Sonic Hedgehog Case Study


Holoprosencephaly (HPE), the most common developmental defect of the forebrain and the face, is genetically heterogeneous. One of the genes involved, Sonic hedgehog (SHH), on 7q36, has been identified as the first HPE-causing gene both in mouse and humans. In order to delineate the phenotype of specific SHH mutations, we described the expression of the SHH gene during early human embryogenesis and investigated the phenotype of novel SHH mutations. In situ hybridization studies were performed on paraffin-embedded human embryo sections at three different development stages. These studies show that SHH is expressed in the notochord, the floorplate, the brain, the zone of polarizing activity and the gut. We also report on the phenotype of four novel mutations identified in 40 HPE families (two in isolated HPE and two in familial HPE). Expressivity ranged from alobar HPE to microcephaly and hypoplasia of the pituitary gland in one family, and from HPE to an asymptomatic form in another family. No SHH mutation was found in six polymalformed cases combining HPE with other defects, such as skeletal, limb, cardiac, anal and/or renal anomalies. This study confirms the genetic heterogeneity of HPE, and further demonstrates that SHH mutations are associated with a broad spectrum of cerebral midline defects.


Holoprosencephaly (HPE; 1:16 000 live births; 1:250 conceptuses) is a common developmental defect affecting both the forebrain and the face (1,2). Clinical expressivity is variable, ranging from a single cerebral ventricle and cyclopia to clinically unaffected carriers in familial HPE. The disease is genetically heterogeneous but additional environmental agents also contribute to the aetiology of HPE (3–5). A previous segregation analysis led to the conclusion that the transmission of isolated HPE was compatible with autosomal dominant inheritance with incomplete penetrance (82%) of the disease (3). Also, a careful analysis of cytogenetic rearrangements in HPE patients led to the identification of at least 12 chromosome regions implicated in the pathogenesis of HPE (5). Among the putative loci for the disease, HPE3 has been shown by linkage analysis of families segregating for the disorder to be localized on chromosome 7q36, and Sonic hedgehog (SHH) has been identified as the first HPE-causing gene both in mouse and human (6–9). Both nonsense mutations and cytogenetic deletions suggest that a loss of function of the SHH gene is causing HPE. More recently, mutations in other genes have been shown to be associated with HPE (10–13; i.e. ZIC2 on 13q32; SIX3 on 2p21; PATCHED on 9q22.3; TGIF on 18p11.3).

SHH belongs to a vertebrate evolutionarily conserved family of genes (including Desert hedgehog and Indian hedgehog) which are related to the Drosophila segment polarity gene hedgehog (14,15). In vertebrates, shh is expressed in the midline central nervous system (CNS), the notochord and the limb bud zone of polarizing activity (ZPA), and the secreted shh protein is an inductive signal in the patterning of the ventral neural tube, the anterior-posterior limb axis and the ventral somites (16–19). However, whether SHH expression is the same in early human development is unknown. The human SHH has three exons (Fig. 1A) and encodes a 462 amino acid polypeptide (7). The protein is synthesized as a precursor molecule that undergoes cleavage of a signal peptide and then autoproteolytic cleavage. This reaction mediated by cholesterol leads to a 19 kDa N-terminal product (SHH-N) with the signalling domain and a C-terminal product of 25 kDa (SHH-C) possessing the cleavage domain closely associated with cholesterol transferase activity (20; Fig. 1B). Mutations of the SHH gene in human HPE are scattered along the whole protein (8,21; Fig. 1A).

In order to understand better the function of SHH and its relationships to the isolated HPE, we studied the expression of the SHH gene during early human embryogenesis. This study of SHH expression in human embryos strongly suggests that, as in other vertebrates, SHH plays an important role in human embryonic patterning. Moreover, we describe the clinical expression of four novel SHH mutations in 40 HPE families. This study, which represents only the second analysis of a large series, helps to establish the range of mutation types causing human HPE and allows SHH mutations to be correlated with a large spectrum of HPE phenotypes.

Figure 1

SHH gene and protein structure and human mutations. The SHH gene is composed of three exons (A) encoding a polypeptide of 462 amino acids (B)which is synthesized as a precursor molecule that undergoes cleavage of a signal peptide and then autoproteolytic cleavage (mediated by cholesterol) into a 19 kDa N-terminal product (SHH-N) and a 25 kDa C-terminal product (13; SHH-C). Reported mutations in the SHH gene are located on the cDNA, including four novel mutations reported in this study (underlined): missense mutations are at the top and nonsense or truncated mutations at the bottom. The localization of the probe used for in situ hybridization studies is represented.

