User:Cjcolon723/sandbox

Duchenne muscular dystrophy is part of a group of inherited disorders that cause muscular debilitation. It is an X-linked recessive disease produced by mutations, most commonly deletions, in the gene responsible for encoding the protein dystrophin. The disease appears in early childhood presenting difficulties walking which progress until loss of ambulation is reached. Patients have a life expectancy of approximately 20 years with cardiac attack being the most common cause of death. There is no known cure but treatments include gene, cell, and drug therapies.

Muscular Dystrophies
Dystrophy, from the Greek dystrophe, means "defective nutrition". Muscular dystrophies are a group of inherited disorders causing muscle debilitation in varying intensities. They affect mainly males. Major muscles than can be affected include: facial, limb, axial, respiratory, cardiac smooth muscle, muscles used for swallowing, and others. Moreover, some organs and tissues can also be attacked, for example: eyes, skin, brain, and inner ear. There are a variety of muscular dystrophies all of which affect the age at which the disorder begins, its stringency, development, prognostication, and the difficulties associated with it. Some of the most severe dystrophies can produce loss of ambulation and cardiac or respiratory failure leading to death.

Types
Muscular dystrophies are categorized by the intensity of muscle debilitation and can be classified into seven categories.

Congenital muscular dystrophy
Congenital muscular dystrophy (CMD) appears at birth, or when a baby is approximately six months old. Patients present some symptoms such as: hypotonia, joint contractures and general weakness. Other symptoms include respiratory insufficiency, rigid spine, distal joint laxity, and muscle hypertrophy. In some cases, the central nervous system (CNS) is compromised, parts that could be affected include: brain stem, cortex, and cerebellum. This can lead to mental deficiency and eventually epilepsy. Some variants of CMD can cause eye problems.

A large percent of children suffering CMD cannot walk by themselves, with the help of physical support structure some can maintain an upright position. The most common form of CMD is Fukuyama congenital muscular dystrophy and it occurs mainly in Japan. CMD is caused by a founder recessive mutation associated with the fukutin gene, the function of which remains unclear. Dr. Yukio Fukuyama was the first to describe this disease in 1960 in Tokyo.

Duchenne and Becker muscular dystrophy
Duchenne muscular dystrophy (DMD) has an incidence of 1 per 3500 in males, making it the most common out of all kinds of muscular dystrophy. Becker muscular dystrophy (BMD) has an incidence of 1 in 17500 in males, it is the least common muscular dystrophy. They are both caused by a mutation of the same gene, and have similar symptoms but BMD has a less severe prognosis.

Regarding DMD, patients present a series of symptoms: the disease appears in early childhood. The child experiments some difficulties to walk, climb stairs, and its knees and hips are weakened. There is a certain extent of mental deterioration, approximately 20% of children with DMD have an intellectual coefficient below 70. Muscle weakness progresses over the years, and around the age of 12 the child with DMD will have lost his capacity to ambulate by himself, and depends on a wheelchair. Around this time, the child will also present respiratory insufficiency and scoliosis. Children diagnosed with DMD begin suffering from cardiac problems in their adolescence and young adulthood. With regards to prognosis, persons affected with DMD die around 20 years old generally victims of cardiac attack.

Patients with BMD present similar symptoms as those with DMD, but they present at a later age and with lower intensity as compared to DMD. Some BMD patients may lose the ability to walk at age16, but some retain this ability even into their adult years. Cardiac muscle is affected in the same way as in DMD but because it occurs with a lower intensity patients have a life expectancy between 50 and 60 years of age.

The cause of both DMD and BMD is a mutation in the gene which codes the dystrophin protein, which exists in the X chromosome.

Emery-Dreifuss muscular dystrophy
Emery-Dreifuss muscular dystrophy is caused by mutations in the STA gene at Xq28 which codes emerin, a protein necessary in the nuclear membrane, which leads to the complete absence of emerin in the muscles. There are a series of symptoms in this kind of disorder: weakness of Achilles tendons, posterior cervical muscles and rigidity in elbows. After that, flexion of neck and entire spine is restricted. If the disease is advanced, limb-girdle muscles waste away and get weakened. In later stages, cardiomyopathy will present, usually around the age of 30, which can lead to sudden death.

Distal muscular dystrophy
Distal muscular dystrophy, as indicated by its name, is characterized by symptoms exhibiting in distal regions (hands and feet). The disease can be due to either autosomal dominant inheritance, patients exhibit symptoms after the age of 40, and autosomal recessive inheritance, patients exhibit symptoms before the age of 30. The cause of this disease is still unknown. The disease can be regarded as a myopathy. It first attacks hands followed by forearms, calves, and finally feet.

This dystrophy is mostly seen in people from Scandinavia. The protect defect is unknown when referring to autosomal dominant inheritance, but in autosomal recessive inheritance it is known to be a defect with dysferlin.

Facioscapulohumeral muscular dystrophy
The prevalence of facioscapulohumeral muscular dystrophy (FSHD) is 1 in 20,000, making it the third most common kind of muscular dystrophy. It is an autosomal dominant disorder. A molecular defect related to FSHD is a deletion of 3.3 kb of DNA repeat fragments in the subtelomeric region of chromosome 4q (D4Z4), but the function of genes involved in FSHD is unclear.

FSHD commonly appears around the age of 30, presenting weakness at facial muscles, shoulder, peroneal muscle, and abdominal muscles. Other symptoms include: scapular winging, hearing loss, and retinal vasculopathy. Interestingly, patients with FSHD do not present cardiac symptoms, inferring that cardiac muscle is not involved or affected by FSHD. Mental capacities and motor functions are not usually compromised, therefore there is independent ambulation after treatment. In the case of older patients a wheelchair may be required after treatment.

