normal growth, very slight dysmorphic facies whereas the phenotype of the younger sister is a little more pronounced with growth at the lower limit and moderate craniofacial dysmorphism. In 2009, Simpson et al. questioned whether the survival through infancy of the patients he described was because of a milder phenotype or aggressive neonatal therapeutic measures performed on his patients and not pursued in the majority of the previously reported cases. Our familial case, and especially the elder sister who did not require prolonged medical care, may represent the mildest form of the Raine syndrome spectrum reported to date. It highlights the very delicate task of prognosis in case of prenatal discovery of features compatible with Raine syndrome such as hypoplasia of the middle facial region and ocular proptosis associated with brain calcification. A major differential diagnosis of Raine syndrome is chondrodysplasia punctata which was first suspected in the eldest sister. Although these two conditions share common features, such as Binder phenotype and ectopic calcifications, brain calcifications and osteosclerosis strongly supports Raine syndrome diagnosis, which is confirmed by FAM20C analysis. Identification of a mutation is of major importance for genetic counseling. It may also help to extend the phenotypic spectrum of Raine syndrome-related phenotypes for the future cases.

Acknowledgements

The authors wish to thank all medical contributors from genetic, pediatric, radiologic and biologic units. They warmly thank the parents of the children for their constant availability.