Genetic cause of the congenital disorder Kabuki Syndrome

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Kabuki Syndrome is a mental retardation malformation syndrome. The main features of the syndrome are mental retardation, post-natal dwarfism and a strange face shape. The faces of children with kabuki syndrome are "characterised by long palpebral fissures, eversion of the lateral third of the lower eyelid, arched eyebrows, depressed nasal tip and large, prominent earlobes" [1].

Fig A: [2] This picture shows the facial characteristics of children with Kabuki at various ages. All of them have the same facial feature combination of a tall forehead with arched eyebrows which is typical in all Kabuki cases. The prominent earlobes and depressed nasal tips are also present.

In 1967 Dr Niikawa from Japan came across a female infant whose features/symptoms didn't fit any existing disorders. It was this encounter that prompted a search to find other people across Japan that had the same/similar features. Observations of 5 unrelated children all with an unrecognised syndrome showed similarities to the first case. Fourteen years later in 1981 after much research, Kabuki syndrome was described by Dr Niikawa and Dr Kuroki (also from Japan but who was working independent of Niikawa). However the aetiology of the syndrome was still unknown at this point.

Originally it was thought that Kabuki syndrome only affected children of a Japanese origin. It wasn't until 1990 that 3 cases of Caucasian children with Kabuki were described [3]. In 1992 a group of scientists studied 16 non-Japanese children with the syndrome from North America and Europe. Whilst they agreed that the facial characteristics were specific across all cases regardless of origin/descent, they concluded that patients from America or Europe had more severe forms of joint hypermobility and neurological dysfunction than people affected from a Japanese origin [4].

The congenital disorder was given its name as people with the syndrome have a facial resemblance to actors wearing special make-up in traditional Japanese Theatre (Kabuki).

In 1988 a study of 62 patients took place to determine the method of inheritance and mutation rate. It was decided Kabuki Syndrome was an autosomal dominant disorder as every patient had a fresh mutation, explaining why the parents didn't show the same traits. Although it seems that in most cases the mutations are sporadic, there were a small number of cases where parent to child transmission has occurred. The rate of mutation was calculated to be 15.6x 106 [5].

Kabuki has a frequency of 1 in every 32,000 newborns in Japan [6]. The ratio of male: female cases is approximately equal. It has been suggested the number is actually higher than this frequency as few health professionals are aware/knowledgeable about the syndrome. Kabuki syndrome characteristics have a very broad spectrum which can also cause complications during diagnosis. CHARGE syndrome is similar to kabuki syndrome and sometimes mistaken for each other during diagnosis. Clinical tests for diagnosing Kabuki are expected to be ready within a year or so. Diagnosis of kabuki is currently based on a patient meeting 4 out of 5 features. The one that always has to be present is the typical facial characteristics. The others can be a combination of the following: skeletal abnormalities (such as brachydactyly, brachymesophalangy, vertebral anomalies and clindactyly), mental retardation, postnatal dwarfism, and dermatoglyphic abnormalities.

Fig B: [7] This shows abnormalities found in Kabuki cases. The first part shows the ear of a baby with Kabuki. Note the preauricular pit circled in red. This is a hole in the ear. The second picture is a toddler's fingers. The toddler has an abnormal index finger. It is short and stubby. The use and movement of the finger is affected. The blue line shows roughly how long the finger should actually be. In the third picture a baby's hand is shown. This baby has persistent foetal finger pads (circled in green).

Immune abnormalities are also frequent in patients with Kabuki. Kabuki patients are 63% more likely to suffer from recurring infections than someone without Kabuki [8]. A study in 2005 of 19 patients showed 16 had a form of decreased antibody levels. The results were similar to patients with CVID (common variable immune deficiency). As the study showed patients with Kabuki are more at risk from hypogammaglobulinemia, all young children upon diagnosis of Kabuki are immediately referred to an immunologist.

Initially research had lead scientist to believe that Kabuki syndrome was caused by either a deletion or an inversion in 8p22-8p23. In 2006 bacterial artificial chromosome-fluorescent in-situ hybridization (BAC-FISH) was used to study this region. However analysis of chromosomes at the metaphase stage showed that there were no deletions or duplications of the BAC probes. FISH was then used to look at the chromosomes during interphase stage. This showed there were no inversions across this region [9].

