Sagittal Craniosynostosis with Uncommon Anatomical Pathologies in a 56-Year-Old Male Cadaver

Sagittal craniosynostosis (CS) is a pathologic condition that results in premature fusion of the sagittal suture, restricting the transverse growth of the skull leading in some cases to elevated intracranial pressure and neurodevelopmental delay. There is still much to be learned about the etiology of CS. Here, we report a case of 56-year-old male cadaver that we describe as sagittal CS with torus palatinus being an additional anomaly. The craniotomy was unsuccessful (cephalic index, CI = 56) and resulted in abnormal vertical outgrowth of the craniotomized bone strip. The histological analysis of the latter revealed atypical, noncompensatory massive bone overproduction. Exome sequencing of DNA extracted from the cadaveric tissue specimen performed on the Next Generation Sequencing (NGS) platform yielded 81 genetic variants identified as pathologic. Nine of those variants could be directly linked to CS with five of them targeting RhoA GTPase signaling, with a potential to make it sustained in nature. The latter could trigger upregulated calvarial osteogenesis leading to premature suture fusion, skull bone thickening, and craniotomized bone strip outgrowth observed in the present case.


Genetic Analysis
The Next Generation Sequencing (NGS) and bioinformatics analysis were performed as previously described [2,3] with the following modifications. DNA was extracted from the left sternocleidomastoid muscle specimen procured from the embalmed cadaveric head and was sequenced to 30x depth of coverage (~4.5 Gb) on the Illumina HiSeq 2500 NGS platform. The 30x depth of coverage fulfills a requirement for the detection of human genome mutations (10x to 30x, Illumina). DNA extraction and exome sequencing were conducted by Omega Bioservices (Norcross, GA). The cumulative exome coverage for > 10x depth of coverage was 82 % indicating that the majority of the exome was probed. The variant call and annotation were performed by Genome Technology Access Center (GTAC, Washington University in St. Louis) using SnpSift varType and ANNOVAR. The resultant data were converted into the Microsoft Excel format and pathologic/deleterious variants were identified through the consecutive filtering steps outlined in [3]. Functional annotation of the remaining variants was performed using UniProtKB Protein, Google Scholar, and PubMed database searches. Figure S1. Craniectomy of the scaphocephalic cadaveric head. An incision was made along the midsagittal plane of the scalp from anterior to posterior. Table S1. Complete list of deleterious (pathologic) genetic variants associated with the current case of sagittal CS.

Gene
Protein Function

ABCD1
ATP Binding Cassette Subfamily D Member 1. Probable transporter. The nucleotide-binding fold acts as an ATP-binding subunit with ATPase activity. This peroxisomal membrane protein is likely involved in the peroxisomal transport or catabolism of very long chain fatty acids. Defects in this gene have been identified as the underlying cause of adrenoleukodystrophy, an X-chromosome recessively inherited demyelinating disorder of the nervous system. ANKRD30B Ankyrin repeat domain-containing protein 30B.

BMP6
Bone morphogenetic protein 6. Teeth development. Cartilage development. Endochondral ossification. Positive regulation of osteoblast differentiation. Positive regulation of bone mineralization. Positive regulation of chondrocyte differentiation.

CACNA1A
Voltage-dependent P/Q-type calcium channel subunit alpha-1A. Voltage-sensitive calcium channels (VSCC) mediate the entry of calcium ions into excitable cells and are also involved in a variety of calcium-dependent processes, including muscle contraction, hormone or neurotransmitter release, gene expression, cell motility, cell division and cell death.

CD101
Immunoglobulin superfamily member 2. Plays a role as inhibitor of T-cells proliferation induced by CD3. Inhibits expression of IL2RA on activated T-cells and secretion of IL2. Inhibits tyrosine kinases that are required for IL2 production and cellular proliferation. Inhibits phospholipase C-gamma-1/PLCG1 phosphorylation and subsequent CD3-induced changes in intracellular free calcium.

CDC27
Cell Division Cycle 27. Component of the anaphase promoting complex/cyclosome (APC/C), a cell cycle-regulated E3 ubiquitin ligase that controls progression through mitosis and the G1 phase of the cell cycle.

CEP162
Centrosomal protein of 162 kDa. Required to promote assembly of the transition zone in primary cilia. Acts by specifically recognizing and binding the axonemal microtubule. Required to mediate CEP290 association with microtubules.

CHRNA7
Neuronal acetylcholine receptor subunit alpha-7. After binding acetylcholine, the AChR responds by an extensive change in conformation that affects all subunits and leads to opening of an ion-conducting channel across the plasma membrane. Calcium ion transport. Positive regulation of cell proliferation.

CNN2
Calponin-2. Thin filament-associated protein that is implicated in the regulation and modulation of smooth muscle contraction. It is capable of binding to actin, calmodulin, troponin C and tropomyosin.

