Acromelic Frontonasal Dysostosis
• Acromelic frontonasal dysostosis
(AFND) is a rare disorder
characterized by:
• frontonasal dysplasia
• interhemispheric lipoma
• agenesis of the corpus callosum
• tibial hemimelia
• preaxial polydactyly of the feet
• intellectual disability
Clinical Features Of Subjects
• Four children with classic features of acromelic frontonasal dysostosis
• Each child had neurocognitive and motor delays, severe symmetric frontonasal dysplasia
associated with median cleft face, carp-shaped mouth, widely spaced nasal alae,
hypertelorbitism, variable parietal foramina, interhemispheric lipoma, and unilateral or bilateral
tibial hemimelia with preaxial polydactyly.
• Variable features included periventricular nodular heterotopia, aplastic or hypoplastic corpus
callosum, absent olfactory bulbs, vertical clivus, patellar hypoplasia, hypopituitarism, and
• The mode of inheritance of AFND was unknown
• In individual 1 the proband’s father had a mild phenotype consisting of broad nasal tip, columella, and
hypertelorism, suggesting autosomal-dominant mutation with either (1) variable expressivity, (2)
dramatically reduced penetrance, (3) and/or possible germline mosaicism
• For individuals 2 and 3, there was no family history of AFND and no reported consanguinity, suggesting
a plausible de novo model of inheritance versus autosomal-recessive inheritance.
• Individual 4 was adopted, and there is no phenotype information available on the biological parents.
• For 15 years, it has been thought that the AFND phenotype was consistent with activation of the
hedgehog pathway; however, candidate-gene screening studies that focused on sonic hedgehog
(SHH pathway members (i.e., GLI3, IHH, and wnt10a) were negative
Whole Exome
Mutation Targets Vs Disorder Frequency
Gilissen, Genome Biol 2011
Whole Genome Or Exome Seq?
• Enabling technologies: NGS machines, open-source algorithms, capture
reagents, lowering cost, big sample collections
• Exomes more cost effective: sequence patient DNA and filter common snps;
compare parents child trios; compare paired normal cancer
• Challenges:
• Still can’t interpret many mendelian disorders
• Rare variants need large samples sizes
• Exome might miss region (e.G. Novel non-coding genes)
• Unsuccessful at using exome-seq to interpret clinical data
Shendure, Genome Biol 2011
Data Analysis
• Heuristic filtering to identify novel genes
for mendelian disorders
Stitziel et al, Genome Biol 2011
chromosome. The reference genome representing the wild-type is shown at the top while the
altered genome found in a sample is shown at the bottom. B) Inversion (INV) has reciprocal join
in opposite orientations. C) Intra-chromosome translocation (ITX) has unilateral join in opposite
orientation. D) Deletion (DEL) has two breakpoints joined in ascending order of genomic
coordinates in the same orientation. E) Insertion (INS) has two breakpoints joined in descending
order of genomic coordinates in the same orientation.
Genomic Structural Variation
Baker et al, Nat Meth 2012
ZSWIM6 As A Novel Target
• Initial supported a dominant model, the assumption being that the father of individual 1 was affected but
with a mild phenotype.
• Were able to detect one de novo variant in individual 1.
• Anything present in the 1000 genomes or the exome sequencing project (ESP) data were excluded
• Also intergenic variants and variants that were flagged as low quality or potential false positives
• The one remaining variant in ZSWIM6, and an identical mutation was also observed in individuals 2 and 3.
• Sanger sequencing of trios confirmed a de novo mutation in individuals 1, 2, and 3.
• Sanger sequencing also confirmed that individual 4 had the variant
• at a 60:40 ratio of wild-type to mutant allele
• suggestive of mosaicism
High Level Of Conservation Of Variant Position
• The variant c.3487C>T is located within a CPG dinucleotide in ZSWIM6 and is predicted to cause
a nonsynonymous coding change (p.Arg1163trp) in this 1216 aa protein.
ZSWIM6 Gene and Protein Structure
ZSWIM6 Structure
• 133.5 kda protein containing a zinc finger SWIM domain
• Sequence analysis predicts an all-α-helical structure with novel fold topology and identifies >75%
conservation across 97 orthologous proteins from residues 269–1215
• One poly-alanine and two poly-glycine repeats occur in the n-terminal 200 residues of the protein
• Residues 248–279 match the stereotypical pattern of the SWIM proteins
• containing SWI2/SNF2 and mudr-type zinc fingers
• thought to carry out protein-protein and protein-dna interactions
Variant Tolerance
Functional Relevance Of Protein Domains in ZSWIM6
• Arginine 1163 occurs in a highly conserved domain from 1148–1215 with similarity to sin3.
