Patterned expression in familial Klippel-Feil syndrome

TERATOLOGY 53152-157 (1996)

Patterned Expression in Familial Klippel-Feil Syndrome RAYMOND A. CLARKE, JOHN H. KEARSLEY, AND DAVID A. WALSH Division of Cancer Services, St George Hospital, Kogarah 221 7, Sydney, Australia (R.A.C., J.H.K.); Mammalian Development, School of Vetinary Science, Sydney University 2006, Australia (Dd. W.)

ABSTRACT Klippel-Feil syndrome (KFS) is characterised by congenital fusion of vertebrae within the rostral spine. The first KFS gene (SGM1) locus identified on chromosome 8 segregates with vertebral fusions and associated vocal impairment within the KF2-01 family (Clarke et al., '94, '95). Here, we describe the unique pattern of variable phenotypic expression within the KF2-01 family. The pattern of anomalies revealed a cumulative, rostrocaudal graded sequence of skipped vertebral fusions. This fusion pattern presents striking similarities with the mutant phenotype and gene expression profile of the Drosophila segment polarity gene engrailed. 0 1996 Wiley-Liss, Inc.

Familial Klippel-Feil syndrome (KFS) is characterised by the congenital fusion of spinal vertebrae in addition to other congenital anomalies, including oto- laryngological and craniofacial abnormalities. Because of apparent heterogeneity, interfamilial KFS comparative studies have been fraught with difficulty. With the exception of the consistency of the C2-3 fusion in the dominant KF2 genetic class of KFS, the very small size of most KFS family pedigrees has, to date, made it difficult to recognise any obvious inter- or intrafamilial patterns of vertebral anomalies. The predominance of sporadic cases of KFS in association with a broader spectrum of developmental anomalies ranging from mild cosmetic deformity to severe disability and lethal- ity has also helped obscure the etiology of this syndrome.

Congenital vertebral blocks of two or more fused vertebrae, principally within the neck are usually apparent from lateral radiographs, after completion of normal spinal ossification in the young child (McBride, '92). However the term fusion in relation to KFS has generally been regarded as a misnomer. This reflects the current view on the developmental etiology of Klip- pel-Feil anomaly as being the result of faulty segmentation of the somites from the segmental plates between the 3rd and 7th weeks of development. The KF2-01 family described in this paper (Fig. 1) provides additional supporting evidence for the segmentation hypothesis.

KF2 is the dominant class of KFS where the C2-3

fusion is always present, with variable expression (sometimes no expression) of more caudal fusions (cervical, thoracic and lumbar fusions). The KF2 genetic class can overlap with Klippel and Feils original clas- sification Types I, I1 and 111 (Klippel and Feil, '12; Gun- derson et al., '67). Approximately 70% of this KF2-01 family have KFS and an abnormal karyotype 46,XX or 46,XY, inv(8)(q22.2q23.3) affecting SGMl gene expression (Clarke et al., '94, '95).

In humans the segmental plates are laid down during the process of gastrulation, appearing on each side of the midline epithelium as the primitive streak regresses along the rostrocaudal axis of the embryo. As development proceeds, neuralation takes place in the midline, and the segmental plates come to flank the neural tube and notochord. Somites segment from the rostral end of each plate in an orderly rostrocaudal sequence (Stern and Keynes, '88). Rostra1 somites result from the segmentation of the existing cell population in the segmental plate (presomitic mesoderm), while the more caudal somites result from a zone of proliferation in a budding like process (Pearson and Elsdale, '79; Tam and Beddington '87; Tam, '88). The sclerotome from somite pairs (caudal of somite 4) migrates medially to surround the neural tube and notochord to eventually form the vertebrae (Stern and Keynes, '88). Therefore, the complete fusion of vertebral bodies with a more rostral/cervical position has been considered indicative of an earlier segmental er- ror of somitogenesis.

The rostrocaudal wave of somite formation does not appear to result from a series of inductive events in which the somite previously formed induces segmentation of the next caudal somite (Deuchar and Burges, '67; Pearson and Elsdale, '79; Meinhardt, '86). Evi- dence indicates that cells in the segmental plate are predetermined for somite formation (Deuchar and Burges, '67). A graded tissue property appears to exist within the segmental plates that induces somite for-

Received June 8, 1995; accepted December 5, 1995. Address reprint requests to Dr. Raymond A. Clarke, Cancer Care Centre, St. George Hospital, Kogarah 2217, NSW, Australia.