Figure 1

SHH gene and protein structure and human mutations. The SHH gene is composed of three exons (A) encoding a polypeptide of 462 amino acids (B)which is synthesized as a precursor molecule that undergoes cleavage of a signal peptide and then autoproteolytic cleavage (mediated by cholesterol) into a 19 kDa N-terminal product (SHH-N) and a 25 kDa C-terminal product (13; SHH-C). Reported mutations in the SHH gene are located on the cDNA, including four novel mutations reported in this study (underlined): missense mutations are at the top and nonsense or truncated mutations at the bottom. The localization of the probe used for in situ hybridization studies is represented.


SHH expression during early human development

To define the temporal and spatial expression of SHH during early human development, we performed in situ hybridization on paraffin-embedded human embryos ranging from Carnegie 12 to 16. The SHH riboprobe was generated by cloning an exon 3 amplification product. Primers and probe sequences (173 bp) were chosen to amplify specifically the SHH gene and hybridize to the SHH mRNAs, respectively (8,14; no homology with human Desert hedgehog, 60% homology with human Indian hegdehog). At Carnegie 12, SHH is strongly expressed in both the notochord and the CNS. Here it is expressed along the entire length of the floorplate, in the spinal cord and the hind-brain (data not shown). SHH signal is also seen in the foregut (Fig. 2A and B), while no expression is detected in the midgut (Fig. 2C and D). At Carnegie 14, SHH expression is still observed in the notochord, the floorplate (Fig. 2K and L) and the foregut. In addition, a strong signal is detected in the hindgut (cloaca, Fig. 2E and F). In the CNS, it extends rostrally to the diencephalon and the prosencephalon (Fig. 2M and N), except in a small region of the ventral diencephalon where no SHH expression is detected. At this stage, posterior mesenchymal cells of the forelimb buds also express SHH (Fig. 2K and L). At Carnegie16, SHH expression is observed all over the CNS midline (Fig. 2O and P), except in the rostral part of the diencephalon where SHH message is located laterally (Fig. 2Q and R). SHH is still strongly expressed along the entire length of the notochord, the floorplate, the foregut and the hindgut. A weak signal is also detected in the lung buds (data not shown). At that stage of limb development, SHH is expressed in the posterior mesenchyme of both fore- and hindlimb buds (Fig. 2I and J). No expression was detected in the developing heart, liver or kidney. Table 1 summarizes SHH expression during early human development.

DNA analysis and patient phenotypes

We identified four novel SHH mutations in four unrelated non-syndromic HPE cases (two familial and two isolated). These four mutations were not observed in 100 control chromosomes. One mutation leads to a premature termination of SHH translation, whereas the three other mutations involved invariant amino acids (Table 2). In addition, no SHH mutation was found in six cases of HPE associated with other malformations.

The second mutation was a 474C→G transversion resulting in a stop codon (Y158X). This mutation abolished an RsaI restriction site. It was identified in a child (Fig. 3, Family 2) presenting with severe microcephaly (−7SD), a nasal pyriform aperture stenosis, a pituitary gland dysgenesis and growth retardation, but no brain malformation. The mutation was inherited from his mother who had hypotelorism and microcephaly (−4SD), a single central incisor, dysgenesis of the corpus callosum and a mild developmental delay. In addition, on the maternal side of the family, two patients died from typical HPE with arrhinia (III5) and alobar HPE (III6).

The third mutation (562G→C), a transversion of the last base of exon 2, resulted in a glutamic acid to glutamine substitution (E188Q) in an invariant amino acid in all of the vertebrate Hedgehog proteins, yet is divergent from the Drosophila sequence. This mutation created a BsaAI restriction site. The male proband (Figs 3 and 4B; Family 3) presented with lobar HPE, microcephaly (−5SD), diabetes insipidus and mental retardation. He was the only affected individual in the family. Surprisingly, the mutation was inherited from the mother who had a normal phenotype. A retrospective family history revealed the existence of several neonatal deaths on the maternal side of the family, but no information on brain or facial malformation was noted. Therefore, a familial form of HPE cannot be excluded. In addition, the causal nature of this mutation should be demonstrated at the functional level.