Oculopharyngeal muscular dystrophy
Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant myopathy, prevalent in French Canada, that affects extraocular, facial, neck, and proximal upper limb muscles. It is characterized by dysphagia, difficulty swallowing, as well as a nasal voice. OPMD presents no cardiac problems.

OPMD is caused by a molecular defect in the 14q chromosome. The molecular defect is due to short GCG expansions of the first exon of the poly(A)-binding protein nuclear 1 gene (PABPN1).

Limb-girdle muscular dystrophy
There are fifteen genetic variants of Limb-girdle muscular dystrophy (LGMD). Therefore, it will exist with both genetic and clinical heterogeneity. Both an autosomal dominant inheritance (LGMD1) and an autosomal recessive inheritance (LGMD2). Most cases of LGMD are reported in Europe. This disease is characterized because it has genetic mutations in multiple genes. Currently, research is being conducted in order to identify more genes responsible for LGMD.

In LGMD the proximal limb-girdle muscles suffer of weakness. One of the concerns for patients with LGMD is cardiac disease, since 92% of patients older than 30 show cardiac failures (dysrhythmias). Therefore, they must be carefully treated. Some cases of mental retardation have been reported. Commonly, patients can ambulate independently.

Symptoms and Prognosis
DMD is a disease that affects males at an incidence rate of 1 in 3500. It is very rare for a woman to present symptoms of DMD because the disease is X-linked. It is estimated that 5-10% of women that suffer from DMD present muscular weakness, usually with less severity than males. They can also be susceptible to cardiomyopathy, even if they don't present muscle weakness.

The disease appears in early childhood with symptoms including difficulty walking and climbing stairs, and knee and hip weakness. Some children may present mental deterioration, approximately 20% of children with DMD have intellectual coefficients below 70. By the age of 12 muscle weakness has progressed to such an extent that the child will most likely not be able to ambulate independently and therefore requires the use of a wheelchair. At this point in the disease's progression the patient also present respiratory insufficiency and scoliosis. Patients of DMD begin suffering from cardiac problems in the adolescence of young adulthood. Patients of DMD have a life expectancy of 20 years, with the most common cause of death being cardiac attacks.

Clinical Diagnosis
In addition to the symptoms of DMD there are some useful clinical matters that have been well studied in order to better identify the disease.

Creatine Kinase Levels
Creatine kinase (CK) levels are unusually elevated, 50 times the normal level, at birth and in the infantile stage. However, there is no exact correlation between CK levels and the severity of the disease. DMD diagnosis can be carried out by measuring the levels of CK, since it provides a very good indication of the existence of DMD during an asymptomatic period.

Transaminase Levels
Transaminase levels should be evaluated via a biochemical analysis if DMD is suspected. Transaminases produces by the liver and muscle cells are particularly relevant in this analysis and so aspartate aminotransferase and alanine aminotransferase are the transaminases that should be evaluated.

Electromyography
Electromyography is an invasive procedure and involves a degree of pain and discomfort and so it is a method that is not frequently employed on children, considering the experience may be frightening. Nonetheless, this technique is very important and useful on adult patients because it provides information on the myopathies being suffered, allowing the determination of cardiac muscle condition. It can also be used to discard the presence of some neurogenic disorders.

Muscle Histology
Muscle histology is a histological search that produces results about some variations in muscle fiber size, necrosis, and macrophages invading the fiber cells. These are important factors that play a critical role when DMD is being studied.

Immunohistochemistry
Immunohistochemistry allows the identification of proteins that are affected by of the presence of DMD, aiding the diagnostic procedure. When a person suffers from DMD the amount of dystrophin, a protein present in muscle cell membranes, is less than the normal value because it is absent from skeletal muscle. For example, a Western Blot analyses on a DMD patient using the dystrophin antibody, will reveal low levels to no dystrophin.

Pathophysiology
Pathophysiology of DMD allows the discovery of drugs and the development of treatments for patients suffering from this condition. Dystrophin is the protein affected in DMD cases, its main function is to establish a link between the internal cytoskeleton and the extracellular matrix. In the sarcolemma there is a dystrophin-associated protein complex (DAPC), and dystrophin binds to it by using its amino-terminus to stabilize DAPC. However, when dystrophin is absent, as is the case with DMD patients, DAPC is destabilized and levels of the member proteins are lowered, leading to gradual muscle fiber damage and membrane breakdown. DAPC is composed of dystroglycans, sarcoglycans, integrins and caveolin. Therefore, any mutation in any of them produces autosomally inherited muscular dystrophies. DAPC also plays a role in signaling that, when not performed, leads to pathogenesis. The process just described leads to muscle weakness, since there is a loss of muscle enzymes, including creatine kinase.

There is a possibility that inter-protein interactions cause increased calcium channel activity, due mainly to conformational changes in their structure. As a result, there is a mitochondrial dysfunction that could induce cell death. In fact, early stages of DMD are characterized by abnormal levels of intracellular calcium.

Treatment
At present, there is no fully effective therapy to treat DMD. Nonetheless, experimental treatment strategies have been developed in three main areas: cell, drug, and gene therapy.

Gene Therapy
Gene therapy is employed to replace the mutated gene. It is based on adeno-associated vectors, which have a very small size and lower immune response. These kinds of vectors are preferred over others. The adeno-associated vector has a limited capacity, and it cannot adapt to the full-size cDNA of dystrophin; however, it can be used to store genes such as sarcoglycans, which have a smaller size.

At present, there are two strategies that have been tested: exon skipping and expression of dystrophin variants of smaller size. The first one uses adeno-associated type 1 viral vectors to form small nuclear U7 RNA targeting exons 6-8, to exclude the mutation of dystrophin. The second strategy has to do with plasmids which have been administered directly by intramuscular injection. These plasmids express human dystrophin and therefore protein expression occurs in the site where the injection was administered.

