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Project goals

Objectives of the project


The objective is to build up cross-border cooperation for the development of early (at newborn) and prenatal (fetal) diagnoses of rare and severe hereditary diseases (phenylketonuria, hypothyroidism, galactosemia, hemophilia, Duchenne’s disease, cystic fibrosis) and for assuring the equal accessibility of the diagnostic system to a wider group of the population. The creation of a joint, complementary diagnostic system in this Euroregion will ensure a health service of high technical level, health gain and equal chances. The project implementation includes the establishment of a new metabolic centre (Arad), the coordinated and complementary development of the existing metabolic and genetics laboratories (Temesvár and Szeged), the creation of a higher-level regional centre in Szeged, the mutual use of the investigative procedures, sharing of experiences, organizing joint professional meetings and delivering public information based on the same principles. Furthermore, the project aims at informing the population, organizing joint training seminars and conferences in order to increase the efficiency of the cooperation and to raise public awareness.


Brief description of the targeted diseases:


Phenylketonuria: Phenylketonuria (PKU) is an autosomal recessive genetic disorder. The genetic defect in the background is localized to chromosome 12. Symptoms develop when both alleles are affected. If both parents carry the disease, the child has a 25% chance to inherit it. While parents are free of symptoms, the child is born with the disease from the so-called “healthy” parents. Any harmful effects on the ovarian cells or sperm cells of the prospective parents are also considered as risk factors. Based on the data of the screening center in Szeged, the incidence of hyperphenylalaninaemia  in Hungary was about 1 : 6,000 live births in 2009.


In case of the disease, the amino acid phenylalanine, which takes part in the build up of proteins, cannot be broken down due to the lack or defective functioning of an enzyme. Thus, phenylalanine accumulates, and the body tries to metabolize it by other metabolic ways. This, however, results in the accummulation of toxic or pathologic metabolites responsible for the organ damages.


The most common symptom is mental retardation. By the end of the first year, the IQ level of the affected children drops to about 50. Later the children become hyperactive, show sudden emotional outbursts, commit self-injury, and seizures may appear.


If,the dietary intake of phenylalanine is restricted, the children’s intelligence may show normal or near-to-normal development. Treatment should be started as early as possible,  ideally within 2 weeks after birth. Early diagnosis may be achieved only through newborn screening (fenilketonuria is screened for in 30 European countries and 50 US states). PKU needs  a life-long strict low-protein intake diet.


Congenital hypothyroidism: is a condition of thyroid hormone deficiency present at birth resulting from the damage of fetal thyroid tissue. Its cause includes developmental disorder of the thyroid gland, v disorders of thyroid hormone synthesis, administration of certain drugs during pregnancy, iodine deficiency, etc. It has a high incidence: 1 in about 3-4,000 newborns is affected.


Although the deficiency of thyroid hormone production appears in the fetal age already, the maternal thyroid hormone supply more or less covers the needs of the fetus. Following birth, the maternal hormone supply is cut, and even though the newborn may get some hormone from the maternal milk, it is absolutely necessary to start the treatment of thyroid deficiency as early as possible. If that does not happen, typical symptom of the congenital disease, delay of the somatic and mental development appears (cretinism). Early treatment is made possible by t newborn screening (performed in 30 European countries and 51 US states). Early treatment of hypothyroidism ensures considerably more favorable physical and mental growth as compared to those treated from a more advanced age.


Galactosemia: Galactosemia is a genetic disorder resulting in a deficient or insufficient activity of the liver enzymes responsible for galactose dmetabolism. Galactose is the degradation product of lactose (a sugar contained in milk) and occurs mostly in dairy products. There are 3 enzymes involved in the conversion of galactose into glucose, and the lack of any of these 3 enzymes may lead to the development of the symptoms. If galactose is not fully degraded, toxic products accumulate and cause symptoms of the disease.


Galactosemia follows an autosomal recessive mode of inheritance, which means that if neither of the parents have symptoms though both of them carry the disease the child has a 25% chance to develop the disorder. If one of the parents carries the disease, and the other is healthy, the child has 50% chance to carry the disease but will never develop the clinical symptoms. Most frequently, galactosemia is caused by galactose-1-phosphate uridylyltransferase (or GALT) deficiency. The gene for this enzyme is located on chromosome 9. The incidence of classic galactosemia is 1:30,000, whereas the milder form, the Duarte variant is more frequent 1:16,000.


