Abnormal vertebral segmentation and the notch signaling pathway in man

AVS Phenotypes Following Mendelian Inheritance

This group includes the distinctive condition known as spondylothoracic dysostosis (STD) and SCD types 1–3, all of which appear to follow autosomal recessive (AR) inheritance. STD is the term best reserved for the distinctive condition, most commonly reported in Puerto Ricans, which is characterized by severe shortening of the trunk and a radiological appearance of the ribs fanning out from their vertebrocostal origin in a “crab-like” manner (Fig. 1). Fusion of the ribs is present posteriorly at their vertebral origin, but otherwise they are usually neatly aligned and packed tightly together. This condition has been well characterized in a recent study (Cornier et al.,2004). Infant mortality is approximately 50% due to restrictive respiratory insufficiency, although the availability of pediatric intensive care greatly improves the prognosis. In early life, the prominence of the vertebral pedicles radiologically has led us to suggest that the term “tramline” sign (Fig. 1) neatly describes the appearance. In addition, in horizontal section on CT scanning, the vertebral bodies conform to a “sickle cell” shape (Cornier et al.,2004). Studies of the ultrastructure at necropsy are scarce. However, one report (Solomon et al.,1978) commented on an increased ratio between cartilage and bone on parasagittal sections of the vertebral bodies, with persistence of cartilage about the midline. The ossification centers were disorganized and varied in size and distribution and in the lateral portion of some vertebral bodies longitudinal clefts of cartilage separated anterior and posterior ossification centers. Although the modeling of the vertebral bodies was severely disorganized, microscopic bone formation and structure were not disturbed.

The STD phenotype, apparently seen most frequently in Puerto Ricans, is sometimes reported as Jarcho-Levin syndrome (JLS) (Lavy et al.,1966; Moseley and Bonforte,1969; Pochaczevsky et al.,1971; Pérez-Comas and García-Castro,1974; Gellis and Feingold,1976; Trindade and de Nóbrega,1977; Solomon et al.,1978; Tolmie et al.,1987; Schulman et al.,1993; McCall et al.,1994; Mortier et al.,1996). Jarcho and Levin (1938) described a Puerto Rican family with two affected siblings, but the radiological phenotype (Fig. 2) differed slightly from STD as illustrated in Figure 1 and it is not certain that they had the same condition. Use of the eponymous term JLS tends to be indiscriminate, and for that reason, we believe should be discontinued. For example, there are other case reports designated as JLS, which are neither very similar to those described by Jarcho and Levin nor consistent with STD (Poor et al.,1983; Karnes et al.,1991; Simpson et al.,1995; Aurora et al.,1996; Eliyahu et al.,1997; Rastogi et al.,2002). Once a gene for STD in the Puerto Rican families is identified, it will be possible to undertake genotype–phenotype studies that will further facilitate classification.

The phenotype for which we prefer to restrict the use of the term spondylocostal dysostosis (SCD) differs from STD by virtue of the ribs being irregularly aligned and manifesting variable points of fusion along their length (Fig. 3). The spine is not usually so severely shortened compared with STD but, as with STD, involvement of the vertebral bodies is extensive, usually contiguous, and often affecting all spinal regions. Nevertheless, the thoracic spine is often more severely affected than other regions. AR inheritance has been regularly reported for this phenotype (Norum and McKusick,1969; Cantú et al.,1971; Castroveijo et al.,1973; Franceschini et al.,1974; Silengo et al.,1978; Beighton and Horan,1981; Turnpenny et al.,1991; Satar et al.,1992), and we now know that SCD can be due to mutations in the Notch pathway genes DLL3 (SCD1), MESP2 (SCD2), or LNFG (SCD3) genes, although it is likely that other gene(s) will be identified in due course. The phenotypic features of SCD types 1–3 are described below in discussion with the molecular aspects of their respective genes. Extensive and contiguous involvement of all spinal regions appears to be a feature that is highly suggestive of a Mendelian form of SCD and is likely to explain some isolated cases reported in the literature, for example the case of Young and Moore (1984). However, the affected twin in a case of monozygotic twins discordant for SCD showed a similar phenotype (Van Thienen and Van der Auwera,1994). and this finding is difficult to explain on this basis.

Additional abnormalities have been reported in some families with SCD apparently demonstrating AR inheritance. For example, urogenital anomalies (Casamassima et al.,1981), congenital heart disease (Delgoffe et al.,1982; Simpson et al.,1995; Aurora et al.,1996), and inguinal herniae in males (Bonaime,1978). Limb anomalies have been described in COVESDEM syndrome (Wadia et al.,1978) but this is believed to be a case of Robinow syndrome.