Cell Therapy
Cell therapy is used to replace affected cells. Some experiments have demonstrated that expression of dystrophin can be achieved by myoblasts transplanted into dystrophic muscle. It is safe, although there are no apparent benefits for muscles, particularly the injected muscles, due mainly to immune response and poor survival and displacement of myoblasts. Another difficulty that arises from local administration has to do with delivering myoblasts systemically into the bloodstream.

Stem cells are being studied, since they are very promising as a treatment for DMD, because they have the ability to self-renew and differentiate into other kinds of cells. Some experiments have been carried out with stem cells from adults, they have been isolated from bone marrow blood and muscles, and used in transplantation experiments.

Drug Therapy
Drug therapy is used mainly to compensate for the absence of dystrophin. To this date, there is a variety of drugs being researched.


 * 1) Utrophin Utrophin was one of the first proteins studied in order to replace dystrophin, because it is a homologue of dystrophin with a molecular weight of 395 kDa. It is similar to dystrophin because it shares 80% of dystrophin's sequence. Patients with DMD have an overexpression of utrophin in their muscles .  It has been demonstrated that utrophin expression increases with age and helps delay the age at which the patient will lose independent ambulation. Some possible drug candidates have been synthesized, and an orally ingestible compound has been developed by Tinsley and co-workers which has been able to enhance levels of utrophin mRNA and the protein itself. This drug, developed by Summit PLC, is safe and has been properly tolerated by voluntary patients.  Corticosteroids are a main aspect of DMD drug therapy and have been used in anti-inflammatory treatments demonstrating the ability to delay the progress of DMD. Clinical studies have demonstrated that corticosteroids can enhance the function and strength of muscles, with little to no sever collateral effects. The corticosteroids primarily used in treatment of DMD are Prednisone/Prednisolone and Deflazacort. Side effects include: gastrointestinal symptoms, nervous system disturbance, metabolic disorders, osteoporosis, and others. However, their benefits have been well documented.
 * 2) Aminoglycosides Gentamicin is an antibiotic drug developed to fight infections by Gram-negative bacteria It interacts with the 40S ribosomal subunit when RNA is being transcribed. Its function is to suppress the termination codons and replace them with another amino acid. Studies carried out with human subjects and mice demonstrated that dystrophin can be expressed in muscle fibers at 20% above normal levels. Nevertheless, there is a controversy about this investigation, because varying results have been obtained regarding diverse studies on DMD patients.  When aminoglycosides are used for a long time and at high dosages, patients can suffer from ototoxicity and renal toxicity. These collateral effects limit their prolonged use.
 * 3) Ataluren Ataluren was synthesized to have the same activity as gentamicin, but with a different structure and a better safety profile. It has been well tolerated by patients, must be taken orally, and has no antibiotic activity.  Research on ataluren found that it bound to the 60S ribosomal subunit, and some studies on mice have found it aids in dystrophin expression in skeletal muscles, diaphragm, and the heart. In other trials, ataluren has been shown to aid dystrophin expression in patients suffering DMD with nonsense mutation. Additionally, it can induce expression of transmembrane conductance regulator (cftr) protein in nonsense mutation to patients suffering cystic fibrosis. The European Union granted conditional marketing approval of ataluren under the trade name Translarna. This is the first drug treatment officially approved for DMD
 * 4) Myostatin Myostatin is a protein that influences regulation of muscle size. It is produced in both blood and muscles. A potential treatment for DMD is the blocking of myostatin. One research regrading clinical trials has been carried out on DMD patients using an antibody against myostatin (MYO-029). It demonstrated that, while the patients didn't suffer from adverse effects, no significant muscle strengthening was achieved  . However, myostatin inhibition is still considered a viable treatment for muscular dystrophies.
 * 5) Histone deacetylase inhibitors Givinostat (ITF2357) is a histone deacetylase inhibitor, which is anti-inflammatory, antio-angiogenic, and anti-neoplastic. In mice it has demonstrated that progress of DMD is delayed by givionstat by increasing the cross-sectional area of myofibers and reducing fibrotic tissue and fatty infiltration. Research demonstrated that is has the capability of muscle regeneration
 * 6) NO-releasing anti-inflammatory agents Flurbiprofen is a non-steroidal anti-inflammatory compound. An NO-releasing derivative of flurbiprofen (HCT1026) has been researched and been found to improve muscle repair by slowing down the progress of DMD, and consequently reducing inflammation and avoiding muscle damage.
 * 7) Vitamin D supplements Patients suffering from DMD tend to have little exposure to sunlight; therefore, synthesis of vitamin D is poor, and they have lower-than-normal levels. Consequences of low vitamin D levels include low bone mineral density, and osteoporosis. Hence, patients could develop pathological fractures which compromise long bones and the spine. Doctors recommend diet with physical exercise and supplements containing calcium and vitamin D for persons with DMD and deficiency of vitamin D.
 * 8) Simvastatin Simvastatin is a drug that belongs to the statin group. In laboratory experiments using mice it was observed that simvastatin improves the muscle function and reduces damage produced by DMD. Simvastatin can also reduce inflammation and enhance the strength of the diaphragm muscle. Its effects are similar to dytrophin gene-based therapies. In short, simvastatin enhances the general health and function of muscles compromised in DMD, by reducing oxidative stress and fibrosis. Clinically, simvastatin and other statins have been approved by the FDA for drug therapy in children older than 10, who are suffering hypercholesterolemia. Therefore, simvastatin and related statins represent a potential safe treatment for DMD.
 * 9) Exondys 51 In September 19th, 2016, the U.S. Food and Drug Administration (FDA) approved Exondys 51 to be administered by injection. This is the first FDA approved drug for treatment of DMD. Exondys 51 is an antisense oligonucleotide recommended for patients who have a positive mutation of the dystrophin gene owed to exon 51 skipping. This mutation occurs in approximately 13% of patients with DMD.  Exondys 51 was approved by the FDA, based on an increase in dystrophin in skeletal muscle, observed in patients treated with this drug. Up to date, the clinical benefits of Exondyn 51 has not been fully established and clinical trials have been ordered by the FDA in order to confirm positively its clinical performance. Among the side effects reported by patients using this drug were balance disorders and vomiting.