Galactosemia does not produce symptoms immediately after birth, however, after feeding jaundice, diarrhea and vomiting may occur and the infant shows no weight gain. If not detected in time, the disease may lead to liver damage, cataract, low blood sugar levels, kidney disease, hemophilia, sepsis, mental retardation, and eventually death.


Galactosemia is usually detected through newborn screening (performed in 6 European countries and 50 US states as part of the NBS).


The treatment for galactosemia is the elimination of lactose and galactose from the diet. Patients with galactosemia have to avoid certain foods. Currently, there is no drug that could supplement the deficient enzyme. Early diagnosis and proper treatment provide good prognosis on the long-term, the mental, somatic and sexual development of the children may reach normal levels.


Congenital adrenal hyperplasia: refers to an autosomal recessive enzyme defect, which causes the absence of hydrocortisone production by the adrenal glands. Incidence: 1:5,000 live births; boys vs. girls = 1:1, however, newborn baby boys die more frequently of this disease, therefore, at the age of 10 girls tend to dominate in this population.


Several types of this disease have been described corresponding to the step of hydrocortisone synthesis blocked because of the deficiency of a specific enzyme (within a family, the same type is inherited).


Symptoms: The symptoms of the non-salt-wasting form in boys are early virilization and short stature. In girls, the external genitals look like male genitalia, and therefore, newborns are mistakenly considered to be boys (pseudo-hermaphroditism). Without treatment virilization continues: male pubic and facial hairs, short body height, male stature.


The symptoms of the salt-wasting form appear at week 1-3 after birth. The disease is usually diagnosed earlier in girls since the uncertain external genitals raise the suspicion of the condition. The newborn progressively looses weight, diarrhea, abdominal pain and recurrent vomiting occur; and shock may develop with neurological symptoms and acidosis, eventually leading to death.


The disease can be detected early due to the abnormal hormone levels in the blood, therefore, the test is part of the NBS in many countries (10 countries in Europe and 32 US states). Screening test:: the elevation of 17-hydroxyprogesterone level is easily detectable from the blood spots, however, in the first 2-3 days of life,  it can also physiologically be elevated. Due to frequent false positive results, the performance of steroid profile analysis as a second-tier test to confirm the diagnosis is unevitable.


Treatment: hormone substitution, which can ensure normal development.


Cystic fibrosis: is an autosomal recessive disease of the exocrine glands (mucous glands, sweat glands). Most frequanely, it affects the lungs, pancreas, liver, intestine, facial and frontal sinuses and the genitals.


Cystic fibrosis (CF) is caused by a mutation in the gene of the cystic fibrosis transmembrane conductance regulator (CFTR). This gene codifies a protein that regulates the in- and outflow of salt and water through the cell wall. In CF patients, this protein does not function efficiently and leads to the production of a sticky and thick mucus, as well as to the very salty sweat which are the hallmarks of the disease. Children inheriting the defective CTFR gene from both parents will develop the disease. Children inheriting one defective CFTR gene from one of the parents will remain symptomless, but will become CF carriers. CF is one of the most common genetic disorders among the Caucasian population, with an incidence of around 1:3,300.


CF symptoms are due to the sticky and thick mucus. The most frequent symptoms are as follows: frequent coughing that brings up thick secretion, frequent respiratory tract and pulmonary infections that lead to permanent damage of the lungs, salty tasting skin and dehydration, infertility, which is typical mostly for male patients, incessant diarrhea and very bad smelling fatty stools, big appetite and no weight gain, which leads to chronic malnutrition as the body is unable to absorb sufficient nutrients from the intestines. CF may cause other health problems, including: parasinusitis, pancreatitis, blocking of the intestines (mostly in newborns), nasal polyps or other overgrowth of the nasal tissue, which require surgery, clubbing of the fingers (since the lungs do not let enough oxygen in the blood), pneumothorax, rectal prolapse (due to the frequent coughing and difficulties in eliminating stools), liver diseases (due to the inflammation or blocking of the bile ducts), diabetes (as the insulin producing pancreas becomes damaged), reduced bone density (as the body can not absorb enough vitamin D). Due to the diseases of various organs CF has a poor prognosis, the patients die in adolescents or at a young adult age.