Families with SCD apparently following autosomal dominant (AD) inheritance have also been reported (Van der Sar,1952; Rütt and Degenhardt,1959; Peralta et al.,1967; Rimoin et al.,1968; Kubryk and Borde,1981; Temple et al.,1988; Lorenz and Rupprecht,1990), but the causative genes in these cases are not known.

The roles of Notch signaling pathway genes in causing SCD (types 1–3) are at least partially understood (see below). However, there are syndromes following Mendelian inheritance that include variable degrees of AVS but the functions of the genes with respect to spinal development is poorly understood. This group includes, for example, ROR2 in Robinow syndrome and CHD7 in CHARGE syndrome. It is not clear what role these genes play in somitogenesis.

AVS Associated With Chromosomal Aberrations

Some clues to genetic causes of AVS may come from patients with axial skeletal defects and chromosomal abnormalities. These cases are relatively rare, and apart from trisomy 8 mosaicism (Riccardi,1977), there is no clear consistency to the group. Deletions affecting both 18q (Dowton et al.,1997) and 18p (Nakano et al.,1977) have been reported, a supernumerary dicentric 15q marker (Crow et al.,1997), and an apparently balanced translocation between chromosomes 14 and 15 (De Grouchy et al.,1963). Genomic haploinsufficiency may be unmasking a Mendelian locus for SCD such that the mechanism is autosomal recessive at the molecular level but the paucity and inconsistency of these cases may indicate that MVSD in association with MCA represents a common pathway of complex pleiotropic developmental mechanisms that are sensitive to a range of unbalanced karyotypes. It is also possible that chromosome mosaicism accounts for some cases where there is marked asymmetry in the radiological phenotype, which would also explain the apparent sporadic occurrence, but currently there is no evidence base for this as skin or tissue biopsy is rarely undertaken.

Delta-like 3 (DLL3)

Autozygosity DNA mapping studies in a large inbred Arab kindred with seven affected individuals was a key breakthrough in identifying the genetic basis of SCD (Turnpenny et al.,1991,1999). The locus identified, 19q13.1, is syntenic with mouse chromosome 7 (Giampietro et al.,1999), which harbors the Dll3 gene. The other key breakthrough was the identification of a mutation in Dll3 as the cause of MVSD in a radiation-damaged mouse known as Pudgy (Grüneberg,1961; Dunwoodie et al.,1997; Kusumi et al.,1998). DLL3 was, therefore, the obvious candidate SCD in the large inbred Arab kindred, and other families, demonstrating linkage to 19q13.1. Sequencing initially identified mutations in three consanguineous families (Bulman et al.,2000).

DLL3 encodes a ligand for Notch signaling and the gene comprises eight exons and spans approximately 9.2 kb of chromosome 19. A 1.9-kb transcript encodes a protein of 618 amino acids. The protein consists of a signal sequence, a Delta–Serrate–Lag2 (receptor interacting) domain, six epidermal growth factor (EGF) -like domains, and a transmembrane domain (Fig. 8). In animal models Dll3 shows spatially restricted patterns of expression during somite formation and is believed to have a key role in the cell signaling processes, giving rise to somite boundary formation, which proceeds in a rostrocaudal direction with a precise temporal periodicity driven by an internal oscillator, or molecular “segmentation clock” (McGrew and Pourquié,1998; Pourquié,1999).

Mutated DLL3 in man results in abnormal vertebral segmentation throughout the entire spine, with all vertebrae losing their normal form and regular three-dimensional shape. The most dramatic changes, radiologically, affect the thoracic vertebrae and the ribs are mal-aligned with a variable number of points of fusion along their length (Fig. 9a). There is an overall symmetry of the thoracic cage and minor, nonprogressive, scoliotic curves that do not require corrective surgery. These features define SCD. In early childhood, before ossification is complete, the vertebrae have smooth, rounded outlines—especially in the thoracic region—an appearance for which we suggested the term “pebble beach” sign (Fig. 9b). We designate this as SCD type 1 (SCD1), although DLL3-associated SCD is an alternative term (Martínez-Frías,2004). Additional anomalies appear to be rare, but in one case abdominal situs inversus was present, although the link with mutated DLL3 is uncertain. In one family, affected siblings homozygous for the exon 8 mutation 1369delCGCTCCCGGCTACATGG (C655M660del17) manifested a form of distal arthrogryposis in keeping with fetal akinesia sequence, and both succumbed in early childhood (C. McKeown, personal communication). This additional phenotype may have been a distinct AR condition segregating coincidentally to SCD, because there was multiple inbreeding in the family. Spinal cord compression and associated neurological features have not been observed in SCD1, and intelligence and cognitive performance are normal. This finding suggests that DLL3 is not expressed in human brain, which contrasts to findings in the mouse, where central nervous system defects have been found (Kusumi et al.,2001), including defects in the neuroventricles of the Pudgy mouse (Kusumi et al.,1998; Dunwoodie et al.,2002).