Genetics of DMD
Duchenne muscular dystrophy was described for the first time by G.B. Duchenne in 1861. DMD is an X-linked recessive disease that is produced by mutations in the gene responsible for encoding dystrophin, that belongs to a family of dystrophin-related proteins. The protein related to DMD (Dp427m) is found at the sarcolemma in the muscle in a membrane-spanning protein complex that links the cytoskeleton to the basal lamina. The function of the dystrophin protein is not known; however, its absence induces destabilization of the membrane that leads to damage of the sarcolemma, loss of calcium homeostasis, and progressive degeneration of muscle fibers.

Detecting mutations in the DMD gene has proven to be difficult primarily because of its length. The reported frequency of different mutations varies widely but according to the Leiden database duplications corresponds to 7% of mutations, point mutations for 20%, and deletions for 72%. Over 1000 mutations have been identified. Most of these deletions occur between exons 44 and 55, which corresponds to the protein's rod domain. These mutations can cause the dystrophin's reading frame to be altered, which leads to out of frame mutations, causing the protein to be truncated and no dystrophin is produced, this leads to development of DMD. If the mutation is "in frame", then an altered dystrophin is produced which can still function and the patient is diagnosed with BMD.

Gene sequence
The DMD gene is found on the X-chromosome in Xp21.2-p21.1 which is the short arm (p arm) of the X chromosome between positions 21.2 and 21.1. Its location was possible thanks to DNA markers. The function of the gene is to encode the protein dystrophin. The DMD gene, isolated in 1986, is one of the largest known genes spanning a genomic range greater than 2 million base pairs (2,241,933 bases). It consists of 89 exons and 88 introns. Duchenne muscular dystrophy is suspected to be caused by deletions, duplications, and point mutations at this gene locus. The genetic sequence can be found here.

mRNA Transcript Variants
The DMD gene produces many distinct mRNA-transcripts which encode various dystrophin proteins containing the same basic sequence but with distinct segments of varying length (isoforms). These transcript variants are due to alternative promoter usage and alternative splicing. The following table lists known transcript variants. For brevity's sake, the mRNA sequences are not included (their length can go over 14000 base pairs) but through the accession numbers both the mRNA sequence and the protein amino acid sequence can be found.

Dp427m Isoform
The dystrophin protein is rod-shaped, has a length of 150nm, 3685 amino acids, and a calculated molecular weight of 427 kDa. DP427m is the main dystrophin found in skeletal muscles and heart muscles. It is involved in both Duchenne and Becker muscular dystrophies and consists of 79 exons. It is coded by an mRNA with a length of 13993 base pairs (Accesion: NM_004006). This protein is found in the sarcolemma in a membrane-spanning protein complex that links the cytoskeleton and the basal lamina. Its function is unknown; but in its absence the membrane destabilizes which leads to sarcolemma damage, loss of calcium homeostasis, and progressive degeneration of muscle fibers.