• Substitution to tryptophan is predicted to have severe effects on protein function.
• Protein modeling of amino acid residues 1148–1215 of the wild-type (left) and mutant (right)
shows that the p.Arg1163trp substitution (arrow) dramatically alters the structure and
hydrophobicity of the protein.
Variant Tolerance Predictions
• Arginine 1163 is in the most evolutionarily conserved part of the protein
• from mammals to invertebrates
• in a sequence within residues 1154–1167 (SRLTHISPRHYSEF), which are conserved in ZSWIM paralogs ZSWIM4 and
• predicted functional importance by a meta-functional signature is >5 SD above the mean (z score
• Relative sequence entropy performed with HMMRE
• Disorder analysis performed with dispro
• Domain boundary prediction performed with dompro
• All together suggest variant is within a well-defined globular domain from residues 1129–1215 that would
maintain biochemical function for in vitro assessment
Variant Tolerance Predictions
• Hereditarily Unfit SNV Computational Yardstick (HUSCY) z score = 4.3
• suggests interactions with other functional residues
• change to tryptophan is the most disruptive substitution that could occur at this position
• The atomic-resolution model of ZSWIM6 residues 1148–1215 predicts that the p.Arg1163 side
chain resides on the protein surface between a nonrandom display of negative and positive
• most likely patterned for protein-protein interactions that would be severely disrupted by the change to
Zebrafish Ortholog
• Zebrafish genome contains a single ortholog of ZSWIM6
• zebrafish zswim6 is highly conserved with human ZSWIM6
• Cloned a zebrafish zswim6cdna product and performed fluorescent RNA in situ hybridization
• used dlx2a and zswim6 cdna probes for in situ hybridization and dlx2a as a reference for expression
Zebrafish ZSWIM6
• Low level of ubiquitous expression in early embryos until 24 hr postfertilization (hpf), when it
exhibits increased, localized expression domains in the telencephalon and in the midbrain
• At 48 hpf, telencephalic expression of zswim6 persisted
• there was increased expression in the midbrain, hindbrain, and retina
• Pattern is consistent with:
• domains seen in mice
• where zswim6would be predicted to function on the basis of the human
IHC Of ZSWIM6 In Mouse Embryo
(A) Immunohistochemistry demonstrating discrete localization of ZSWIM6 in fiber cells of
the developing lens of an E13.5 mouse embryo
(B) Neighboring-section control of (A) without primary antibody.
(C) Postnatal day 0 mouse expression of ZSWIM6 in stereocilia of the outer hair cells of
the cochlea
(D) Subpopulation of cells in the outer root sheath of the hair follicle
(E) Odontoblasts and ameloblasts of the tooth
(F) Variable expression in skeletal muscle
Role Of Sonic Hedgehog Pathway
• Vargas et al. (1998) thought AFND could involve the sonic hedgehog (SHH) pathway
• Doublefoot (dbf) mice have median cleft face, preaxial polydactyly and tibial hemimelia associated
with dysregulation of indian hedghog homolog (IHH) signaling and altered gli3 processing.
• gli3 is a zinc-finger DNA-binding transcription factor that mediates downstream SHH signaling
• gli3xt mice have craniofacial defects and preaxial polydactyly
• Multiple individuals with heterozygous deletions spanning ZSWIM6 do not have AFND, likely gainof-function.
Activation Of The Hedgehog Pathway By ZSWIM6
In Summary
• Utilized whole-exome sequencing to identify an identical de novo mutation in zswim6(c.3487C>T)
in three unrelated probands and one isolated proband
• Variable phenotypic expression suggests that mosaicism or other gene modifiers are involved in
the phenotype
• New data of expression of ZSWIM6 using zebrafish and mouse models
• QRT-PCR data support the previous hypothesis that AFND is caused by dysregulation of the
hedgehog pathway
Future Work
• Lack of information on the function of ZSWIM6 and other ZSWIM family members suggests that
this discovery might introduce more candidates for additional conditions
• frontonasal dysplasia
• preaxial polydactyly
• tibial hemimelia
• Experimental model systems to evaluate the developmental pathways impacted
• Proof of causation:
• More than one unrelated family member with deleterious mutations in the same gene with the same
• Variant is not found in controls from a similar ethnic background
• Not found in unaffected family members
• Protein networks support role in disease process
• Model organism with similar phenotype
• Expression studies are compatible with mechanism of action
• Functional studies to show downstream effects of altered protein/halpoinsuffciency
• Variant is deleterious: evolutionary conservation, predicted domains, tolerance for particular outcomes
(nonsense vs missense)

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