To date, 24 mutations (Table 3) have been identified in SCD patients from 26 families (Table 4). Only five of these families are definitely nonconsanguineous, and mutations have been identified in a wide range of ethnic groups. Eighteen mutations truncate the protein and six are missense. Some missense mutations may give rise to a slightly milder phenotype and protein modeling studies may help explain these subtle phenotypic differences in due course. With the one exception of a mutation in the transmembrane domain, all mutations affect the extracellular domain of the gene and are clustered in exons 4–8. Three mutations have been identified in more than one family. The 945delAT (T315C316del2) mutation was found in two ethnic Pakistani kindreds originating from Kashmir, and DNA marker analysis flanking the DLL3 locus has identified a common haplotype, supporting a common ancestry. Similarly, the 614insGTCCGGGACTGCG (R205ins13) mutation was found in two consanguineous families, one ethnic Lebanese Arab and the other ethnic Turkish. Haplotype analysis again supports a common ancestry for these two Levant kindreds. The 599-603dup mutation is present in the original Arab kindred, homozygous in those affected, but in a Spanish family, the affected child is heterozygous for the same mutation. Haplotype analysis on these two pedigrees does not support a common ancestry, and the 599-603dup mutation is, therefore, believed to be recurrent, occurring as it does within a region of the gene with multiple repeat GCGGT sequences. Slipped mispairing during DNA replication is the likely explanation of this insertion mutation.

The missense mutation G504D was found in a Northern European family originally reported as demonstrating autosomal dominant SCD (Floor et al.,1989) but recently shown to be an example of pseudodominant inheritance (Whittock et al.,2004a). At present, there is no confirmed case of AD SCD due to mutated DLL3. However, in the large Arab family reported by Turnpenny et al. (1991), one female heterozygous for the 599-603dup mutation had a mild thoracic scoliosis, but no associated segmentation abnormality in the thoracic region, and a very localized segmentation anomaly in the lower lumbar vertebrae. It is possible her scoliosis and lumbar segmentation anomaly were coincidental to her DLL3 carrier status, as no other obligate carrier in the kindred is known to have similar features. It is possible she carried a mutation in a separate somitogenesis gene and was, therefore, a manifesting “double heterozygote,” an example of which is known in an animal model (Kusumi et al.,2003).

In general, we have observed a remarkable consistency in the radiological phenotype in mutation-positive cases (Turnpenny et al.,2003), and with experience, scrutiny of the radiograph is usually possible to identify those patients who will prove to have DLL3 mutations. Importantly, DLL3 mutations have not been found in the wide variety of more common, although diverse, phenotypes that include MVSD and abnormal ribs (Maisenbacher et al.,2005; Giampietro et al.,2006). Therefore, there is remarkably little clinical heterogeneity for the axial skeletal malformation due to mutated DLL3, which has significant implications for the application of genetic testing in the clinical setting.

In relation to defects of the axial skeleton in man, identification of the DLL3 gene in SCD has represented a breakthrough in understanding the causative basis of this group of malformations, as well as highlighting another example of cross-species biological homology. It has become the paradigm for searching for the genetic basis of other SCD phenotypes, which led directly to the identification of MESP2, and later LNFG, in cases of SCD.

Mesoderm Posterior 2 (MESP2)

The identification of mutated MESP2 in association with SCD arose from the study of two small SCD families in whom no DLL3 mutations were found and linkage to 19q13.1 was excluded. In these families, the radiological phenotype was similar but subtly different to SCD type 1. Thoracic vertebrae were severely affected but the lumbar vertebrae were only mildly, as shown by magnetic resonance imaging (MRI; Fig. 10).

