The Genetic Factors Of Human Holoprosencephaly Biology Essay

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Holoprosencephaly (HPE) is a 'less known' but common disease that cause incomplete cleavage of prosencephalon (forebrain) into two lobes of cerebral hemispheres (telencephalon). This review will explain about genetic abnormalities in HPE caused by mutation of specific major genes or even the minor gene and how HPE is identified in new patients. Despite being a minor HPE causation (25 to 50% of HPE cases), studies in genetic factor will help in gaining updated information about HPE and the discovery of new HPE candidate genes.


HPE affects 1 fetus in 200-250 pregnancies and 1 in 16 000 live-born infants in the world (Roessler et al., 1996). HPE can be classified into 4 types which are alobar, semi lobar, lobar and middle interhemispheric variant (MIHV). The most severe is alobar HPE, which can be characterized by incomplete cleavage of brain into hemisphere due to the absence of interhemispheric fissure.

The moderate HPE is semi lobar that can be detected by partially divided brain when it is fused in the front but separated in the rear. The mild lobar HPE's patients have well disjoined brain but some fusions on the ventral telencephalon do exist. The patient diagnosed with MIHV HPE will has incomplete division of the middle brain in between parietal lobes and posterior frontal in the telencephalon.

Symptoms that can be observed in the patient are as severe as cyclopia, having a single central eye and proboscis, which the nose is located on the forehead to the mild type, of having single inaxillary central incisor that refers to an upper central tooth (Dubourg et al., 2007). Epilepsy, mental retardation and endocrine abnormalities are common diseases affecting the patients with HPE (Dubourg et al., 2007).

Besides genetic factor that contributes to 20 to 50% of HPE (based on number of patients taking part in previous research), most HPE cases were caused by maternal factors. Mother suffering from diabetes mellitus, infection occurring during pregnancy such as herpes, rubella and syphilis or even pregnancy loss and bleeding in the first trimester, all can lead to HPE (Johnson and Rasmussen, 2010). Teratogen consumption, for example, retinoic acid and alcohol can affect the embryo development too (Johnson and Rasmussen, 2010).





Figure 1. The 4 types of HPE with the brain structure and facial deformities formed. A. Brain of alobar HPE and patient with cyclopia and proboscis. B. Brain of semi lobar HPE and patient with cleft lip. C. Brain structure of lobar HPE and patient with normal facial appearance. D. Brain of MIHV HPE and patient with normal face appearance. Pictures obtained from Muenke and Gropman, 2000.

Genetic Etiology of Holoprosencephaly

25% of HPE cases are from well recognized syndromes such as Smith- Lemli- Opitz Syndrome and Pallister-Hall Syndrome (Bendavid et al., 2010). Meanwhile, 10 to 15% of it is caused by unidentified genetic alteration and environmental causes since no identification of specific genetic cause can be traced to nonsyndromic and nonchromosomal HPE (Dubourg et al., 2007). Other times, HPE is caused by specific autosomal dominant genes which mutate in 12 different loci (Wallis and Muenke, 2000). The chromosomal rearrangement can exist as inherited autosomal dominant or de novo mutation (Dubourg et al., 2007).

Inherited Autosomal Dominant

HPE can be an inherited autosomal dominant disorder with the gene variably expressed and penetrate incompletely (Mercier et al., 2010) (Pineda- Alvarez et al., 2010). In a research done among 30 families experiencing autosomal dominant HPE, 5 of them showed different SHH heterozygous mutation, 2 faced nonsense mutation, while others showed different genotype features (Roessler et al., 1996).

Inherited HPE may re-occur at high risk but it is hard to predict the functional consequences since most of the patients have unique family specific mutation (Mercier et al., 2010). However, Mercier et al., 2010 concluded that although the parent suffered from severe HPE, it did not indicate HPE will be passed to the foetus since only 7 out of 15 pregnancies carried the inherited mutation and only 3 of them had to be terminated (Mercier et al., 2010). Inherited HPE is influenced by mutation type, pedigree, carrier parent's microform presence as well as maternal factor (Mercier et al., 2010).