0 1996 WILEY-LISS, INC.

PATTERNED EXPRESSION IN FAMILIAL KFS 153

KFl - 01 FAMILY PEDIGREE I 1 2 3

1

LEGEND @El TOItICOIIS 0 not examined

em vertebral fusion n chromosomally normal

@B vocal lmpalrment inv paracentric lnverslon Bq

malformed ears spontaneous abortlon

Fig. 1. Family pedigree of the KF2-01 kindred. Of the 4 living who were not assessed via spinal radiograph, IV-14 and 16 had restricted neck movement and vocal impairment; while 111-1 showed no signs of the KF2-01 phenotype. Deceased individuals classified as affected were either obligate carriers of this dominant disease or voice impaired (11-5, 111-4) or both (1-2, 11-2, -4, and -6); or deaf and mentally retarded with scoliosis (IV-7). The pedigree is complete except for the three unaffected children of unafTected female 111-1.

mation in a temporally regulated segmenting sequence, which is completed much earlier in the rostral end of the embryo. The now well-established pattern of Homeobox gene expression patterns along the developing axis and the apparent implication of Hox genes in vertebral specification lend additional support for the implication of gene expression gradients in development of the bony spine (Hunt et al., '91, Hunt and Krumlauf, '91; Kessel and Gruss, '91; McGinnis and Krumlauf, '92).

Here we report the first description of a patterned sequence of vertebral fusions within the Klippel-Feil KF2-01 family. This pattern includes both gradient and skipping components, suggestive of a close association between phenotype and perturbations to patterned SGMl gene expression.

METHODS Radiography

Radiographs were taken of anterior, posterior, and lateral views of the cervical and upper thoracic spine, with the neck flexed and extended (Fig. 1).

RESULTS KF2-01 family study

The pedigree of the KF2-01 family is shown (Fig. l) , where 71% of living family members radiographed had

fused vertebrae. Of the entire family (living and deceased), 74% were affected with 64% males and 84% females. Previous medical and family histories were also assessed relative to KFS with no evidence of con- sanguineous marriages. All affected tested had a paracentric inversion on chromosome 8q (Clarke et al., '95).

Fusion profile From radiological findings it was possible to recog-

nise a unique pattern in the position and frequency of vertebral fusions in the KF2-01 family:

1. Fusion was restricted to the cervical region of the spine. Most fused vertebrae retained their general cervical identity with occasional malalignments of vertebral bodies and processes (Fig. 2a).

2. Fusion occurred across the alternate or skipped vertebral interspaces C2-3, C4-5, and C6-7.

3. Fusions were cumulative; always adding from the rostral (C2-3) end (Fig. 3).

4. Fusion frequency diminished caudally as a gradient; all those who tested positive for vertebral fusion via spinal radiographs (100%) had the C2-3 fusion, 70% the (32-3 and C4-5 fusions, and 20% (2 individuals, namely 111-7 and IV-6) had the C2-3, C4-5, and C6-7 fusions (Fig. 3).

5. Fusion frequency appeared to correlate with voice impairment, in that all those with two or more fusions were vocally impaired, in addition to male proband IV-3, who had the single C2-3 fusion.

All affected individuals tested via spinal radiography exhibited vertebral fusion(s), including fusion of the 2nd and 3rd cervical vertebrae to form a continuous C2-3 block vertebra (Fig. 2a). Such vertebral block formation was often associated with the thinning of adjacent vertebral interspaces. However, thinning of adjacent interspaces was never classified in this study as fusion, except when the intervertebral space, that nor- mally exists between the main bodies of adjacent vertebrae, was absent or moderately ossified. The single exception to the skipped profile rule was presented by proband 111-6, who had all three of the skipped fusions listed in addition to fusion of C5-6, resulting in block formation of C4-7, in conjunction with gross deformation of the most rostral C2-3 block vertebra (Fig. 2b). Other evidence suggests that this C5-6 fusion may be explained by progressive ossification around this severely restricted joint. All affected suffered from tor- ticollis; however, with the possible exception of deceased child IV-7 abnormal vertebral configurations did not result in any notable spinal instability or pro- nounced neurological deficit. Individuals 111-7 and IV-6 had shorter than normal necks. Individual IV-11 had 13 pairs of ribs.