In 2007 high resolution chromosome banding analysis showed that a girl aged 13 with Kabuki had a 45,X/46,X,r(X) [10]. Scientists believed that Kabuki was caused by a ring X chromosome. A ring chromosome forms when the chromosome arms fuse together to create a ring shape. Most people with this karyotype have Uner-Tan syndrome. Ring X syndrome also has the same karyotype. It has been found in a small number of Kabuki cases. However it was determined that the mechanism must be elsewhere in the genome because of the dissimilarity of phenotypes in the 3 conditions. This prompted the search for other causes.

A study was conducted looking at a 2 year old Romanian boy with Kabuki. Cytogenetic tests showed a normal karyotype of 46,XY. FISH tests ruled out a 22q11 deletion (causes Velo-cardial-facial syndrome) and a 16q deletion (causes Rubinstein-Taybu syndrome) as these have phenotypic similarities to Kabuki [11].

On 15th August 2010, the National Human Genome Research Institute announced that scientists at the University of Washington had discovered the gene responsible for causing around 70% of Kabuki cases. Researchers at the university used a second generation sequencing technique to identify the mutation in the MLL2 gene. They sequenced only the exome (protein-coding gene regions of human genome). The exome makes up approximately 1-2% of the human genome. This makes it fast and cheap to sequence, making it a popular place to look for mutations.

Before an exome is sequenced, enrichment strategies are used to select the DNA regions that are of interest in the genome. Examples of enrichment techniques are PCR, hybridization, molecular inversion probes and in solution captures. In this particular case of exome sequencing, hybridization was used. This involves the hybridizing shotgun fragments of DNA to microarrays. Hybridization is when two or more complementary strands of nucleotides interact and combine to form a single hybrid. This hybrid is then known as a duplex. DNA, RNA or oligonucleotides may be used as complementary strands. Shotgun sequencing is the process of randomly breaking up long pieces of DNA into small segments. These segments are then sequenced using chain termination to obtain reads. Several rounds of fragmentation and sequencing take place in order to provide lots of overlapping reads. Special computer programmes them assemble the reads into a whole continuous sequence [12]. The University of Washington geneticists sequenced 6.3 gigabases of DNA 40 times [13]. They did so much in order to get as accurate information as possible and know that it wasn't just down to chance.

The exomes of 10 probands were sequenced and compared against Single Nucleotide Polymorphism databases. In order to make it fair, all the cases were unrelated to each other and a variety of origins were used (7 European, 2 Hispanic and 1 Haitian). At first there was no evidence for any candidate genes. Kabuki syndrome is heterogeneous. This means that potentially multiple genes could be involved in the causal. This would explain why not all Kabuki cases have the MLL2 mutation. It is also the most likely reason for failing to find any compelling candidate genes. The criteria were then lowered slightly. It was at this point that phenotypic and genotypic stratification showed MLL2 to be the potential cause [14]. In 7 out of the 10 cases, a mutation was found in MLL2. Later Sanger sequencing was used to detect MLL2 mutations in two of the three remaining probands. A follow up study of 43 probands showed the same mutations in 26 of these cases confirming it was the cause. As a control chromosomes were analysed from 190 individuals without Kabuki. None of these showed the same MLL2 mutations.

All of the mutations found in MLL2 were either frame-shift or nonsense mutations. A frame-shift mutation is the result of the insertion/deletion of a base in the DNA sequence. A codon is 3 bases long. If the number of bases inserted/deleted is not a multiple of 3 then the entire reading frame will be altered and every codon gets changed. The effect of this is the amino acid sequence of the encoded protein gets changed. This results in a different protein being coded for. A nonsense mutation is a point mutation. It changes an amino acid codon into a stop codon. This causes premature termination of mRNA translation. The protein produced is truncated and suffers from a loss of function. An external file that holds a picture, illustration, etc. Object name is nihms224006f1.jpg Object name is nihms224006f1.jpg

Fig C: [15] This diagram shows the gene structure and allele spectrum of the mutations found in MLL2 causing Kabuki. MLL2 consists of a total of 54 exons. It shows the location of the 32 different mutations found in the 53 patients in total that were studied. Out of 32 mutations, 20 were nonsense, 7 were insertions/deletions and 5 were substitutions. The exon in orange codes for a untranslated region. The other 53 in blue show code for protein sequences. These code for the SET domain (shown in red) and 7 PHD(Plant Homeon Domain) fingers shown in yellow.