De Novo Mutation and Other Possible Mutations

Based on results obtained from array CGH, 17% of HPE patients have de novo mutation that is not heredity (Bendavid et al., 2009). There are also cases where a few siblings or relatives were affected even though the parent was not a carrier. This finding leads to an idea of autosomal recessive inheritance, yet it has not been approved as no event has been identified on other allele and all mutations mapped are heterologous (Mercier et al., 2010). Besides exonal mutation causing HPE, there is also raising evidence that HPE may be caused by non-coding portion of HPE gene and gene regulatory elements (Jeong et al., 2008).

Genes Causing Holoprosencephaly

Specific gene causing HPE can be distinguished by karyotype approach which helps in defining a minimal critical region (Bendavid et al., 2010). 4 major genes discovered in relatively high occurrence in HPE patients are Sonic Hedgehog, SHH (37% of inherited cases), ZIC 2 (9% of HPE patients), SIX 3(5% of patients) and TGIF (approximately 1% of HPE patients) (Gripp et al., 2000).

There are also other genes contibuting to HPE such as GLI, PATCHED-1, DISP-1, FOXH1, NODAL and TGDF1 (de la Cruz et al., 2002) (Roessler et al., 2008). However, they were reported to occur rarely in less than 1% of HPE cases and usually relate with HPE microform (Roessler et al., 2008) (Pineda- Alvarez et al., 2010) (Bendavid et al., 2010). Besides, individual gene gives minor overall effect due to insignificant function to cause HPE, therefore requires few mutant genes (Roessler et al., 2008) (Bendavid et al., 2010) (Roessler and Muenke, 2010).

Figure 2. Location of responsible gene causing HPE in specific loci. From this karyotype mapping, we know that SIX3(HPE2) have mutation at 2p21, ZIC2 (HPE5) at 13q32, SHH (HPE3) at 7q36 while TGIF (HPE4) alter locus of 18p11.3 (Brown et al., 1998) (Roessler et al., 1996) (Gripp et al., 2000). This diagram also tells about other deletions including microdeletions and microarrangements besides the localization of HPE minor genes mentioned earlier (Bendavid et al., 2010).

Major Genes Causing Holoprosencephaly


SHH is a ligand protein from the hedgehog family which consists of IHH (Indian Hedgehog and DHH (Desert Hedgehog). SHH has three exons of which 96% of its N-terminal fragment (SHH-N) is on the first two exons (Chiang et al., 1996) (Roessler et al., 1996). 19kD amino terminus, (SHH-N) and a 25 kD carboxy terminus, (SHH-C) are produced by autoproteolytic cleavage which is very crucial for all biological processes (Porter et al., 1995) (Chiang et al., 1996). Let if there are eight mutations happen on SHH gene, 62.5% of it will be caused by disruption on SHH-N and the rest is stimulated by interference on SHH-C, although the later plays small role in patterning activity (Wallis and Muenke, 2000).

Studies on homozygous mutant SHH gene in mice indicated that SHH is responsible for the midline in CNS development, besides plays important roles in eyes and craniofacial structures formation (Chiang et al., 1996) (Wallis and Muenke, 2000). SHH is also functional in signal peptide dissociation besides encoding proteins that will be secreted for microsome post-trafficking (Roessler et al., 1996). Moreover, it was then approved that SHH is expressed within ventral midline neural tubes, notochords and Hensen's nodes (Roessler et al., 1996).

Haploinsufficiency of SHH occurs when the anterior ventral midline lacks SHH signaling, disabling the ventral midline to develop normally and leads to shortening of prosencephalon's width (Scotting, 2009). Reduction of cleavage leads to fusion of lobes and may contribute to cyclopia since SHH also changes the expression of transcription factors specific for eye development, Pax 2 and Pax 6 (Scotting, 2009). Cyclopia also have been reported in sheep consumed cyclopamine, an SHH inhibitor in its diet (Scotting, 2009).

