Greig Cephalopolysyndactyly Syndrome

Greig Cephalopolysyndactyly Syndrome

Akpene Gbegnon, MS4Department of Pathology

June 5th , 2008

Outline

Greig syndrome Genetic abnormalities in GCPS GLI3 – mutated gene in GCPS Association of GLI3 with limb development Case with GCPS & brain tumor Relationship between GLI3 and Sonic

hedgehog Greig syndrome and medulloblastomas

Greig Cephalopolysyndactyly Syndrome (GCPS)

First described by David Greig in 1926 The incidence is less than 10/1,000,000 It is an autosomal dominant mutation

Phenotype can include: Craniofacial abnormalities

Hypertelorism (increased inter-pupillary distance) Macrocephaly with frontal bossing

Limb abnormalities Syndactyly (webbed fingers or toes) Polydactyly (extra digits)

Developmental delay (uncommon)

Craniofacial and limb findings vary significantly

Cases of Greig Syndrome

1926 – Greig describes digital malformations of mother and daughter with polysyndactyly and high forehead

1978 – McKusick studied a family in which 10 members of 4 generations were affected in a pattern consistent with a fully penetrant autosomal dominant trait.

1981 – Merlob reported a female infant with polydactyly, syndactyly, and craniofacial dysmorphism with frontal bossing.

The father had a high forehead and mild hypertelorism

Genetic Abnormalities Found in Greig Syndrome

Translocation t(3;7) (p21.1;p13) Translocation t(6;7) (q27;p13) Deletion of 7p13-p11.2 Deletion of 7p14-p12.3

Greig syndrome results from a mutation in chromosome 7p13

GLI3 is a gene on chromosome 7p13 that is mutated in Greig syndrome

(1991) GLI3 considered candidate gene since it had been mapped to chromosome 7p13

(1991) Vortcamp demonstrated that 2 of 3 translocations found to be associated with Greig syndrome interrupt the GLI3 gene.

(2000) Infant with Greig syndrome (syndactyly, macrocephaly) with a

father and grandfather with digital abnormalities.

Infant had 2 dominant mutations in the GLI3 gene which was also present in the father and grandfather.

Mutations of the GLI3 gene is associated with Greig syndrome

What is GLI3?

The GLI3 gene: Is one of the three zinc-finger GLI family proteins

(Gli1,Gli2,Gli3) is located on chromosome 7p13 functions as a transcription factor

Is a negative regulator of Sonic hedgehog (Shh) signaling

Is there an association between GLI3 and limb development?

Yes A mouse model of Greig Cephalopolysyndactyly syndrome: the

extra-toes mutation contains an intragenic deletion of the GLI3 gene. Hui et al. Nature Genetics. 1993.

Mouse model is called: extra-toes (Xt)

Phenotype: Craniofacial defects (enlarged interfrontal bone) Polydactyly and syndactyly

Heterozygotes are viable and breed freely Homozygous lethal (polydactyly, syndactyly, malformation of

brain)

Mouse Model of Greig Syndrome

Results The extra-toes (Xt) mutants have decreased

GLI3 expression secondary to a deletion in the 3’ end.

In the early embryo of mice, GLI3 expression is most prominent in the developing limb buds and facial primordia.

In situ hybridization analysisof GLI3 transcript in developing limb.Idm = interdigital mesenchymeIzm = interzonal mesenchyme

Mouse Model of Greig Syndrome

Conclusion Structures affected in the mouse mutant and human

syndrome correlate with the expression of GLI3.

This supports the hypothesis that GLI3 plays a role in limb development, and its mutation leads to the abnormal limb development seen in Greig syndrome.

In situ hybridization analysisof GLI3 transcript in developing head and limbNt = neural tubeHlb = hindlimb bud.

Mouse Model of Greig Syndrome(Litingtung et al., 2002; Welscher et al., 2002)

This supports the hypothesis that GLI3 plays a role in limb development, and its mutation leads to the abnormal limb development seen in Greig syndrome.

Skeleton morphology

GLI3 and limb development(Lallemand et al, 2006)

In the limb, although the three Gli genes are expressed, only Gli3 appears to be necessary for patterning

Case (cont.)

