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Researchers from Ulm identify congenital genetic defect

Researchers led by the Ulm paediatrician Holger Cario have identified a rare, congenital genetic defect that leads to the dysregulation of the folic acid metabolism, resulting in aenemia, epilepsy and learning difficulties in children. All aspects of the disease can be treated quite successfully with the exception of the cramps associated with the defect.

At the same time as the German researchers discovered this mutation, English researchers discovered another mutation of the same gene (dihydrofolate reductase (DHFR)) in three Pakistani patients from two different families. Both reports were published in the renowned American Journal of Human Genetics.

Holger Cario believes that the importance of the work is not just related to the newly discovered gene mutation. He believes that the finding is also important because it provides further information on the function of dihydrofolate reductase, an enzyme involved in the folic acid metabolism, which is not yet understood in detail and which might also play a role in the treatment of cancer.

It all began during the haematology consultation hour

It all started with enlarged blood cells. © Cario

Holger Cario is especially interested in non-malignant haematological diseases such as leukaemia and other blood diseases. The senior physician works in the oncology day hospital of the Department of Paediatric and Adolescent Medicine at Ulm University Hospital.

It took Cario and his colleagues several years to identify a point mutation in the DHFR gene (p.Asp153Val) on the long arm of chromosome 5 as the cause of the disease symptoms with which they had been presented.

Cario started the research on the DHFR gene around six years ago when a then five-year-old boy presented with severe anaemia that Cario was able to effectively treat with vitamin B12 and folic acid.

Holger Cario discovered a rare gene defect © University of Ulm

Three years later, the sister of the anaemia patient began to suffer severe cramps. The doctors knew from previous examinations that the girl did not have anaemia, but that she did have enlarged red blood cells. In addition, the girl developed neurological symptoms and the doctors considered initiating folic acid therapy. Shortly after, the girl's older brother also presented with cramps leading the physicians to conclude that the two were suffering from the same disease, and that folic acid alone would not be an effective treatment for the disease.

Cario's investigations revealed that the liquor of the two patients lacked folates but that the same molecules were detected in the patients' plasma. Folates are biochemical intermediaries of folic acid that are reduced by the enzyme dihydrofolate reductase (DHFR).

DHFR catalyses the conversion of dihydrofolate (DHF) into tetrahydrofolate (THF) and, to a lesser extent, folic acid into DHF. The enzyme plays a key role in maintaining the intracellular folate homoeostasis and is an important target for cytostatic drugs.

When the researchers looked at the parental ancestors, they found six distantly related generations who carried the genetic defect. This finding suggested that Cario's hypothesis of a recessive disease picture, according to which the siblings might have inherited a defective gene copy from both parents, was plausible.

When the researchers started to look for homozygous regions in the genome of the siblings as well as for factors involved in the metabolism and transport of folic acid, Cario and his colleagues discovered the DHFR gene that codes for the enzyme dihydrofolate reductase.

Nucleotide exchange in all three siblings

Sequence analyses finally revealed a homozygous mutation affecting only one nucleotide of the nucleic acid. The researchers also identified the same point mutation in the older brother. Although this brother did not display any clinical symptoms at the time when the mutation was identified, he still had enlarged red blood cells in his blood (macrocytosis). An electroencephalogram revealed that he had similar modifications to his siblings, without displaying clinical symptoms.

Complex investigations on the expression of RNA, the expression of proteins in immunoblots and a number of functional investigations carried out with partners such as the Ulm-based Institute for Clinical Transfusion Medicine and Immunogenetics (Klaus Schwarz) showed that the mutation of the gene led to defective enzyme function. In addition, Cario found that the binding of the enzyme to the substrate methotrexate was defective. Methotrexate is used for the treatment of cancer in order to inhibit the synthesis of the enzyme dehydrofolate reductase.

Cario succeeded in treating the haematological symptoms of his patients with folinic acid, a folic acid precursor that spontaneously forms folic acid in vivo without relying on the enzyme DHFR. The severe learning and concentration problems of the first patient improved when treated with folinic acid. Affected patients need to take folinic acid for the rest of their life. The neurological disorders of the two siblings could not however be remedied with folinic acid.

New insights into the enzyme

In addition to the purely scientific effect of the gene defect that was discovered, Holger Cario believes that the results are important as they confirm the crucial role of the enzyme in transporting folates from the plasma into the liquor (cerebrospinal fluid). Cario explained that the findings also showed that intracellular folate metabolism disorders inhibit the transport of folates into the central nervous system.

These new findings might also be used to obtain more detailed insights into the range of adverse effects caused by methotrexate, which is used for the treatment of cancer because it not only inhibits the growth of cancer cells, but also blocks the transport of folate into the liquor.

For clinicians, the findings mean that they can consider the possibility of one more disease when they are faced with patients suffering from macrocytary and megaloblastary anaemias. Cario believes that it might be worth taking into account a DHFR defect when treating patients with neurological symptoms that are difficult to explain, especially severe forms of epilepsy that cannot be grouped into classical schematics as well as unclear developmental disorders, in particular in cases where they are associated with alterations (even small ones) of the blood picture.

To clarify whether a patient has symptoms caused by DHFR gene defects, Cario recommends measuring the concentration of folate in the liquor and in the red blood cells as this defect is characterised by lower folate concentrations in both liquor and red blood cells despite normal values in the plasma.

Can the hypothesis of harmful waste be confirmed?

Cario has no intention of starting research into folate. Nevertheless, he still wants to work with his partners from Ulm, Zurich, Munich and Amsterdam in order to answer some unanswered questions. He would very much like to substantiate his hypothesis that oxidised folate metabolites that cannot be reduced remain as waste products in the cell, where they might potentially cause further damage. This would then explain why current therapies are ineffective against neurological disorders associated with DHFR defects.

Insufficient amounts of folic acid are harmful

Fruit, vegetables, wholemeal products – people who eat sufficient quantities of these foods, and hence take up sufficient quantities of folates, do not suffer from folic acid deficiency. © Lilo Kapp/pixelio.de

Folic acid is a water-soluble B vitamin that occurs naturally in many foods. It can also be produced synthetically. Folic acid is involved in a large number of metabolic processes and is therefore important for all cell division and growth processes. Insufficient amounts of folic acid lead to abnormal metabolic processes which can result in diseases such as anaemia, digestive disorders and disorders of the mucous membranes. Unborn babies require folic acid to prevent neural tube defects from developing.

Cario et al., Dihydrofolate Reductase Deficiency Due to a Homozygous DHFR Mutation Causes Megaloblastic Anemia and Cerebral Folate Deficiency Leading to Severe Neurologic Disease, The American Journal of Human genetics (2011), doi:10.1016/j.ajhg.2011.01.007


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