Another role of SHH is inducing somites' ventral specification and patterning of limb bud development (Chiang et al., 1996). In that context, haploinsufficiency in human SHH is insufficient to induce long-range effects, such as limb formation although it is adequate to disrupt neurogenesis (Roessler et al., 1996) (Scotting, 2009). Neurogenesis of neurons in forebrain and spinal cord were triggered when floor plate is induced by SHH (Chiang et al., 1996). The fact is, different SHH concentration requirement varies among species. For example, chicks need 5 times SHH-N concentration for induction of its floor plate s compared to its motor neuron (Roelink et al., 1995, cited in Roessler et al., 1996).

SHH becomes the most pathogenic HPE gene because a single allele without SHH is enough to cause HPE in humans (Chiang et al., 1996) (Roessler and Muenke, 2010). 10 out of 300 HPE cases that were identified had SHH mutations and 7 of them were autosomal dominant (Roessler et al., 1996). The SHH mutations involved in the cases were 4 missense mutations, 2 nonsense mutations, and 1 for both deletion and insertion (Roessler et al., 1996).

HPE caused by inherited SHH mutation is more common than sporadic cases although principally, the frequency of de novo mutation is higher than the familial event in HPE cases (Wallis and Muenke, 2000). In a research demonstrated, it showed that 14 out of 23 HPE patients suffered from inherited SHH mutation while sporadic cases occurred in others (Roessler et al., 1996).


The second major gene is ZIC2, a nuclear zinc finger which was first discovered in mouse but has quite a different location for expression and perhaps, a different role in humans (Brown et al., 1998). ZIC2 is expressed only in foetal brain and this has been proved in two abnormal bands found in the foetal brain's DNA in two patients with small base deletion (Brown et al., 1998). Moreover, mutant human ZIC2 do not cause digital abnormalities since no HPE patients who suffered from ZIC2 mutation have abnormal fingers (Brown et al., 1998).

ZIC2 is a dosage-sensitive gene and can cause happloinsuffiency because 3 HPE patients were reported to have lost only one of their ZIC2 alleles (Brown et al., 1998). Interestingly, patients diagnosed with ZIC2 mutation in severe HPE do not have severe facial deformities (Brown et al., 1998).


SIX3 acts as transcriptional factor and has two functional regions which are crucial for specific DNA binding (Wallis and Muenke, 2000). SIX3 is also an activator of SHH in rostral diencephalon ventral midline and works synergistically with SHH (Geng et al., 2008). 5 missense mutations reported in SIX3 caused haploinsufficiency, but did not affect its protein or mRNA (Geng et al., 2008).

Loss of one SHH alleles in the presence of hypomorph SIX3 will lead to semilobar HPE while SIX3 haploinsufficiency will inhibit SHH expression (Geng et al., 2008). The reason is, to organize anterior brain, SIX3 needs to bind to SHH brain enhancer (SBE2). Thus, the inhibition process occurs both ways since SIX3 is unable to bind to SBE2 if either SBE2 or SIX3 is mutated (Jeong et al., 2008). Besides functions in brain development, SIX3 is required for eye formation too (Wallis and Muenke, 2000).


TGIF stands for TG- interacting factor and is a human homeodomain protein (Gripp et al., 2000) (Ferrand et al., 2007). TGIF mutation occurs rarely since in 268 DNA samples from blood or lymphoblastoid cells, only 4 missense mutations were found in TGIF loci (Gripp et al., 2000). Detection of TGIF can be performed using FISH which will have no FISH signal if the TGIF is mutated and vice versa (Gripp et al., 2000).

It contains 4 exons and the last 3 carry coding sequence (Gripp et al., 2000). HPE disturbs the function of TGIF as a transcriptional co-repressor of SMAD2 (Gripp et al., 2000) (Ferrand et al., 2007). Repression of transcription will take place if increasing concentration of TGIF is targeted through multiple TGIF-binding sites to a promoter (Gripp et al., 2000). For binding to SMAD2, TGIF has to compete with p300 histone acetylases in SMAD2 and modification of TGF-beta family may be established (Gripp et al., 2000) (Ferrand et al., 2007).

Minor Genes Causing Holoprosencephaly

GLI2 gives microform symptom of HPE in anterior pituitary of the proband (Roessler et al., 2003). Nevertheless, there are specific pituitary gland anomalies, such as pituitary hormones' low secretion that can occur with or without extensive brain abnormalities and craniofacial deformities in HPE (Roessler et al., 2003). GLI2 works as a transcription factor in human SHH signalling pathway too(Roessler and Muenke, 2010).