Patient with Greig syndrome and pilocytic astrocytoma

Brain

Tumor (2.7cm X 2.0cm) involving the left cerebellopontine angle, dorsal medulla, and middle cerebellum

Hemorrhage at the resection site

Tumor is present in the cerebellum Asymmetry of the

cerebellum

Shift from left to right side

Left cerebellum is enlarged

Dentate nucleus seen on the right, but not on the left

Upper part of the left also looks enlarged

Left Right

FS of brain tumor showing Pilocytic Astrocytoma

increased cellularity, elongated nuclei fibrillary processes present Rosenthal fibers present

Is there an association between Greig syndrome and tumors?

Unclear…..

There are <200 articles published on Greig syndrome to date < 20 of those discuss Greig syndrome AND cancer

Cases: Medulloblastoma (2) Acute lymphoblastic leukemia (1)

Homer-wright rosettes

Medulloblastoma, WHO grade IV

Tumor of the cerebellumDense cellularityCircular arrangement of cells in rosettesSmall blue cell tumor

GLI3, Shh, and medulloblastomas

Sonic hedgehog (Shh) plays a major role in the developing cerebellum

Activation of the Shh pathway is associated with hereditary and sporadic medulloblastomas.

GLI3 is a negative regulator of Shh signaling

GLI3 and Sonic Hedgehog (Shh)

In Drosophila, hedgehog signaling is mediated by the Cubitus interruptus (Ci) protein. Ci75 is a hedgehog repressor Ci155 is a transcription activator

In birds and mammals, the orthologues of Cubitus interruptus are the three member Gli family (Gli1, Gli2, and Gli3)

GLI3 and Sonic hedge hog

Truncated form of GLI3 (GLI3R) is a repressor of Shh

Another form is possibly an activator

Complicated interaction between GLI3 and Shh, involving other proteins including Hand 2, Gremlin, Bmps, etc.

The relationship between GLI3 and Shh(Robert et al, 2006)

GLI3 is a negative regulator of Shh signaling

Is GLI3 involved in medulloblastomas?

GLI3 is not mutated commonly in sporadic Medulloblastomas. Erez et al. ACS. 2002.

Question: Are mutations in the GLI3 gene associated with medulloblastomas?

Why? Because there were 2 patients with Greig syndrome who had developed medulloblastomas.

Is GLI3 involved in medulloblastomas?

GLI3 is not mutated commonly in sporadic Medulloblastomas. Erez et al. ACS. 2002.

Method: The authors sequenced the GLI3 gene, including all exon-intron boundaries in: 10 medulloblastomas samples from 10 patients 2 human medulloblastoma cell lines 30 control DNA blood samples from healthy donors 1 blood sample from a patient with Greig syndrome and

medulloblastoma. (tumor sample was not available)

Is GLI3 involved in medulloblastomas? Results:

Authors found several new polymorphisms on exon 5 and 15 of GLI3 (CGCCAC, CTGCCG, etc)

Those polymorphisms also occurred in normal individuals with a frequency of > 25%

There were no tumor-associated mutations in the sporadic tumors

Authors found a new nonsense germline mutation (introduction of a stop codon) leading to a severely truncated GLI3 protein in the child with Greig syndrome and medulloblastoma

Conclusion:

GLI3 is “rarely” mutated in medulloblastoma

Summary

Greig syndrome is a rare genetic disorder Patients with Greig syndrome have hypertelorism,

macrocephaly, syndactyly and polydactyly Greig syndrome is caused by a mutation in the GLI3

gene, which is a gene located on chromosome 7p13 GLI3 is a transcription factor expressed in the limb and

skull during development GLI3 is a suppressor of the Sonic hedge hog pathway The association between GLI3 and neoplasms is not

well understood.

References A mouse model of Greig Cephalopolysyndactyly syndrome: the

extra-toes mutation contains an intragenic deletion of the GLI3 gene. Hui et al. Nature Genetics. 1993.

GLI3 is not mutated commonly in sporadic medulloblastomas. Erez et al. ACS. 2002.

Acute Lymphoblastic Leukemia in a Patient with Greig Cephalopolysyndactyly and Interstitial Deletion of Chromosome 7 del(7)(p11.2 p14) Involving the GLI3 and ZNFNIAI Genes. Mendoza et al. Genes, Chromosomes, & Cancer. 2005.