Dp427m mRNA sequence

1 tcctggcatc agttactgtg ttgactcact cagtgttggg atcactcact ttccccctac 61 aggactcaga tctgggaggc aattaccttc ggagaaaaac gaataggaaa aactgaagtg 121 ttactttttt taaagctgct gaagtttgtt ggtttctcat tgtttttaag cctactggag 181 caataaagtt tgaagaactt ttaccaggtt ttttttatcg ctgccttgat atacactttt 241 caaaatgctt tggtgggaag aagtagagga ctgttatgaa agagaagatg ttcaaaagaa 301 aacattcaca aaatgggtaa atgcacaatt ttctaagttt gggaagcagc atattgagaa 361 cctcttcagt gacctacagg atgggaggcg cctcctagac ctcctcgaag gcctgacagg 421 gcaaaaactg ccaaaagaaa aaggatccac aagagttcat gccctgaaca atgtcaacaa 481 ggcactgcgg gttttgcaga acaataatgt tgatttagtg aatattggaa gtactgacat 541 cgtagatgga aatcataaac tgactcttgg tttgatttgg aatataatcc tccactggca 601 ggtcaaaaat gtaatgaaaa atatcatggc tggattgcaa caaaccaaca gtgaaaagat 661 tctcctgagc tgggtccgac aatcaactcg taattatcca caggttaatg taatcaactt 721 caccaccagc tggtctgatg gcctggcttt gaatgctctc atccatagtc ataggccaga 781 cctatttgac tggaatagtg tggtttgcca gcagtcagcc acacaacgac tggaacatgc 841 attcaacatc gccagatatc aattaggcat agagaaacta ctcgatcctg aagatgttga 901 taccacctat ccagataaga agtccatctt aatgtacatc acatcactct tccaagtttt 961 gcctcaacaa gtgagcattg aagccatcca ggaagtggaa atgttgccaa ggccacctaa 1021 agtgactaaa gaagaacatt ttcagttaca tcatcaaatg cactattctc aacagatcac 1081 ggtcagtcta gcacagggat atgagagaac ttcttcccct aagcctcgat tcaagagcta 1141 tgcctacaca caggctgctt atgtcaccac ctctgaccct acacggagcc catttccttc 1201 acagcatttg gaagctcctg aagacaagtc atttggcagt tcattgatgg agagtgaagt 1261 aaacctggac cgttatcaaa cagctttaga agaagtatta tcgtggcttc tttctgctga 1321 ggacacattg caagcacaag gagagatttc taatgatgtg gaagtggtga aagaccagtt 1381 tcatactcat gaggggtaca tgatggattt gacagcccat cagggccggg ttggtaatat 1441 tctacaattg ggaagtaagc tgattggaac aggaaaatta tcagaagatg aagaaactga 1501 agtacaagag cagatgaatc tcctaaattc aagatgggaa tgcctcaggg tagctagcat 1561 ggaaaaacaa agcaatttac atagagtttt aatggatctc cagaatcaga aactgaaaga 1621 gttgaatgac tggctaacaa aaacagaaga aagaacaagg aaaatggagg aagagcctct 1681 tggacctgat cttgaagacc taaaacgcca agtacaacaa cataaggtgc ttcaagaaga 1741 tctagaacaa gaacaagtca gggtcaattc tctcactcac atggtggtgg tagttgatga 1801 atctagtgga gatcacgcaa ctgctgcttt ggaagaacaa cttaaggtat tgggagatcg 1861 atgggcaaac atctgtagat ggacagaaga ccgctgggtt cttttacaag acatccttct 1921 caaatggcaa cgtcttactg aagaacagtg cctttttagt gcatggcttt cagaaaaaga 1981 agatgcagtg aacaagattc acacaactgg ctttaaagat caaaatgaaa tgttatcaag 2041 tcttcaaaaa ctggccgttt taaaagcgga tctagaaaag aaaaagcaat ccatgggcaa 2101 actgtattca ctcaaacaag atcttctttc aacactgaag aataagtcag tgacccagaa 2161 gacggaagca tggctggata actttgcccg gtgttgggat aatttagtcc aaaaacttga 2221 aaagagtaca gcacagattt cacaggctgt caccaccact cagccatcac taacacagac 2281 aactgtaatg gaaacagtaa ctacggtgac cacaagggaa cagatcctgg taaagcatgc 2341 tcaagaggaa cttccaccac cacctcccca aaagaagagg cagattactg tggattctga 2401 aattaggaaa aggttggatg ttgatataac tgaacttcac agctggatta ctcgctcaga 2461 agctgtgttg cagagtcctg aatttgcaat ctttcggaag gaaggcaact tctcagactt 2521 aaaagaaaaa gtcaatgcca tagagcgaga aaaagctgag aagttcagaa aactgcaaga 2581 tgccagcaga tcagctcagg ccctggtgga acagatggtg aatgagggtg ttaatgcaga 2641 tagcatcaaa caagcctcag aacaactgaa cagccggtgg atcgaattct gccagttgct 2701 aagtgagaga cttaactggc tggagtatca gaacaacatc atcgctttct ataatcagct 2761 acaacaattg gagcagatga caactactgc tgaaaactgg ttgaaaatcc aacccaccac 2821 cccatcagag ccaacagcaa ttaaaagtca gttaaaaatt tgtaaggatg aagtcaaccg 2881 gctatcaggt cttcaacctc aaattgaacg attaaaaatt caaagcatag ccctgaaaga 2941 gaaaggacaa ggacccatgt tcctggatgc agactttgtg gcctttacaa atcattttaa 3001 gcaagtcttt tctgatgtgc aggccagaga gaaagagcta cagacaattt ttgacacttt 3061 gccaccaatg cgctatcagg agaccatgag tgccatcagg acatgggtcc agcagtcaga 3121 aaccaaactc tccatacctc aacttagtgt caccgactat gaaatcatgg agcagagact 3181 cggggaattg caggctttac aaagttctct gcaagagcaa caaagtggcc tatactatct 3241 cagcaccact gtgaaagaga tgtcgaagaa agcgccctct gaaattagcc ggaaatatca 3301 atcagaattt gaagaaattg agggacgctg gaagaagctc tcctcccagc tggttgagca 3361 ttgtcaaaag ctagaggagc aaatgaataa actccgaaaa attcagaatc acatacaaac 3421 cctgaagaaa tggatggctg aagttgatgt ttttctgaag gaggaatggc ctgcccttgg 3481 ggattcagaa attctaaaaa agcagctgaa