PATCHED-1 or PTCH1, a transmembrane protein is referred to as a receptor of SHH (Bendavid et al., 2010). It works by inhibiting the pathway of SHH when SHH is absent, thus preventing SHH signalling (Muenke and Gropman, 1993-2000). Mutation in PTCH1 will disrupt its inhibition on SHH pathway by enhancing the repression. As a result, SHH signalling will decrease. Mutated PTCH1 will also lose its ability to bind to SHH (Dubourg et al., 2007).

The third gene, NODAL regulates the development of body axes during gastrulation (Gripp et al., 2010). NODAL has a close link with TGIF as TGIF connects the NODAL signalling pathway to the forebrain and helps to establish ventral midline structures (Gripp et al., 2000). NODAL signalling is a pathway crucial for development of left-right axis, midline and forebrain (Dubourg et al., 2007).

TGDF1 (CRIPTO) and FOXH1 are also involved in NODAL signalling and act as transcription factors of it (Roessler et al., 2008). TGDF1 functions in development of the forebrain and midline (de la Cruz et al., 2002). Meanwhile, FOXH1 has a domain essential for DNA binding and another domain that acts together with SMAD proteins (Roessler et al., 2008). Therefore, mutation in FOXH1 will interrupt these two domains (Dubourg et al., 2007).

Lastly, DISP1, also known as DISPATCHED, is one of the transcription factors involved in SHH signalling pathway beside PTCH1 and GLI2 (Roessler and Muenke, 2010). It releases and transports cholesterol-anchored hedgehog ligands (Roessler and Muenke, 2010).

Research is still being carried out to identify new HPE genes, for example CFC1 (CRYPTIC), a gene in the same EGF-CFC family with TGDF1 (de la Cruz et al., 2002). Nonetheless, it can only be acknowledged as HPE gene if point mutation or microdeletion was detected in the patient's karyotype analysis (Bendavid et al., 2010). Tests to investigate if HPE is caused by the minor genes should only be performed on selective patients or the patient is referred only to experts in specialized testing centres (Pineda- Alvarez et al., 2010).

Holoprosencephaly Detection and Techniques Used

Prenatal Detection

HPE will start to develop at the early stage, during the first few weeks of pregnancy (Bendavid et al., 2010). Therefore, ultrasound examination and MRI (Magnetic Resonance Imaging) can be demonstrated to check for any feature deformities after 16 weeks of gestation (Mercier et al., 2010). MRI will provide better picture of any brain abnormalities, but it can only be carried out at the later stages of pregnancy (Hahn and Barnes, 2010, cited in Mercier at al., 2010). When the child is born, Computed Tomography and MRI analysis of head will help in confirming the results (Mercier et al., 2010). Unfortunate foetuses usually will undergo foetal autopsy for the neuropathological examination.

For detecting chromosomal abnormalities, amniocentesis can be used by drawing out small samples of amniotic fluid during embryogenesis. This procedure is perfect for detecting Trisomy 18 (Edward Syndrome) or close-related Trisomy 13 (Patau Syndrome) since 70% of the Trisomy 13 patients suffer from HPE too (Mercier et al., 2010). Chorionic villus sample can also be analysed.

Postnatal Detection

Figure 3. The procedures to examine new HPE patients (Pineda-Alvarez et al., 2010).

Gene mutation screening is applied for testing HPE at the gene level (Pineda-Alvarez et al., 2010). GeneDx is the only lab that offers HPE testing commercially in the world. However, the cost is very high, around USD2,000 for detecting mutation in the major genes of HPE (The Carter Centers for Brain Research in HPE and Related Malformation, 2010). National Institute of Health, on the other hand, conducted gene screening for research.