Voice impairment Ninety percent of all affected individuals (76% of the

living affected) were obviously vocally impaired (Fig.

154 R.A. CLARKE ET AL.

Fig. 3. Vertebral fusion profile. Skipped fusion position versus per- centage frequency *The only exception to the skipped pattern of fusions was presented by proband 111-7, who had fusion across the C2-3, C4-5, and C6-7 fusions, in addition to the only one fusion across the C5-6 interspace. There is limited evidence to suggest that the C5-6 fusion here may be the result of degenerative change within the cervical spine of 111-7 due to severe immobility. This resulted in block formation of (34-7; III-7 has concurrent gross deformation of the C2-3 block.

1) and were given a comparative ranking relative to an extensive speech analysis carried out previously on proband 111-6 (Clarke et al., '94). Those individuals with severe voice impairment were essentially aphonic and communicated with a soft, hoarse whisper which was relatively ineffective, except in very quiet back- ground noise conditions. Even those aaFected individuals with a voice judged to be mildly impaired still had a soft and hoarse voice. The severest cases of voice impairment were male and reports suggest deterioration of voice as they approached puberty. All individuals without vertebral fusion were judged to have a com- pletely normal voice. The affected status of deceased individuals 11-5 and 111-4 was reliant on the history of voice impairment.

The previous clinical case report on aphonic proband 111-6 reviewed severe voice impairment relative to malformed laryngeal cartilages and determined that no common dysplasia could account for both cervical fusions (C2-3 and (24-5) and laryngeal abnormalities. Laryngeal examination also revealed absent right vocal cord movement and restricted left vocal cord mo- tion. Consequently the vocal cords failed to adduct to the midline for phonation and vocal cord vibration was absent (Clarke et al., '94).

Deceased individuals classified as affected were either obligate carriers of this dominant disease or re- ported as severely vocally impaired (n-5 and 111-4) or both (1-2, 11-2, -4, and -6) (Fig. 1). Two living, vocally impaired individuals (IV-14 and -16) who were un- available for radiography were also assumed to have KFS.

Fig. 2. vertebral fusions. Lateral cenrical ra&ographs. a: c2-3 fusion from IV-11 with slight degenerative changes around (23-4. b The most extensive sequence of fusion in the KF2-01 family was from 111-7, in addition to gross deformation of his C2-3 block. Radiographs were compiled over a 3-year period.


Other anomalies All affected members in the KF2-01 family (100%)

had microtia, while some had a previous history of mild conductive hearing impairment or had low set and/or underdeveloped ears (Clarke et al., '94) and bilaterally restricted supination and elbow flexion of the fore- arms. Tendons at the back of the foot were often tight with a tendency to walk on the ball of the foot. While vertebral fusion was not confirmed for the deceased child IV-6 (14 years), she presented with KFS-associated anomalies, including scoliosis, congenital deafness, and profound mental retardation. This child also presented with extra pyramidal disease in association with microcephaly and somatic stunting and with min- imal right facial paralysis, Cheyne-Stoke respirations and bradycardia and involuntary movements (possibly myoclonic jerks) of the upper limbs and face, particu- larly the fingers, hands, and tongue, consistent with a severe bilateral dysfunction of the basal ganglia.

DISCUSSION This report establishes the position and sequence of

vertebral fusions as being highly informative of KFS genotype. In contrast with many other KFS families in which penetrance appears to be incomplete, the KF2- 01 family appears to manifest complete penetrance (74% affected) in association with variable expression. However, consistencies within the KF2-01 abnormal phenotype, including the C2-3 vertebral fusion (evident in 100% of positive spinal radiographs) and voice impairment (90% of afYected family) always segregat- ing with the chromosome 8 inversion indicate a single genellocus effect.