MLL2 codes for a Trithorax-group histone methyltransferase [16]. MLL2 encodes a protein that is 5,262 residues long [17]. That is a very large protein. Typically bacterial proteins are around 300 residues and human proteins are normally approximately 500 residues long. This protein is part of the SET family of proteins. MLL2's SET domain shows very strong histone-3-lysine-4-methyltransferase activity [18]. This activity is important for the epigenetic control of gene expression. They act as chromatin modifiers and control the organisation of chromatin.

A study showed a murine loss of MLL2 increased apoptosis, slowed growth and retarded development. This is partly because of mis-regulation for the expression of the homeobox gene [19]. The MLL2 mutations found in Kabuki cases are thought to truncate the polypeptide chain that translates for the SET domain. It is not yet certain how Kabuki arises. However it is thought to be as a result of haploinsuffiency at MLL2. This is something that needs to be researched further.

Most of the MLL2 mutations found in Kabuki cases are positioned in the parts of MLL2 that code for C-terminal domains. This is a possible explanation as to why the severity of cases varies. Kabuki cases that have mutations in other regions of MLL2 might be either lethal, normal, or a better/worse tolerance. Again this is something that needs to be researched more.

Now scientists have identified a definite gene that explains the cause of a large proportion of kabuki cases a clinical test can be developed to detect cases in the future. There are a small number of cases that don't have this specific mutation and therefore it is very likely that it is a different gene that is the problem. Even now scientists are still conducting studies looking for other genes.

Although clinical tests can now be developed there is still no "cure" for kabuki syndrome. There isn't currently an effective treatment just management strategies to help try and limit the effect it has on the patients' lives. These strategies also aim to support the child's family as well [20].

An "early intervention programme" is used from birth to school entry. It provides a support system for families with a developmentally delayed child. Professionals work alongside the parents to identify each individual's needs before creating a plan and implanting this to ensure they receive the right therapy in order to help them. Children with Kabuki syndrome can attend integrated schools but in almost all cases will require a carer/assistant. They may also have some special lessons to improve some of their skills. Music stimulates the learning and motivation of a large number of children with Kabuki.

Nearly all children with Kabuki syndrome have speech problems relating to delays, acquisition of language and abnormal pitch. It is thought oral hypotonia is the main cause behind this, although facial shape such as jaw misalignment also plays a part. Speech therapy helps them to develop their language skills and improve their oral articulation. Some children with kabuki will need to use sign language in order to communicate.

Occupational therapy helps to develop the fine motor skills of the patient. It improves their co-ordination and strength. It will also identify any aids the patient might need around the house or around school/the workplace. Such things include walking sticks, modified bathrooms and a motor arm. Physical therapy helps muscle development/growth. This increases the child's strength to enable them to crawl/walk. Physical therapy is important as people with Kabuki syndrome are affected by hypotonia and many suffer from ligament problems.

Sensory integration is a process that occurs in our brains. It is the way humans organise the information that we get from our environment during everyday life. Normally during childhood SI will happen naturally. However for some, particularly children with Kabuki syndrome it won't. This leads to problems with behaviour, learning and development. Sensory Integration therapy is a specialised type of occupational therapy. Special exercises are designed to improve the patients balance, proprioceptive skills and their sense of touch. Therapy is started in the early stages of childhood and continues for as long as necessary including into adulthood.

Even though the main cause of Kabuki has been found, the research is not yet over. Other gene mutations that cause Kabuki are still being looked for. Even though the MLL2 mutation was found it's not yet definite how this leads to the features of Kabuki syndrome. It is also uncertain as to why some cases/symptoms are more severe than others and as to what part the gene plays in this. Clinical tests are in the process of being developed. More effective methods of treatment are being looked in particular growth hormone therapy. Scientists are hoping over the next few years that they will be able to work with and analyse further the MLL2 gene and human genome to find the answers to these questions.