acagtgcaga cttttagtca gtgatattca 3541 gacaattcag cccagtctaa acagtgtcaa tgaaggtggg cagaagataa agaatgaagc 3601 agagccagag tttgcttcga gacttgagac agaactcaaa gaacttaaca ctcagtggga 3661 tcacatgtgc caacaggtct atgccagaaa ggaggccttg aagggaggtt tggagaaaac 3721 tgtaagcctc cagaaagatc tatcagagat gcacgaatgg atgacacaag ctgaagaaga 3781 gtatcttgag agagattttg aatataaaac tccagatgaa ttacagaaag cagttgaaga 3841 gatgaagaga gctaaagaag aggcccaaca aaaagaagcg aaagtgaaac tccttactga 3901 gtctgtaaat agtgtcatag ctcaagctcc acctgtagca caagaggcct taaaaaagga 3961 acttgaaact ctaaccacca actaccagtg gctctgcact aggctgaatg ggaaatgcaa 4021 gactttggaa gaagtttggg catgttggca tgagttattg tcatacttgg agaaagcaaa 4081 caagtggcta aatgaagtag aatttaaact taaaaccact gaaaacattc ctggcggagc 4141 tgaggaaatc tctgaggtgc tagattcact tgaaaatttg atgcgacatt cagaggataa 4201 cccaaatcag attcgcatat tggcacagac cctaacagat ggcggagtca tggatgagct 4261 aatcaatgag gaacttgaga catttaattc tcgttggagg gaactacatg aagaggctgt 4321 aaggaggcaa aagttgcttg aacagagcat ccagtctgcc caggagactg aaaaatcctt 4381 acacttaatc caggagtccc tcacattcat tgacaagcag ttggcagctt atattgcaga 4441 caaggtggac gcagctcaaa tgcctcagga agcccagaaa atccaatctg atttgacaag 4501 tcatgagatc agtttagaag aaatgaagaa acataatcag gggaaggagg ctgcccaaag 4561 agtcctgtct cagattgatg ttgcacagaa aaaattacaa gatgtctcca tgaagtttcg 4621 attattccag aaaccagcca attttgagca gcgtctacaa gaaagtaaga tgattttaga 4681 tgaagtgaag atgcacttgc ctgcattgga aacaaagagt gtggaacagg aagtagtaca 4741 gtcacagcta aatcattgtg tgaacttgta taaaagtctg agtgaagtga agtctgaagt 4801 ggaaatggtg ataaagactg gacgtcagat tgtacagaaa aagcagacgg aaaatcccaa 4861 agaacttgat gaaagagtaa cagctttgaa attgcattat aatgagctgg gagcaaaggt 4921 aacagaaaga aagcaacagt tggagaaatg cttgaaattg tcccgtaaga tgcgaaagga 4981 aatgaatgtc ttgacagaat ggctggcagc tacagatatg gaattgacaa agagatcagc 5041 agttgaagga atgcctagta atttggattc tgaagttgcc tggggaaagg ctactcaaaa 5101 agagattgag aaacagaagg tgcacctgaa gagtatcaca gaggtaggag aggccttgaa 5161 aacagttttg ggcaagaagg agacgttggt ggaagataaa ctcagtcttc tgaatagtaa 5221 ctggatagct gtcacctccc gagcagaaga gtggttaaat cttttgttgg aataccagaa 5281 acacatggaa acttttgacc agaatgtgga ccacatcaca aagtggatca ttcaggctga 5341 cacacttttg gatgaatcag agaaaaagaa accccagcaa aaagaagacg tgcttaagcg 5401 tttaaaggca gaactgaatg acatacgccc aaaggtggac tctacacgtg accaagcagc 5461 aaacttgatg gcaaaccgcg gtgaccactg caggaaatta gtagagcccc aaatctcaga 5521 gctcaaccat cgatttgcag ccatttcaca cagaattaag actggaaagg cctccattcc 5581 tttgaaggaa ttggagcagt ttaactcaga tatacaaaaa ttgcttgaac cactggaggc 5641 tgaaattcag cagggggtga atctgaaaga ggaagacttc aataaagata tgaatgaaga 5701 caatgagggt actgtaaaag aattgttgca aagaggagac aacttacaac aaagaatcac 5761 agatgagaga aagcgagagg aaataaagat aaaacagcag ctgttacaga caaaacataa 5821 tgctctcaag gatttgaggt ctcaaagaag aaaaaaggct ctagaaattt ctcatcagtg 5881 gtatcagtac aagaggcagg ctgatgatct cctgaaatgc ttggatgaca ttgaaaaaaa 5941 attagccagc ctacctgagc ccagagatga aaggaaaata aaggaaattg atcgggaatt 6001 gcagaagaag aaagaggagc tgaatgcagt gcgtaggcaa gctgagggct tgtctgagga 6061 tggggccgca atggcagtgg agccaactca gatccagctc agcaagcgct ggcgggaaat 6121 tgagagcaaa tttgctcagt ttcgaagact caactttgca caaattcaca ctgtccgtga 6181 agaaacgatg atggtgatga ctgaagacat gcctttggaa atttcttatg tgccttctac 6241 ttatttgact gaaatcactc atgtctcaca agccctatta gaagtggaac aacttctcaa 6301 tgctcctgac ctctgtgcta aggactttga agatctcttt aagcaagagg agtctctgaa 6361 gaatataaaa gatagtctac aacaaagctc aggtcggatt gacattattc atagcaagaa 6421 gacagcagca ttgcaaagtg caacgcctgt ggaaagggtg aagctacagg aagctctctc 6481 ccagcttgat ttccaatggg aaaaagttaa caaaatgtac aaggaccgac aagggcgatt 6541 tgacagatct gttgagaaat ggcggcgttt tcattatgat ataaagatat ttaatcagtg 6601 gctaacagaa gctgaacagt ttctcagaaa gacacaaatt cctgagaatt gggaacatgc 6661 taaatacaaa tggtatctta aggaactcca ggatggcatt gggcagcggc aaactgttgt 6721 cagaacattg aatgcaactg gggaagaaat aattcagcaa tcctcaaaaa cagatgccag 6781 tattctacag gaaaaattgg gaagcctgaa tctgcggtgg caggaggtct gcaaacagct 6841 gtcagacaga aaaaagaggc tagaagaaca aaagaatatc ttgtcagaat ttcaaagaga 6901 tttaaatgaa tttgttttat ggttggagga agcagataac attgctagta tcccacttga 6961 acctggaaaa gagcagcaac taaaagaaaa gcttgagcaa gtcaagttac tggtggaaga 7021 