Malformations of both vertebral and otolaryngeal structures, and KFS facial dysmorphology generally, are consistent with maldevelopment early in embryo- genesis (Tucker and Tucker, '75; Hunt and Krumlauf, '91; Kessel and Gruss, '91; Fjosse et al., '92; Ang and Rossant, '931, possibly prior to any visible signs of structural segmentation in the developing embryo (3rd to 4th week). Classical embryology has shown that three distinct embryonic lineages contribute to the early skeleton. The neural crest gives rise to the branchial arch derivatives of the craniofacial skeleton, the sclerotome generates most of the axial skeleton, and the lateral plate mesoderm forms the appendicular skeleton. Transplantation studies have indicated that the information regarding the number and anatomic identity of derived skeletal elements resides in these lineages by the time of their appearance and long be- fore skeletogenesis.

Five of the seven vertebrae that make up the cervical spine (Cl-7) are very similar in appearance. However, vertebral morphology and formation are not uniform along the rostrocaudal axis (Meinhardt, '86). Somito- genesislsegmentation progresses caudally and ossification of vertebral bodies progresses rostrally along

the developing cervical spine (Morgan et al., '89; Couly et al., '93). This lack of uniformity relates to differences and patterns in developmental gene expression (Deutsch et al., '88; Hunt et al., '91; McGinnis and Krumlauf, '92). Here, we will outline the recurring theme of "skipped periodicity" and "gene expression gradients" as they pertain to the regulation and interpretation of axial patterninghewentation generally and their re-emergence here, in the KF2-01 phenotype.

Skipped gene expression gradients within segmenting tissues

Here, the phenomenon of skipped and graded gene expression during axial development implies the su- perimposition of two distinct expression patterns of the same gene along the rostrocaudal axis: (1) graded expression in (2) skipped (alternate) spatial domains.

The developmental requirements for establishing unique vertebrae from somites, along the rostrocaudal axis, can be approached in terms of differences in Hox gene expression codes (Stern and Keyne, '88; Kessel and Gruss, '91; Morgan and Tabin, '93). A number of the same Hox genes express in both the segmenting tissues of the presomitic mesoderm and developing hindbrain (Kessel and Gruss '91; McGinnis and Krum- lauf, '92; Krumlauf, '94). In animal models (e.g., mouse and chicken), altering the pattern of Hox gene expression by chemicals and "gene knockout" has coincided with homeotic transformations of the vertebrae and developmental defects of facial structures, including laryngeal and otic cartilage, the cranium, and hindbrain (Kessel and Gruss, '91; Morris et al., '91; Chisaka and Capecchi, '91; Chisaka et al., '92; Jegalian and Rober- tis, '92). It is apparent, however, that different mechanisms are used in patterning the head and trunk.

It has been established that homeotic Hox genes often express in caudally diminishing gradients within segment restricted domains along the developing vertebrate axis (Keynes and Stern, '84, Stern and Keynes, '88). The full implications of this are unknown. In Dro- sophila, both principal axes of the embryo are established by a positional information system encoded by many protein gradients (St. Johnston and Nusslein- Volhard, '92).

Vertebrate somitogenesis reveals an obvious half somite rostrocaudal periodicity (Meinhardt, '86) com- patible with the theory of resegmentation. The double segment skipping in the KF2-01 fusion profile predicts a double somitehegment rostrocaudal periodicity. Such double segment periodicity is now a common theme in developmental genetics. For example, in vertebrates the anterior borders of Hox expression are often coin- cident with future segment boundaries (Hunt et al., '91; Hunt and Krumlauf, '91; Kessel and Gruss, '91). In addition, these anterior borders of expression, of the different members of Hox gene groups (e.g., Hoxa, b and d), are often periodically staggered along the developing vertebrate axis (Hunt et al., '91; Hunt and

156 R.A. CLARKE ET AL.

Krumlauf, '91; McGinnis and Krumlauf, '92). These staggeredlskipped patterns often have a double segment periodicity (e.g., Hoxb-2,b-3 and b-4). In vertebrates, the regulation and downstream implications of these skipped patterns of Hox gene expression remains largely undefined. However, in Drosophila, the estab- lishment of a segmented body plan during embryogen- esis is known to require the sequential activation of segmentation genes prior to homeotic gene expression (e.g., the segment polarity gene engrailed). Segmenta- tion genes such as engrailed demarcate future segment boundaries and homeotic genes (eg., Hox) appear to interpret the patterned signals of the segmentation genes engrailed achieves this level of regulation inpart by having its own stripedhkipped pattern of early gene expression that also has a double segment periodicity (Akam, '87; Ingham, '88; Martinez-Arias and White, '88). An analogous double segment (skipping) periodicity is again evident here with the KF2-01 (SGM1) fusion profile.