COL3A1
Collagen alpha-1(III) chain. Collagen type III occurs in most soft connective tissues along with type I collagen. Involved in regulation of cortical development. It Is the major ligand of ADGRG1 in the developing brain and binding to ADGRG1 inhibits neuronal migration and activates the RhoA pathway by coupling ADGRG1 to GNA13 and possibly GNA12.

CROCC
Rootletin. Major structural component of the ciliary rootlet, a cytoskeletal-like structure in ciliated cells which originates from the basal body at the proximal end of a cilium and extends proximally toward the cell nucleus (By similarity). Required for the correct positioning of the cilium basal body relative to the cell nucleus, to allow for ciliogenesis.

CRYGD
Gamma-crystallin D. Crystallins are the dominant structural components of the vertebrate eye lens.

CYP4V2
Cytochrome P450 4V2. Omega-hydroxylase that oxidizes medium-chain saturated fatty acids and polyunsaturated omega-3 fatty acids, and which plays a role in fatty acid and steroid metabolism in the eye.

DCAF16
DDB1-and CUL4-associated factor 16. This protein is involved in the pathway protein ubiquitination, which is part of protein modification. May function as a substrate receptor for CUL4-DDB1 E3 ubiquitin-protein ligase complex. DDX54 ATP-dependent RNA helicase DDX54. Has RNA-dependent ATPase activity.
Represses the transcriptional activity of nuclear receptors.

DENND2A
DENN domain-containing protein 2A. Guanine nucleotide exchange factor (GEF) which may activate RAB9A and RAB9B. Promotes the exchange of GDP to GTP, converting inactive GDP-bound Rab proteins into their active GTP-bound form.
May play a role in late endosomes back to trans-Golgi network/TGN transport.

DNAH11
Dynein heavy chain 11, axonemal. Force generating protein of respiratory cilia. Produces force towards the minus ends of microtubules. Dynein has ATPase activity; the force-producing power stroke is thought to occur on release of ADP.

EIF4E1B
Eukaryotic translation initiation factor 4E type 1B. Recognizes and binds the 7methylguanosine-containing mRNA cap during an early step in the initiation of protein synthesis and facilitates ribosome binding by inducing the unwinding of the mRNAs secondary structure.

ENDOG
Endonuclease G, mitochondrial. Cleaves DNA at double-stranded (DG)n.(DC)n and at single-stranded (DC)n tracts. In addition to deoxyribonuclease activities, also has ribonuclease (RNase) and RNase H activities. Capable of generating the RNA primers required by DNA polymerase gamma to initiate replication of mitochondrial DNA (By similarity).

FANCC
Fanconi anemia group C protein. DNA repair protein that may operate in a postreplication repair or a cell cycle checkpoint function. May be implicated in interstrand DNA cross-link repair and in the maintenance of normal chromosome stability. Upon IFNG induction, may facilitate STAT1 activation by recruiting STAT1 to IFNGR1.

FPR2
N-formyl peptide receptor 2. Low affinity receptor for N-formyl-methionyl peptides, which are powerful neutrophils chemotactic factors. Binding of FMLP to the receptor causes activation of neutrophils. This response is mediated via a Gprotein that activates a phosphatidylinositol-calcium second messenger system. The activation of LXA4R could result in an anti-inflammatory outcome counteracting the actions of proinflammatory signals such as LTB4 (leukotriene B4).

GBP6
Guanylate-binding protein 6. Binds GTP, GDP and GMP. GRAMD1A GRAM domain-containing protein 1A. May play a role in tumor progression.

GUCY2F
Retinal guanylyl cyclase 2. Probably plays a specific functional role in the rods and/or cones of photoreceptors. It may be the enzyme involved in the re-synthesis of cGMP required for recovery of the dark state after phototransduction.

HAS3
Hyaluronan synthase 3. Catalyzes the addition of GlcNAc or GlcUA monosaccharides to the nascent hyaluronan polymer. Therefore, it is essential to hyaluronan synthesis a major component of most extracellular matrices that has a structural role in tissues architectures and regulates cell adhesion, migration and differentiation.

IGF2R
Cation-independent mannose-6-phosphate receptor. Transport of phosphorylated lysosomal enzymes from the Golgi complex and the cell surface to lysosomes. This receptor also binds IGF2. Acts as a positive regulator of T-cell co-activation, by binding DPP4.

INADL
InaD-like protein also known as PATJ. Negative regulator of Wnt signaling. Blocks DFz1 activity in the planar cell polarity pathway (PCP) in cooperation with atypical PKC. Fzd/PCP pathway represents the non-canonical Wnt signaling.

MAP7D3
MAP7 domain-containing protein 3. Promotes the assembly and stability of microtubules.