Genome wide homozygosity mapping in one family of Lebanese Arab origin revealed 84 homozygous markers scattered throughout the genome, of which 6 were concentrated in a block on 15q (D15S153 to D15S120) and 5 were concentrated in a block on 20q (D20S117 to D20S186). Subsequent mapping excluded linkage to the 20q region but demonstrated linkage to the 15q markers D15S153, D15S131, D15S205 and D15S127. Fine mapping using additional markers demonstrated a 36.6 Mb region on 15q21.3-15q26.1, between markers D15S117 and D15S1004, with a maximum two-point lod score of 1.588, at θ = 0 for markers D15S131, D15S205, D15S1046, and D15S127. The region between markers D15S117 and D15S1004 contains in excess of 50 genes and is syntenic to mouse chromosome 7 that contains the Mesp2 gene. The Mesp2 knockout mouse manifests altered rostrocaudal polarity, resulting in axial skeletal defects (Saga et al.,1997). The predicted human gene, MESP2, comprises two exons spanning approximately 2 kb of genomic DNA at 15q26.1. Direct sequencing of the MESP2 gene in the two affected siblings demonstrated a homozygous 4-bp (ACCG) duplication mutation in exon 1, termed 500-503dup (Whittock et al.,2004b). The parents were shown to be heterozygous and the unaffected sibling homozygous normal, consistent with the duplication segregating with SCD in the family. Fluorescent polymerase chain reaction excluded this mutation from 68 ethnically matched control chromosomes. Analysis of the genomic structure of the MESP2 gene highlighted a discrepancy between the Sanger Centre and NCBI human genomic assembly databases. In the latter, there is an additional short intron located after base 502 of the MESP2 coding region. This finding does not appear in the Ensembl gene prediction and, due to a lack of consensus splice sites within this proposed intronic sequence, the Exeter group concluded that the intron does not exists. However, the presence or absence of such an intron did not effect the conclusion concerning the 4-bp insertion on MESP2 protein production, which is predicted to interrupt splicing and lead to a frameshift at the same point in the MESP2 protein. Having confirmed mutated MESP2 as a cause of AR SCD, we designated this as SCD type 2, or MESP2-associated SCD.

In the second, nonconsanguineous family under study, haplotype data were consistent with linkage to the MESP2 locus, but sequencing of the gene failed to identify mutations. The MESP1 gene, which is located upstream of MESP2 on 15q, was also sequenced without any alteration being identified. It remains to be seen whether a mutation in one of the promoter regions, or some other position effect, might be responsible.

The MESP2 gene is predicted to produce a transcript of 1,191 bp encoding a protein of 397 amino acids. The human MESP2 protein has 58.1% identity with mouse MesP2, and 47.4% identity with human MESP1. Human MESP2 amino terminus contains a basic helix–loop–helix (bHLH) region encompassing 51 amino acids divided into an 11 residue basic domain, a 13 residue helix I domain, an 11 residue loop domain, and a 16 residue helix II domain. The loop region is slightly longer than that found in homologues such as paraxis. The length of the loop region is conserved between mouse and human MESP1, MESP2, Thylacine 1 and 2, and chick mesogenin. In addition, both MESP1 and MESP2 contain a unique CPXCP motif immediately carboxy-terminal to the bHLH domain (Fig. 11). The amino- and carboxy-terminal domains are separated in human MESP2 by a GQ repeat region also found in human MESP1 (2 repeats) but expanded in human MESP2 (13 repeats). Mouse MesP1 and MesP2 do not contain GQ repeats but they do contain two QX repeats in the same region: mouse MesP1 QSQS, mouse MesP2 QAQM. Hydrophobicity plots indicate that MesP1 and MesP2 share a carboxy-terminal region that is predicted to adopt a similar fold, although MesP2 sequences do contain a unique region at the carboxy-terminus.

Sequence analysis of 20 ethnically matched and 10 nonmatched individuals revealed the presence of a variable length polymorphism in the GQ region of human MESP2, beginning at nucleotide 535. This region contains a series of 12-bp repeat units. The smallest GQ region detected contains two type A units (GGG CAG GGG CAA, encoding the amino acids GQGQ), followed by two type B units (GGA CAG GGG CAA, encoding GQGQ) and one type C unit (GGG CAG GGG CGC, encoding GQGR). Analysis of this polymorphism in the matched and nonmatched controls revealed allele frequencies that were not significantly different, statistically, between the two groups.