In common with dominant SGMl mutants, engrailed mutants are also characterised by the fusion of alternate or skipped segments (Nusslein-Volhard et al., '84). In contrast to the Hox genes in Drosophila (and in vertebrates), the level of engrailed gene expression is also functionally critical; accounting for the dominance of the mutant phenotype of engrailed. This is not to say that SGMl is in any way related to the engrailed gene. What appears certain, however, is that SGMl is impli- cated in spinal patterning, if not patterning of the axial mesoderm.

One obvious interpretation of the KF2-01 fusion frequency gradient would be that it quite likely reflects critical reductions of SGMl gene expression, via a haploinsufficiency of SGM1, due to inactivation of the SGMl gene at one of the chromosome 8 inversion breakpoints. The SGMl mutation appears to have peturbed the function of SGMl or an associated mole- cule that regulates morphological patterning of the axis, possibly downstream of somitogenesis. As such, even ubiquitous reductions in SGMl expression would not be uniform within different fields of SGMl expression or in their phenotypic effects. The high end of any gene expression gradient may be more likely to expe- rience greater comparative reductions due to gene inactivation than the low end of the same gradient. In this way, a haploinsufficiency of SGMl may account for the higher proportion of more rostral fusions in the KF2-01 family. Note that such gene copy number de- pendent regulation of RNA and protein levels within the rostrocaudal gradient of bicoid (the primary rostral determinant), also determines the caudal range of bicoid influence within the developing Drosophila embryo. Consistent with this hypothesis is an increased incidence of voice impairment concurrent with an in- crease in the frequency of more caudal cervical fusions, along with increased deformation of the rostral C2-3 vertebral block (Fig. 2B, proband 111-7).

Craniofacial abnormalities In vertebrates, the lower face and throat arise from

paired segmental structures ie. the branchial arches and pharyngeal pouches. Cartilaginous and connective tissues of the head and neck are derived from multipo- tential neural crest cells that migrate ventrally into the adjacent branchial arches, from specific hindbrain rhombomere segments (Hunt et al., 1991; Hunt and Krumlauf, '91; McGinnis and Krumlauf, '92). Bran- chial arches 1, 2, and 3 are filled by neural crest from the alternatehkipped rhombomeres 2 ,4 and 6 respec- tively (Hunt and Krumlauf, '91). This skipped pattern of cell migration also has a double segment periodicity within the rhombomeres during facial development. Organisation of the neural crest is thus related by the processes of immigration to the intrinsic mechanisms that segment the neuroaxis (Lumsden et al., '91).

Prepatterned neural crest from the rhombomeres migrates to the branchial arches generating cartilage for facial structures. Therefore, in the KF2-01 family, laryngeal malformation, microstomia, underdeveloped ears, microtia, bilaterally restricted movement of the upper limbs, and the periodic respirations and myoclonic jerks of proband IV-6, could all conceivably be linked to patterning/segmentation anomalies of the hindbrainlneural crest. In addition, the apparent cor- relation between the degree of vertebral fusion and the extentheverity of associated anomalies appears to indicate a single genelcause effect.

Whereas most KFS affected families have been too small to reveal such intrafamilial patterning as re- ported here for family KF2-01. Some of the larger families (e.g., Sheffield, '82) do reveal consistent reoccur- ring periodicity between fusions, albeit not double segment periodicity. What is certain, however, is that the human rostocaudal axis segments and develops un- der the influence of many developmental gene expression patterns including that of various periodicities and gradients. Whether KFS genes such as SGMl es- tablish and/or interpret such patterns remains to be investigated.

ACKNOWLEDGMENTS The authors thank Dr. Merle De Silva for radiologi-

cal interpretation and Linda Clarke for artwork.

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Patterned expression in familial Klippel-Feil syndrome