The fourth mutation was a 6647G→A transition changing an aspartic acid to an asparagine (D222N), 26 amino acids from the cleavage site between SHH-N and SHH-C. This mutation segregated over four generations. The female proband (Fig. 3, Family 4) had a semilobar HPE, whereas the great-grandmother had hypotelorism with unilateral cleft lip and palate. The proband's mother (III9) and brother (IV5) both had microcephaly and hypotelorism. In addition, many severe forms of HPE (II3,II4, III3, III6, III7, III11,IV1) and at least one clinically unaffected carrier (III10) were observed in this family.

Figure 2

SHH expression in human embryos. Bright-field (A, C, E, G, K, M, O and Q) and dark-field (B, D, F, H, J, L, N, P and R) illumination. (A-D) Transverse sections through a Carnegie 12 embryo showing SHH expression in the stomach (St) and duodenum (Duod) but not in the midgut (MG). (E and F) Transverse section through the caudal region of a Carnegie 14 embryo showing SHH expression in the cloaca. SHH is also expressed in the forelimb buds by Carnegie 14 (G and H) and in the hindlimb buds by Carnegie 16 (I and J). (K-N) Sagittal sections through a Carnegie 14 embryo showing a strong SHH signal in the notochord (NC), the floorplate (Ventral NT) and the ventral midline of the brain. (O-R) Transverse sections through the head of a Carnegie 16 embryo showing the midline expression of SHH in the CNS in the mesencephalon (Mes, O-R), and lateral expression in the diencephalon (Di, Q and R). Ao, aorta; H, heart; NC, notochord; NT, neural tube. Magnification: (A-D, H and J) ×80; (E and F) ×60; (I) ×35; (O and P) ×25; (G and K-N) ×20.

Figure 2

SHH expression in human embryos. Bright-field (A, C, E, G, K, M, O and Q) and dark-field (B, D, F, H, J, L, N, P and R) illumination. (A-D) Transverse sections through a Carnegie 12 embryo showing SHH

Sonic Passion: A case study on idiots
by Lord Shmeckie, Master of AFTER, and Sway

We all know the world wide web is not always a pretty place. Sure, there are plenty of normal web sites for people to visit -- web sites that allow us to book vacations or shop from the comfort of our homes, web sites that provide us with great free entertainment, web sites that allow us to get our news for free, and web sites that allow us to interact and connect with others across the globe, for example, but beneath the web's bright and shiny exterior lies a seamy underbelly full of insane conspiracy theories, sexual depravity, disturbing pictures, and just about anything else you don't want to see. The bright side of this underbelly is that it is an endless source for humor and entertainment in the form of mockery and satire. In other words, some of the stuff people are into is just plain hilarious.

One such hilarious web site is Sonic Passion, a forum for people who are into something called "robophilia". Just what is robophilia, you ask? The preferred definition seems to be a sexual attraction or love for an artificially intelligent, human-like robot. So if you were attracted to Data from Star Trek (if he were to exist in real life), for example, that would be robophilia. The Sonic Passion forum members have misconstrued this word to mean a sexual attraction to, believe it or not, video game characters. And we're not talking Lara Croft or Kasumi or Yuna. These people are primarily attracted to characters from the Sonic The Hedgehog series of games. Yeah, you read right. These people would like nothing better than to bend Sonic over a chair and fuck him right up his little blue asshole. Or maybe they'd love to pound into Amy Rose while she beats them senseless with her giant mallet. And those are the least insane fantasies you'd find on this forum. These sexual deviants can combine fur fetish, scat fetish, pedophilia, rape fantasies, bestiality, voyeurism, slash fiction, and some other shit no one's ever even heard of into a horrific cocktail of anti-erotic madness that will leave you wondering whether you should be laughing or crying. The forum members also make it clear they despise society and real people and feel a connection only with video game characters and other real people like them.

Chief among the offenders is the forum's administrator, Alix Henriol, who has taken her sexual obsession with Sonic so far beyond rationality that it makes the rest of the members seem perfectly normal by comparison. Most of the following screencaps feature her in some way or another, because she really is the most entertaining out of all of them. She is willing to kill herself over Sonic, as she has readily claimed time and time again, and after reading some of her forum posts, you'll see there's no reason to doubt her sincerity. Is this the craziest forum on the web? In all honesty, probably. So on that note, onto the show!

Further proof of the popular scientific theory that Sonic's semen transcends sexual orientation.