gttgcccctg cgccagggaa ttctcaaaca attaaatgaa actggaggac ccgtgcttgt 7081 aagtgctccc ataagcccag aagagcaaga taaacttgaa aataagctca agcagacaaa 7141 tctccagtgg ataaaggttt ccagagcttt acctgagaaa caaggagaaa ttgaagctca 7201 aataaaagac cttgggcagc ttgaaaaaaa gcttgaagac cttgaagagc agttaaatca 7261 tctgctgctg tggttatctc ctattaggaa tcagttggaa atttataacc aaccaaacca 7321 agaaggacca tttgacgttc aggaaactga aatagcagtt caagctaaac aaccggatgt 7381 ggaagagatt ttgtctaaag ggcagcattt gtacaaggaa aaaccagcca ctcagccagt 7441 gaagaggaag ttagaagatc tgagctctga gtggaaggcg gtaaaccgtt tacttcaaga 7501 gctgagggca aagcagcctg acctagctcc tggactgacc actattggag cctctcctac 7561 tcagactgtt actctggtga cacaacctgt ggttactaag gaaactgcca tctccaaact 7621 agaaatgcca tcttccttga tgttggaggt acctgctctg gcagatttca accgggcttg 7681 gacagaactt accgactggc tttctctgct tgatcaagtt ataaaatcac agagggtgat 7741 ggtgggtgac cttgaggata tcaacgagat gatcatcaag cagaaggcaa caatgcagga 7801 tttggaacag aggcgtcccc agttggaaga actcattacc gctgcccaaa atttgaaaaa 7861 caagaccagc aatcaagagg ctagaacaat cattacggat cgaattgaaa gaattcagaa 7921 tcagtgggat gaagtacaag aacaccttca gaaccggagg caacagttga atgaaatgtt 7981 aaaggattca acacaatggc tggaagctaa ggaagaagct gagcaggtct taggacaggc 8041 cagagccaag cttgagtcat ggaaggaggg tccctataca gtagatgcaa tccaaaagaa 8101 aatcacagaa accaagcagt tggccaaaga cctccgccag tggcagacaa atgtagatgt 8161 ggcaaatgac ttggccctga aacttctccg ggattattct gcagatgata ccagaaaagt 8221 ccacatgata acagagaata tcaatgcctc ttggagaagc attcataaaa gggtgagtga 8281 gcgagaggct gctttggaag aaactcatag attactgcaa cagttccccc tggacctgga 8341 aaagtttctt gcctggctta cagaagctga aacaactgcc aatgtcctac aggatgctac 8401 ccgtaaggaa aggctcctag aagactccaa gggagtaaaa gagctgatga aacaatggca 8461 agacctccaa ggtgaaattg aagctcacac agatgtttat cacaacctgg atgaaaacag 8521 ccaaaaaatc ctgagatccc tggaaggttc cgatgatgca gtcctgttac aaagacgttt 8581 ggataacatg aacttcaagt ggagtgaact tcggaaaaag tctctcaaca ttaggtccca 8641 tttggaagcc agttctgacc agtggaagcg tctgcacctt tctctgcagg aacttctggt 8701 gtggctacag ctgaaagatg atgaattaag ccggcaggca cctattggag gcgactttcc 8761 agcagttcag aagcagaacg atgtacatag ggccttcaag agggaattga aaactaaaga 8821 acctgtaatc atgagtactc ttgagactgt acgaatattt ctgacagagc agcctttgga 8881 aggactagag aaactctacc aggagcccag agagctgcct cctgaggaga gagcccagaa 8941 tgtcactcgg cttctacgaa agcaggctga ggaggtcaat actgagtggg aaaaattgaa 9001 cctgcactcc gctgactggc agagaaaaat agatgagacc cttgaaagac tccaggaact 9061 tcaagaggcc acggatgagc tggacctcaa gctgcgccaa gctgaggtga tcaagggatc 9121 ctggcagccc gtgggcgatc tcctcattga ctctctccaa gatcacctcg agaaagtcaa 9181 ggcacttcga ggagaaattg cgcctctgaa agagaacgtg agccacgtca atgaccttgc 9241 tcgccagctt accactttgg gcattcagct ctcaccgtat aacctcagca ctctggaaga 9301 cctgaacacc agatggaagc ttctgcaggt ggccgtcgag gaccgagtca ggcagctgca 9361 tgaagcccac agggactttg gtccagcatc tcagcacttt ctttccacgt ctgtccaggg 9421 tccctgggag agagccatct cgccaaacaa agtgccctac tatatcaacc acgagactca 9481 aacaacttgc tgggaccatc ccaaaatgac agagctctac cagtctttag ctgacctgaa 9541 taatgtcaga ttctcagctt ataggactgc catgaaactc cgaagactgc agaaggccct 9601 ttgcttggat ctcttgagcc tgtcagctgc atgtgatgcc ttggaccagc acaacctcaa 9661 gcaaaatgac cagcccatgg atatcctgca gattattaat tgtttgacca ctatttatga 9721 ccgcctggag caagagcaca acaatttggt caacgtccct ctctgcgtgg atatgtgtct 9781 gaactggctg ctgaatgttt atgatacggg acgaacaggg aggatccgtg tcctgtcttt 9841 taaaactggc atcatttccc tgtgtaaagc acatttggaa gacaagtaca gatacctttt 9901 caagcaagtg gcaagttcaa caggattttg tgaccagcgc aggctgggcc tccttctgca 9961 tgattctatc caaattccaa gacagttggg tgaagttgca tcctttgggg gcagtaacat 10021 tgagccaagt gtccggagct gcttccaatt tgctaataat aagccagaga tcgaagcggc 10081 cctcttccta gactggatga gactggaacc ccagtccatg gtgtggctgc ccgtcctgca 10141 cagagtggct gctgcagaaa ctgccaagca tcaggccaaa tgtaacatct gcaaagagtg 10201 tccaatcatt ggattcaggt acaggagtct aaagcacttt aattatgaca tctgccaaag 10261 ctgctttttt tctggtcgag ttgcaaaagg ccataaaatg cactatccca tggtggaata 10321 ttgcactccg actacatcag gagaagatgt tcgagacttt gccaaggtac taaaaaacaa 10381 atttcgaacc aaaaggtatt ttgcgaagca tccccgaatg ggctacctgc cagtgcagac 10441 tgtcttagag ggggacaaca tggaaactcc cgttactctg atcaacttct ggccagtaga 10501 ttctgcgcct gcctcgtccc ctcagctttc