Sweat testing is the most useful assay in setting the CF diagnosis. Usually, this test is performed twice and the repeated high salt level is indicative of CF. Further tests: blood test to detect the abnormal CFTR gene that may confirm CF, chest X-ray, which may reveal the scars due to the inflammation, pulmonary function tests (how much air goes into the lungs, how fast can the patient exhale, how fast can the lungs transport oxygen into the blood and how fast can it remove carbon dioxide from the blood). Prenatal (before birth) genetic tests may reveal whether the baby has CF or not. Such tests are the following: amniocentesis or chorionic villus sampling. In many countries (e.g. in Europe: in UK, France, Italy, Spain, Austria, Poland, and the Czech Republic, and in the USA and Australia) all newborns have their blood screened for CF.


CF can not be cured; however, the treatment of the disease may improve considerably the quality of life and the prognosis of the condition. Therapy aims at the prevention and early therapy of pulmonary infections, loosening and removing the sticky and thick mucus from the lungs, prevention of the blocking of the intestines and provision of proper nutrition. Prenatal genetic diagnostics performed for relevant families may help positive family planning, and reduce the incidence of the disease as demonstrated by the French example.


Hemophilia: is a hereditary genetic disorder that impairs coagulation. Its estimated prevalence is 30-100 in 1 million people, varying from country to country. As of March 14, 2007, there were 838 hemophilia A and 192 hemophilia B patients registered in Hungary.


In hemophilia A patients, the production of an important clotting protein called factor VIII is deficient, reduced or defective. Hemophilia B patients have similar alteration with regard to the production of blood clotting factor IX. In about two thirds of the cases the disease follows a recessive, sex-linked X chromosome mode of inheritance, where positive family history is present. The rest of the cases are sporadic as a result of spontaneous mutations of the X chromosome, and in such cases no hemophilia occurred in these families before. As boys have only one X chromosome (XY), they only have one copy of the gene of factors VIII and IX. If that gene is defective, factor VIII and IX production is altered and the patient will develop hemophilia. Girls have two X chromosome (XX), therefore, they have two genes responsible for the production of factor VIII and IX. This means that even if a girl has a defective gene, due to the other gene she will still be able to produce normal factor VIII and IX, and no hemophilia will develop.


Hemophilia is considered "severe" if the activity of the relevant clotting factor is less than 1% of the normal level. In such cases spontaneous bleeding often occurs. The "mild" form means that the activity is above 5% of the normal level and "moderate" is when the activity of the clotting factor is between these values. The majority of the hemophilia patients have the moderate or the severe form and they require treatment if spontaneous bleeding or injuries occur. The frequency of the treatment may range from several sessions per month to a few sessions per year.


Severe hemophilia presents itself in the first years of life. When the child starts moving around independently, bleeding occurs in the joints (especially in areas where the physical load is higher such as the knee, ankle or hip area). Bleedings are associated with severe pain and often lead to joint damage that result in deformities and inability to move. Life-threatening or limb-threatening bleedings may develop in the muscular or soft tissue; occasionally severe gastrointestinal and peritoneal bleeding may occur. Bleeding in the cranial cavity poses also a permanent risk. Superficial injuries, tooth extractions or other minor surgeries as well as major surgical interventions can cause unstoppable bleeding.


Treatment of hemophiliacs and patients with other blood clotting factor deficiencies is based on the substitution of the deficient factor. Unfortunately, hemophilia is one of the rare diseases with extremely high therapeutic costs (prophylactic, preventive therapy of a severe case costs USD 300,000 in the US). Accordingly, 75-80% of hemophiliacs worldwide still do not obtain any therapy and therefore, they do not reach adult age. Prenatal genetic testing may promote positive family planning and reduce the incidence of this severe disease.


Duchenne muscular dystrophy: Duchenne muscular dystrophy (DMD) is a genetic disorder characterized by progressive muscle cell degeneration. Fatty and conjunctive tissue unable to perform muscular activity take over the place of muscle cells and that gradually weakens the patient, eventually rendering them unable to take care of themselves.