Robert B, Lallemand Y.Anteroposterior patterning in the limb and digit specification: contribution of mouse genetics. Dev Dyn. 2006 Sep;235(9):2337-52. Review.

Acknowledgement

Dr. Tim Rand Dr. Han Lee Dr. Scott Oakes

Dr. Philip Ursell

And..

All of the awesome path residents and fellows and attendings…

Extra Slides

.

What happens when you knockout Shh?

In the Shh null mutant, no digit is detectable in the forelimb and

a single digit

remains in the hindlimb.

This digit hasa digit1 (hallux)

identity according to molecular markers

Thus, despite the absence of Shh, the limb phenotype of the Shh:Gli3 double null mutants is indistinguishable from that of single Gli3 homozygotes.

This finding suggests that,in the limb, the main if not exclusive role of Shh is to prevent Gli3 processing

What happens if you have a Shh:Gli3 double knock-out?

Greig Syndrome and Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia in a patient with Greig cephalopolysyndactyly and interstitial deletion of

chromosome 7 del(7)(p11.2 p14) involving the GLI3 and ZNFNIAI Genes. Mendoza et al. Genes,

Chromosomes, & Cancer. 2005.

Case: 9 y/o boy with Greig syndrome with recurrent ALL.

Greig Syndrome and Acute Lymphoblastic Leukemia

Question: Is GLI3 related to ZNFNIAI?

ZNFNIAI a lymphoid-restricted zinc finger transcription factor which

has been associated with childhood leukemia

Decrease in ZNFNIAI activity leads in mice leads to T-cell hyperproliferation, aberrant expansion of thymic clones, and T-cell neoplasia

Mice that express non-DNA binding ZNFN1A1 isoforms develop an aggressive form of lymphoblastic leukemia

Results: Karyotype showed deletion of 7p11.2p14 in ~ 40% of

cells taken from a skin biopsy, or bone marrow aspirates FISH analysis shows the GLI3 gene was deleted in 74%

of cells

FISH (fluorescence in situ hybridization) of bone marrow cells using a GLI3 gene probe (red) showed normal hybridizationin 26% of the cells (b) and deletion in 74% of the cells (c).(Green labels the centromere of chromosome 7)

Present 26% Absent 74%

Patient with Greig syndrome and ALL has chromosome deletion that includes both GLI3 and ZNFNIAI

FISH (fluorescence in situ hybridization) of bone marrow cells using a ZNFNIAI gene probe (red) confirmed that the gene was within the deleted segment.

(Green labels the centromere of chromosome 7)


Conclusion: The deletion of a region in chromosome 7

resulted in Greig Syndrome (deletion of GLI3) and predisposed the patient to developing leukemia (deletion of ZNFN1A1).


The story of Sonic hedgehog

The zone of polarizing activity is a region of the limb bud mesenchyme and specifies the identity of each digit by threshold values of a morphogen

That morphogen is Sonic hedgehog Shh is a secreted protein Shh is expressed in the ZPA Cells expressing Shh can induce mirror-image

duplications of the digits Mice bearing a null mutation of Shh have forelimbs

that lack all digits in the forelimb and a single digit in the hindlimb

The story of Sonic Hedgehog

Mouse mutations leading to polydactyly are characterized by an ectopic Shh expression domain in the limb These mice include:

Hemimelic extra-toes, Sasquatch, Strong’s Luxoid (Alx4), extra-toes (Gli3)


SHH and GLI3 In Drosophila, hedgehog signaling is mediated

by the Cubitus interruptus (Ci) protein. Ci155 is a transcription activator Ci75 is a repressor

In birds and mammals, the orthologues of Cubitus interruptus are the three member Gli family (Gli1, Gli2, and Gli3)

In the limb, although the three Gli genes are expressed, only Gli3 appears to be necessary for patterning


SHH and GLI3 One form of GLI3 is a repressor of Shh Another form is possibly an activator Complicated interaction between GLI3 and

Shh, involving other proteins including Hand 2, Gremlin, Bmps, etc.

Technology

Greig Cephalopolysyndactyly Syndrome