acacgatgat actcattcac gcattgaaca 10561 ttatgctagc aggctagcag aaatggaaaa cagcaatgga tcttatctaa atgatagcat 10621 ctctcctaat gagagcatag atgatgaaca tttgttaatc cagcattact gccaaagttt 10681 gaaccaggac tcccccctga gccagcctcg tagtcctgcc cagatcttga tttccttaga 10741 gagtgaggaa agaggggagc tagagagaat cctagcagat cttgaggaag aaaacaggaa 10801 tctgcaagca gaatatgacc gtctaaagca gcagcacgaa cataaaggcc tgtccccact 10861 gccgtcccct cctgaaatga tgcccacctc tccccagagt ccccgggatg ctgagctcat 10921 tgctgaggcc aagctactgc gtcaacacaa aggccgcctg gaagccagga tgcaaatcct 10981 ggaagaccac aataaacagc tggagtcaca gttacacagg ctaaggcagc tgctggagca 11041 accccaggca gaggccaaag tgaatggcac aacggtgtcc tctccttcta cctctctaca 11101 gaggtccgac agcagtcagc ctatgctgct ccgagtggtt ggcagtcaaa cttcggactc 11161 catgggtgag gaagatcttc tcagtcctcc ccaggacaca agcacagggt tagaggaggt 11221 gatggagcaa ctcaacaact ccttccctag ttcaagagga agaaataccc ctggaaagcc 11281 aatgagagag gacacaatgt aggaagtctt ttccacatgg cagatgattt gggcagagcg 11341 atggagtcct tagtatcagt catgacagat gaagaaggag cagaataaat gttttacaac 11401 tcctgattcc cgcatggttt ttataatatt catacaacaa agaggattag acagtaagag 11461 tttacaagaa ataaatctat atttttgtga agggtagtgg tattatactg tagatttcag 11521 tagtttctaa gtctgttatt gttttgttaa caatggcagg ttttacacgt ctatgcaatt 11581 gtacaaaaaa gttataagaa aactacatgt aaaatcttga tagctaaata acttgccatt 11641 tctttatatg gaacgcattt tgggttgttt aaaaatttat aacagttata aagaaagatt 11701 gtaaactaaa gtgtgcttta taaaaaaaag ttgtttataa aaacccctaa aaacaaaaca 11761 aacacacaca cacacacata cacacacaca cacaaaactt tgaggcagcg cattgttttg 11821 catccttttg gcgtgatatc catatgaaat tcatggcttt ttcttttttt gcatattaaa 11881 gataagactt cctctaccac cacaccaaat gactactaca cactgctcat ttgagaactg 11941 tcagctgagt ggggcaggct tgagttttca tttcatatat ctatatgtct ataagtatat 12001 aaatactata gttatataga taaagagata cgaatttcta tagactgact ttttccattt 12061 tttaaatgtt catgtcacat cctaatagaa agaaattact tctagtcagt catccaggct 12121 tacctgcttg gtctagaatg gatttttccc ggagccggaa gccaggagga aactacacca 12181 cactaaaaca ttgtctacag ctccagatgt ttctcatttt aaacaacttt ccactgacaa 12241 cgaaagtaaa gtaaagtatt ggattttttt aaagggaaca tgtgaatgaa tacacaggac 12301 ttattatatc agagtgagta atcggttggt tggttgattg attgattgat tgatacattc 12361 agcttcctgc tgctagcaat gccacgattt agatttaatg atgcttcagt ggaaatcaat 12421 cagaaggtat tctgaccttg tgaacatcag aaggtatttt ttaactccca agcagtagca 12481 ggacgatgat agggctggag ggctatggat tcccagccca tccctgtgaa ggagtaggcc 12541 actctttaag tgaaggattg gatgattgtt cataatacat aaagttctct gtaattacaa 12601 ctaaattatt atgccctctt ctcacagtca aaaggaactg ggtggtttgg tttttgttgc 12661 ttttttagat ttattgtccc atgtgggatg agtttttaaa tgccacaaga cataatttaa 12721 aataaataaa ctttgggaaa aggtgtaaaa cagtagcccc atcacatttg tgatactgac 12781 aggtatcaac ccagaagccc atgaactgtg tttccatcct ttgcatttct ctgcgagtag 12841 ttccacacag gtttgtaagt aagtaagaaa gaaggcaaat tgattcaaat gttacaaaaa 12901 aacccttctt ggtggattag acaggttaaa tatataaaca aacaaacaaa aattgctcaa 12961 aaaagaggag aaaagctcaa gaggaaaagc taaggactgg taggaaaaag ctttactctt 13021 tcatgccatt ttatttcttt ttgattttta aatcattcat tcaatagata ccaccgtgtg 13081 acctataatt ttgcaaatct gttacctctg acatcaagtg taattagctt ttggagagtg 13141 ggctgacatc aagtgtaatt agcttttgga gagtgggttt tgtccattat taataattaa 13201 ttaattaaca tcaaacacgg cttctcatgc tatttctacc tcactttggt tttggggtgt 13261 tcctgataat tgtgcacacc tgagttcaca gcttcaccac ttgtccattg cgttattttc 13321 tttttccttt ataattcttt ctttttcctt cataattttc aaaagaaaac ccaaagctct 13381 aaggtaacaa attaccaaat tacatgaaga tttggttttt gtcttgcatt tttttccttt 13441 atgtgacgct ggaccttttc tttacccaag gatttttaaa actcagattt aaaacaaggg 13501 gttactttac atcctactaa gaagtttaag taagtaagtt tcattctaaa atcagaggta 13561 aatagagtgc ataaataatt ttgttttaat ctttttgttt ttcttttaga cacattagct 13621 ctggagtgag tctgtcataa tatttgaaca aaaattgaga gctttattgc tgcattttaa 13681 gcataattaa tttggacatt atttcgtgtt gtgttcttta taaccaccaa gtattaaact 13741 gtaaatcata atgtaactga agcataaaca tcacatggca tgttttgtca ttgttttcag 13801 gtactgagtt cttacttgag tatcataata tattgtgttt taacaccaac actgtaacat 13861 ttacgaatta tttttttaaa cttcagtttt actgcatttt cacaacatat cagacttcac 13921 caaatatatg ccttactatt gtattatagt actgctttac tgtgtatctc aataaagcac 13981 gcagttatgt tac