MESP2 is a member of the bHLH family of transcriptional regulatory proteins essential to a vast array of developmental processes (Massari and Murre,2000). Murine Mesp1 and Mesp2, located on chromosome 7, are separated by approximately 23 kb. They are positioned head to head and transcribed from the interlocus region (Saga et al.,1996). At least two enhancers are involved in the expression of these genes in mouse (Haraguchi et al.,2001): one in early mesoderm expression and the other in presomitic mesoderm (PSM) expression. In addition, a suppressor responsible for the rostrally restricted expression in the PSM has been identified (Haraguchi et al.,2001). These enhancers are essential to the specific and coordinated expression of the MesP proteins. The expression of MesP1 and Mesp2 is first detected in the mouse embryo at the onset of gastrulation (∼6.5 days post coitum [dpc]) and is restricted to early nascent mesoderm (Saga et al.,1996; Kitajima et al.,2000). At this stage, the expression domain of Mesp1 is broader than that of Mesp2, and lineage analysis of Mesp2-expressing cells shows that they contribute to cranial, cardiac, and extraembryonic mesoderm (Saga et al.,1999). Expression is then down-regulated as Mesp transcripts are not detected later in development. A second site of Mesp expression is detected at 8.0 dpc immediately before somitogenesis. A pair of MesP-expressing bands appear on each side of the embryonic midline, at the anterior part of the PSM where the somites are anticipated to form (Saga et al.,1997,1999). During somitogenesis MesP1 and MesP2 continue to be expressed in single bilateral bands in the anterior PSM. MesP2 expression within the PSM continues until approximately the time when somite formation ceases (∼13.5 dpc; Saga et al.,1999), after which Mesp expression is rapidly down-regulated as transcripts are not detected in newly formed somites.

Alignment of MesP2 homologues from human, mouse, Xenopus, and chick demonstrates a highly divergent carboxy-terminus between species (Fig. 11), and it is unknown whether functional domains are similarly arranged in all Mesp2 orthologues, though this is well characterized for the Xenopus Mesp2 orthologue, Thylacine (Sparrow et al.,1998). If the carboxy-terminus in human MesP2 is required for transcriptional activation, then the mutant form of the protein described here, lacking the carboxy-terminus, would lose this function. Mouse pups lacking MesP2 die within 20 minutes of birth, presenting with short tapered trunks and abnormal segmentation, affecting all but a few caudal (tail) vertebrae (Saga et al.,1997). This finding resembles Dll3-null mice, where the rib and vertebral architecture is disturbed along the entire axis.

Unlike the mouse null MesP2 allele, the human mutant protein retains its bHLH region and, although truncated, could still dimerize, bind DNA, and act in a dominant-negative manner. However, in the affected family the heterozygous parents demonstrated no axial skeletal defects, from which we deduce that this MESP2 mutation is recessive. Similarly, heterozygous mice are normal and fertile (Saga et al.,1997). It is possible that a single functional carboxy-terminus is sufficient to activate transcription but alternatively the MESP1 protein may compensate for the absence of MESP2 function, just as MesP1 can rescue the axial skeletal defects in MesP2-deficient mice in a dosage-dependent manner (Saga,1998). Another possibility is that the mutant MESP2 transcript, containing a premature stop codon at 1099–1101 bp, is degraded by nonsense-mediated RNA decay, with no truncated protein being produced (Culbertson,1999).

In murine somitogenesis, MesP2 has a key role in establishing rostrocaudal polarity by participating in distinct Notch-signaling pathways (Takahashi et al.,2000,2003). Mesp2 expression is induced by Dll1-mediated Notch signaling (presenilin1-independent) and Dll3-mediated Notch signaling (presenilin1-dependent), while inhibition of Mesp2 expression is achieved through presenilin1-independent Dll3-Notch signaling. Because Mesp2 can inhibit Dll1 expression, this complex signaling network results in stripes of Dll1, Dll3, and Mesp2 gene expression in the anterior PSM.

The extent to which this process directly correlates with somitogenesis in man is unknown, except that the phenotypes of the Mesp2 and Dll3 mutant mice bear a resemblance to human SCD (Saga et al.,1997; Kusumi et al.,1998; Dunwoodie et al.,2002). The findings in this one family provided the first evidence that MESP2 is critical for normal somitogenesis in man. A second affected family with the identical mutation was presented at the 2005 meeting of the International Skeletal Dysplasia Society (L. Bonafé, personal communication), Martigny, Switzerland, and manifests a similar phenotype. Whether there is common ancestry between the two families is not known.