MED1
Mediator of RNA polymerase II transcription subunit 1. Component of the Mediator complex, a coactivator involved in the regulated transcription of nearly all RNA polymerase II-dependent genes. Ligand-dependent nuclear receptor binding. Ligand-dependent nuclear receptor transcription coactivator activity.

MFGE8
Lactadherin. Plays an important role in the maintenance of intestinal epithelial homeostasis and the promotion of mucosal healing. Promotes VEGF-dependent neovascularization (By similarity).

MROH2B
Maestro heat-like repeat-containing protein family member 2B. May play a role in the process of sperm capacitation.

MST1
Hepatocyte growth factor-like protein. Negative regulation of gluconeogenesis.

MTCH2
Mitochondrial carrier homolog 2. The substrate transported is not yet known. Induces mitochondrial depolarization.

MUC5B
Mucin-5B. Gel-forming mucin that is thought to contribute to the lubricating and viscoelastic properties of whole saliva and cervical mucus.

MYO15A
Unconventional myosin-XV. Myosins are actin-based motor molecules with ATPase activity. Unconventional myosins serve in intracellular movements.

NCOR2
Nuclear receptor corepressor 2. Transcriptional corepressor. Mediates the transcriptional repression activity of some nuclear receptors by promoting chromatin condensation, thus preventing access of the basal transcription.

NOS3
Nitric oxide synthase, endothelial. Produces nitric oxide (NO) which is implicated in vascular smooth muscle relaxation through a cGMP-mediated signal transduction pathway.

NUMBL
Numb-like protein. Plays a role in the process of neurogenesis. Required throughout embryonic neurogenesis to maintain neural progenitor cells, also called radial glial cells (RGCs), by allowing their daughter cells to choose progenitor over neuronal cell fate. Negative regulator of NF-kappa-B signaling pathway.

PARD3B
Partitioning defective 3 homolog B. Putative adapter protein involved in asymmetrical cell division and cell polarization processes. May play a role in the formation of epithelial tight junctions.

PCDHB3
Protocadherin beta-3. Potential calcium-dependent cell-adhesion protein. May be involved in the establishment and maintenance of specific neuronal connections in the brain.

PCNXL2
Pecanex-like protein 2. May play a role in tumorigenesis of colorectal carcinomas with high microsatellite instability (MSI-H).

PIEZO1
Piezo-type mechanosensitive ion channel component 1. Pore-forming subunit of a mechanosensitive non-specific cation channel. Plays a key role in osteogenesis. Its activation commits mesenchymal stem cells to osteogenic differentiation. Phospholipase B-like 1. In view of the small size of the putative binding pocket, it has been proposed that it may act as an amidase or a peptidase (By similarity).

PSMB11
Proteasome subunit beta type-11. The proteasome is a multicatalytic proteinase complex which is characterized by its ability to cleave peptides with Arg, Phe, Tyr, Leu, and Glu adjacent to the leaving group at neutral or slightly basic pH.

RASAL1
RasGAP-activating-like protein 1. Probable inhibitory regulator of the Ras-cyclic AMP pathway. Plays a role in dendrite formation by melanocytes.

REC8
Meiotic recombination protein REC8 homolog. Required during meiosis for separation of sister chromatids and homologous chromosomes.

RNF213
E3 ubiquitin-protein ligase RNF213. Involved in the non-canonical Wnt signaling pathway in vascular development: acts by mediating ubiquitination and degradation of FLNA and NFATC2 downstream of RSPO3, leading inhibition of the non-canonical Wnt signaling pathway and promoting vessel regression.

RPP40
Ribonuclease P protein subunit p40. Component of ribonuclease P, a protein complex that generates mature tRNA molecules by cleaving their 5'-ends.

SCARF2
Scavenger receptor class F member 2. Probable adhesion protein, which mediates homophilic and heterophilic interactions. In contrast to SCARF1, it poorly mediates the binding and degradation of acetylated low density lipoprotein (Ac-LDL)

SYT15
Synaptotagmin-15. May be involved in the trafficking and exocytosis of secretory vesicles in non-neuronal tissues.

TBC1D2
TBC1 domain family member 2A. Acts as GTPase-activating protein for RAB7A. Signal effector acting as a linker between RAC1 and RAB7A, leading to RAB7A inactivation and subsequent inhibition of cadherin degradation and reduced cellcell adhesion.

TBXAS1
Thromboxane-A synthase. Oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen. Heme binding. Iron ion binding.

TNS4
Tensin-4. May be involved in cell migration, cartilage development and in linking signal transduction pathways to the cytoskeleton (By similarity). May promote apoptosis, via its cleavage by caspase-3.

ZP3
Zona pellucida sperm-binding protein 3. The mammalian zona pellucida, which mediates species-specific sperm binding, induction of the acrosome reaction and prevents post-fertilization polyspermy, is composed of three to four glycoproteins, ZP1, ZP2, ZP3, and ZP4. ZP3 is essential for sperm binding and zona matrix formation.