Since HPE is caused by chromosome micro rearrangement, special screenings are established and they are successful in detecting deletion and point mutation of few genes in 8% of gene anomalies (Dubourg et al., 2007) (Bendavid et al. 2010). They are Multiple Ligation Dependent Probe Amplification (MLPA) and Quantitative Multiple PCR of Short Fluorescent Fragment (QMPSF) (Bendavid et al., 2009) (Pineda-Alvarez et al., 2010). MLPA is very beneficial for analysing subtelomeric loss or gain and it contributes to identification of 4.3% aberrations in 181 HPE cases (Ledbetter and Martin, 2007) (Dubourg et al., 2007) (Bendavid et al., 2007). It increases the diagnosis effectiveness from 18 to 25% too (Bendavid et al., 2010).

However, a few tests have to be carried out to identify the presence of different dosages, for example real-time quantitative PCR (qPCR) and FISH (fluorescent in situ hybridization) (Pineda-Alvarez et al., 2010). There are also high-resolution DNA melting(HRM) which is a screening process of high sensitivity and specificity amplicons which can be achieved in approximately 2 hours (Pineda-Alvarez et al., 2010).

Besides analysis in the progeny, gene screening on the parent DNA has been done as involvement of two molecular event may occur and assist in recognition of mutation and micro rearrangement associated in the same proband (Nanni et al., 1999, cited in Pineda-Alvarez et al., 2010) (Bendavid et al., 2009).

FISH can be an additional analysis of proband's karyotype anomalies of known HPE gene as well as identify any cryptic-balanced chromosomal changes (Ledbetter and Martin, 2007) (Bendavid et al., 2006). With the help of array CGH, anomaly imbalance can be identified since all of 111 nonsyndromic patients examined have HPE phenotype but 25% of them were discovered to have unbalanced chromosomes (Bendavid et al., 2009) (Mercier et al., 2010). QMPSF and qPCR are needed in FISH (Bendavid et al., 2010).

Array CGH

Mainly, the DNA in the blood sample of both proband and child will undergo bi-directional DNA sequencing of exon, an excellent procedure to date (Pineda-Alvarez et al., 2010). Simultaneously, targeted array CGH analysis with exon-level resolution, so called ExonArrayDx in GeneDx is executed (Mercier et al., 2010) (GeneDx, 2010). Assessment of exons' duplication or deletion helps to recognize new HPE regions and thus suggests that genomic rearrangement occurs at a high rate in HPE (Bendavid et al., 2009) (Mercier et al., 2010). Array CGH uses high resolution technique and it can detect up to approximately 8kb of DNA using high-density oligonucleotide arrays (Ledbetter and Martin, 2007) (Bendavid et al., 2010). High resolution chromosome analysis can identify more than 550 band stage of chromosome condensation (Pineda-Alvarez et al., 2010).

Genotype-Phenotype Correlation

HPE is the utmost multi-phenotypic disease and have different specific symptoms in different individuals, caused by different genes and this is approved by brain abnormalities characterization (Pineda-Alvarez et al., 2010). Correlation of genotype-phenotype helps the researchers minimize the time to identify genes that responsible in causing HPE in the patients. If members in the same family suffer several microforms and severe facial malformation, SHH should be checked first (Brown et al., 1998). If members in the family suffer severe brain abnormalities, syntelencephaly or neural tube defects but generally have no major face deformities, except for several eye abnormalities, analysis on ZIC2 should be carried out first (Brown et al., 1998) (Wallis and Muenke, 2000). If there is abnormal development of brain and eyes, SIX3 may be the factor (Mercier et al., 2010). For TGIF, usually it is uncommon to cause significant phenotypic features since it occurs at a low rate (Mercier et al., 2010).


HPE still remains as a tragedy for most parents if they do not gain enough information about the survival chance for their children since most of HPE effects are uncertain and the result obtained from analysis is best interpreted by genetic counselling (Pineda-Alvarez et al., 2010). There are efforts made by the public such as Families for HoPE Organization which gather families suffering from HPE and not forgotten, The Carter Centers for Brain Research in HPE and Related Malformation (Pineda-Alvarez et al., 2010).

Since HPE is common, parents are urged to join in HPE research so that the data sharing and sample test will help in better understanding of HPE phenotypes and aid in discovery of new HPE genes (Pineda-Alvarez et al., 2010).