Defective or deficient production of a protein called dystrophin causes the disease. As a result calcium ions flow in unlimited amounts among the muscle cells, leading to cell death. The disorder is caused by the mutation of a recessive gene located on the X chromosome. Therefore, though females may be carriers, they will not develop the disease since the normal gene of the other X chromosome will take over the role of the defective gene. However, males who inherit the defective gene will develop the disease as they only have one X chromosome. DMD affects 1 in 3,600 live birth male children. In about 30% of the cases the disease develops newly as a result of a harmful environmental effect (ionizing radiation, etc.).


The disease is characterized by progressive, gradually increasing muscle weakness and a decline of the intellectual capabilities. Symptoms usually do not appear in newborns; however, in some cases slight weakness of the muscles can be observed: sometimes the difficulty of holding up the head may be indicative of the disease. Usually children learn in time to turn, sit up and walk, and the disease becomes evident at around the age of 3 when due to the weakness of the muscles around the hip, the child can only stand up if taking support on his knees. Inability to ascend staircases and waddling occur. As the child ages the muscle degeneration spreads to more and more muscle fibers. The muscles of the wrist, lower arm and hand remain fit for a longer time, so that the patient can eat, drink and use the computer. Unfortunately, the ability to walk rapidly progresses and most patients become wheelchair dependent after the age 10.


The symptoms of muscular weakness also afflict the internal organs: due to the weakness of the pharyngeal and respiratory muscles patients often experience difficulty in swallowing, have less force to cough, and thus, are less able to bring up secretions from the lung, which leads to frequent upper respiratory tract infections and pneumonia. As sphincters are sluggish and weak, inability to hold back stool and urine may rarely also occur. Wheelchair dependent children face growing difficulties with breathing due to chest deformities and worsening curvature of the spine. Life expectancy depends on the severity of the affliction of respiratory and heart muscles. At a later stage the patient will require mechanical ventilation due to the respiratory distress.


There is no known cure for the disease and treatment is generally aimed at controlling the symptoms and to improve quality of life. This involves drug therapy, physical therapy and surgery. Prevention is possible through prenatal diagnostics, i.e. amniocentesis will help establish whether the unborn baby inherited the disease or not. Similarly to cystic fibrosis and hemophilia this may promote positive family planning and reduce the incidence of this devastating condition.


In summary it may be concluded that early diagnosis and therapy may ensure prevention of serious permanent damage and death, slow down the progression of pathologic processes (secondary prevention), improve the quality of life of patients and their families, whereas fetal (prenatal) diagnosis may promote positive family planning and reduce the incidence of such severe diseases. This recognition has lead worldwide to the intorduction of newborn screening and genetic testing, and this is why this project too aims at expanding early diagnosis (newborn screening), introduction of second-tier tests, and the development genetic test methods as well as ensuring the equal availability to the population of all 3 counties in the region.


Screening of newborns for hypothyroidism and phenylketonuria will start in Arad in June 2011 and will last for all the duration of the project and beyond. These tests are already available in Timis and Csongrád counties. The Arad screening center will provide screening tests for around 6,000 children during the project. Samples will be collected from 3 maternity

wards in Arad and around the county, including the cities of Lippa, Kisjenő, Gurahont, and Borosjenő. In Arad county there are 3,200-3,500 children born annually, who have not had access so far to newborn screening tests. 


Newborn screening for galactosemia and congenital adrenal hyperplasia will be set up in Timisora and will be performed for newborns in Arad and Timis counties. This test is not yet available for newborns nor in Arad neither in Timis counties. Screening test for galactosemia is available in Csongrád county since 1975. There is no newborn screening for congenital adrenal hyperplasia in Csongrád county, and there are no plans as yet to take up this test into the newborn screening panel. However, as a result of the project (setting up of steroid profiling) the selective testing will become available for newborns in Csongrád county who are suspected of the disease.


Justification for the need of second-tier testing in case of CAH: For the diagnosis of congenital adrenal hyperplasia, an ELISA method will be introduced in Timişoara and shall be used subsequently for the screening of newborns. This is the screening method used worldwide, though it yields a high rate of false positive results (see: description of the disease). Therefore samples giving a positive result must undergo a confirmation test (second-tier test), which in this case is the steroid profile analysis. The analysis will be performed with H-UPLC-MS/MS following the procurement of the instrument and establishment of the method. In the future, this instrument will make the measurement of further metabolites present in only minute amounts in the blood and of diagnostic importance possible. This will ensure the further development of the diagnostic system set up during the project.