Lunatic Fringe

The identification of Notch pathway genes as causes of human SCD inevitably suggests the hypothesis that other genes of the pathway may be implicated in other forms of AVS/MVSD. Among these, the Lunatic Fringe (LNFG) gene was considered a strong candidate because the Lnfg-null mouse has a nonlethal phenotype that includes costovertebral abnormalities. Initially, however, mutation screening in a series of affected subjects with diverse radiological phenotypes failed to identify any positive cases. LFNG encodes a glycosyltransferase that post-translationally modifies the Notch family of cell surface receptors, a key step in the regulation of this signaling pathway (Haines and Irvine,2003). The LFNG protein is a fucose-specific β-1,3-N-acetylglucosaminyltransferase (Bruckner et al.,2000; Moloney et al.,2000) that functions in the Golgi to post-translationally modify the Notch receptors, altering their signaling properties (Haines and Irvine,2003). Earlier studies have shown that Lfng gene expression is severely disregulated in Dll3-null mice, suggesting that Lfng expression is dependent on Dll3 function (Dunwoodie et al.,2002; Kusumi et al.,2004).

The proband under study was an adolescent boy originating from northern Lebanon, the second of five children born to consanguineous parents. He had a short neck and a short trunk at birth and at 15 years had marked shortening of the thorax with a pectus carinatum deformity and kyphoscoliosis. A spinal MRI confirmed MVSD in the cervical and the thoracic spine with a serpentine curve in the cervical spine and concavity to the right in the upper spine and concavity to the left inferiorly. The thoracolumbar spine showed multiple hemivertebrae (Fig. 12), and there were multiple rib anomalies. The spinal cord was normal with no evidence of a syrinx. At 15, he had a markedly short trunk with a height of 155 cm (5th percentile), lower segment of 92.5 cm and a span of 186.5 cm. The span:height ratio is close to unity in individuals with normal proportions, but the measurements suggest that his foreshortened trunk reduced his stature by approximately 30 cm. At birth, he had been noted to have a contracture of the left index finger and, at age 15, he had hypoplasia of all the distal interphalangeal joints of the fingers, which were long and slender. Radiographs of the wrists and ankles were normal and the link between the spinal malformation and mild digital contractures remains speculative.

On sequencing the entire coding region and splice sites of the LFNG gene, a homozygous missense mutation (c.564C>A) in exon 3 was detected (Sparrow et al.,2006), resulting in substitution of leucine for phenylalanine (F188L). The proband's parents, with normal spinal anatomy, were both heterozygous for the mutant allele. The phenylalanine residue substituted is highly conserved (Correia et al.,2003) and close to the active site of the enzyme. The mutation created a novel MseI restriction enzyme site, which was used to confirm the sequencing results in the pedigree. This variant was not found in 34 ethnically matched control subjects (68 chromosomes), and the underlying base substitution was not present in the NCBI SNP database (www.ncbi.nlm.nih.gov). Examination of the mutation within a fringe model based on solved glycosyltransferase structures showed the conserved phenylalanine residue (F188) to be located in a helix that packs against the strand containing Mn²⁺-ligating residues, rather than being directly involved in UDP-N-acetylglucosamine or protein binding.

Further evidence of causality was provided by functional assays of the LFNG mutant. Two F187L mutations in mouse Lfng that correspond to F188L in human LFNG were generated: a c.564C>A mutation that encodes the rare leucine codon (TTA) observed in the proband, and the [c.562T>C + c.567C>G] mutation encoding the most common human leucine codon (CTG). In addition, a previously characterized enzymatically inactive form of Lfng (D202A) was created that disrupts the conserved DDD Mn²⁺-binding active site (Chen et al.,2001). Protein expression studies showed that both F187L mutant Lfng proteins were expressed at higher levels than wild-type or D202A Lfng forms, indicating that both translation efficiency and protein stability were not adversely affected by the F187L amino acid change. As Lfng protein is normally present in the Golgi apparatus (Fig. 7), intracellular protein localization was examined using immunofluorescence. Wild-type and D202A mutant Lfng were localized predominantly to the Golgi, whereas the F187L mutant Lfng did not colocalize with a marker. It was concluded that the F187L mutant form of Lfng is expressed but mislocalized within the cell.

This single case of SCD due to mutated LNFG has provided further evidence that proper regulation of the Notch signaling pathway is an absolute requirement for correct patterning of the axial skeleton, at the same time defining SCD type 3, or LNFG-associated SCD.