Alix's Father: Tell me again why you wanted me to buy this extremely expensive digital camera for you?
Alix: It's for... umm... school.

Dear lord, this is Alix's full signature. Basically all of that boils down to: "I plan on dying alone."

How far gone are you when circles are more arousing that actual human beings? Anthropomorphic animals like Sonic, I can at least understand the why/how, but these are fucking circles with eyes.


Screw any good reviews it gets from respected magazines and web sites, this post would make a great quote for the box cover of the next Sonic game!

Damn woman, even your fellow "robophiles" think you're a frothing nutcase. Go get yourself some medication. And then resize that fucking gigantic avatar.


I believe there are about 14,035 pictures done by 12 furries that say otherwise...

The Grim Reaper and Satan are your role models in life? That's not normal. Oh, right, you want to fuck video game characters.

Here's a picture of Shadow #1 fan. A gay goth who admires the religious representation of evil, the manifestation and symbol of death and the circle of life, and a cartoony hedgehog from a video game. He's a joke in itself, folks.

Throw in some dialogue and we got ourselves material for!

Well now you're just ruining it for everybody.

"Why won't she respond top my marriage proposal? Is it because I'm already married to the letter 'D'?! It's fake, Amy! My marage certifercit is a fake!"

No dude, I totally think Sonic Team was all like: "We need more sex appeal in our games, and since the voluptuous, flirtatious bat with huge tits didn't do it for anybody, let's include a prepubescent rabbit girl in the next game to really get the fanboys drawing those naked pictures of our characters!" Makes perfect sense.

Can't we just cuddle?

Nice to see Kramer's "bro" caught on in Sonic's world.

What the hell is it with you people wanting to ejaculate all over everything!

Fantasizing about a girl who isn't alive anymore? Eww dude that's gross! Quit talking about all this gross stuff and go back to picking which video game character you want to have anal sex with.

Suddenly, just not having a date doesn't really seem that pathetic anymore.

HOW COULD YOU BASE YOUR MARRIAGE TO A VIDEO GAME CHARACTER ON A LIE?! CHEATING ON SHADOW?! THE NERVE! I mean, really, cheating on video game characters... how can you even face yourself in the mirror?

Someone should tell her that marriage certificate isn't legally binding, just to feed my curiosity as to which method of suicide she'll attempt. My guess is cutting. It's not terribly adventurous, but it's a tried and true method endorsed by assloads of teenagers.

Chaosangel sat at her computer and repeatedly pressed the refresh button on her browser for days, eagerly awaiting a reply from her dear Shadow-chan, until finally she starved to death and was later discovered when her parents decided to sweep out the basement. You folks at home will get the whole story when the movie version comes out.

I wonder why there are no replies...

Here we can see the wide spectrum of sexual insanity that exists on these forums. Mmmm, that Bomberman is a piece of ass.

You know why people can't understand that there are people in this world in love with Sonic and therefore have a sexual attraction toward him? Because that's fucking retarded!

Ah, yes, proof on the internet that you married Sonic, that'll impress a lot of people. Could this day possibly get any more romantic? And you're crying, too. That's insane.

Yeah, heaven forbid you ruin the sanctity of video game marriage by allowing polygamy! It's forbidden in almost all countries on Earth, unlike fucking video game characters, which is so utterly obtuse and ridiculous they haven't even begun to think about considering making a law against it! Thanks, Alix! Anything goes, as long as it's done to ONE "person", dammit!

Tough break... And what really sucks is that if you're away from your child for that long, the Video Game Child Services people might declare you an unfit mother and take your beloved Rubi away. Oh my god, what would you do if that happened and the Video Game Courts wouldn't give you custody again? What would you tell Knuckles? Oh my god. But seriously, when a seven-year-old kid has a better grasp of reality than you, it's time to die.

Alix's Sonic sex toy collection. If you think the imagery is bad, just imagine the stench.

Wouldn't it have been great if he never woke up?

Huge monkeys throwing barrels!? Holy shit, I just came in my pants!

Well, that's it. This was just a small sample of what goes on at Sonic Passion. We hope you laughed as hard reading this as we did working on it. Be sure to visit the Sonic Passion Forum for more -- way more. You can open up any topic there and 95% of the time you will be treated to some laugh-out-loud insanity courtesy of one of the members there.

Just... be sure to turn images off in your browser beforehand. Seriously.

Sonic Online


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