Genetic analysis in the Methylmalonic Acidurias

By David S. Rosenblatt, MD

Professor of Human Genetics, Medicine, Paediatirics and Biology

McGill University 687 Pine Ave West H5-63 Montreal, Canada H3A IAI

Phone (514) 842-1231 x 5571

Fax (514) 843-1712

E-mail MC74@Musica.McGill.CA

Extracted from OAA News December 1997 with permission

My laboratory often receives cultured fibroblasts (skin tissue) from patients around the world who excrete methylmalonic acid in their urine.  The approach that we use combines the disciplines of biochemical, somatic cell, and molecular genetics.

Clinically, there are a number of important questions that we want answered before we undertake our diagnostic studies.  The first relates as to whether there are elevated levels of the chemical homocystine in the blood or urine in addition to methylmalonic acid.  The second is whether the levels of the metabolites (homocystine and methylmalonic acid) in the blood and urine goes down in the patients after treatment with vitamin B12 (Cobalirnin).  Usually we prefer that hydroxycobalamin (a specific form of vitamin B 12) be used to see if there is a response.  Of course we want to know about other patients in the family who have had methylmalonic acid excretion in the urine.

It is not uncommon for us to receive skin cells from infants who have transient excretion of methylmalonic acid because they were exclusively breast fed by mothers who themselves were vitamin B12 deficient.  These infants will have abnormally low levels of vitamin B12 in their blood.  They also may excrete homocystine in their urine.  These children improve very quickly on vitamin B12 treatment, but may have long-term effects if not treated early.  They do not have a genetic disease and study of their cultured cells will be normal.

  The tests described below are used to determine what form of MMA the patient has and to see which type of treatment is desired, low protein diet and/or vitamin B12 supplementation.  For patients who excrete both homocystine and methylmalonic acid in their urine, we look in cultured skin cells at the incorporation of radio labelled propionic acid (see note A) as a marker for the methylmalonyl coA mutase enzyme (functional deficiency of the mutase results in methylmalonic aciduria) and at the incorporation of radio labelled methyltetrahydrofolate (see note B) as a marker for the methionine synthase enzyme (functional deficiency of the methionine synthase results in homocystinuria).  Patients with a pure methylmalonic aciduria usually have low incorporation of propionate and normal incorporation of methyltetrahydrofolate.

If the incorporation of propionate is low in cultured cells, we re-do the study in the presence of vitamin B12 in the culture medium.  Patients with cblA and cbIB types of vitamin B12 responsive methylmalonic aciduria will increase their propionate uptake in the presence of the vitamin, but not to normal levels.  Patients with muto methylmalonic aciduria do not increase their levels of propionate uptake in the presence of vitamin B 12, and patients with muto- methylmalonic aciduria have a slight increase of propionate uptake in the presence of vitamin B 12.

The classification of patients into mut, cblA or cbIB types of methylmalonic aciduria is based on complementation studies.  Briefly, cells from an undiagnosed patient with low propionate incorporation are grown together in culture with cells from patients with known diagnoses in the presence of a chemical (PEG) which causes cells to fuse together.  Cells from a patient from a different complementation class will correct the defect in propionate incorporation in the patient, but cells from a patient with the same defect will not.

These studies in cultured cells can be useful in predicting outcomes in groups of patients.  For example, as a group the cblA patients are the mildest followed by the cbIB, the mutant then the muto patients.

The genes responsible for the cblA and cbIB forms of methylmalonic aciduria are not known.  However patients with mut methylmalonic aciduria have mutations in the mut gene on the short arm of chromosome 6. Many of the patients with mut- methylmalonic aciduria have mutations in the vitamin B₁₂ binding region of the mutase gene.  A few more than 20 mutations have been identified to date.  With the exception of two mutations (one in 6 Japanese patients and one in 5 African or African-American patients), most mutations are not seen in multiple patients with methylmalonic aciduria. (In other words, most patients with methylmalonic aciduria have unique mutations).  When both mutations are known in a family, molecular analysis can be done for carrier detection or prenatal diagnosis.  This is still done only by a few research laboratories.  For most families, prenatal diagnosis is still performed by looking at propionate incorporation in cultured amniotic fluid cells.

It is to be hoped that, as we know more ~ the mutations causing methylmalonic aciduria, we will be in a better position to adjust therapy and improve outcomes.

Note A: Propionic acid is connected to a compound called succinyl coA by the enzyme methylmalonyl coA mutase and this then goes on to make energy for the body.  If this enzyme does not function properly, then the propionic acid will change into methylmalonic acid, which accumulates in blood and urine.

Note B: Methyltetrahydrofolate is needed to activate the enzyme methionine synthase, which converts homocystine to the protein methionine.  If this process does not work, then the homocystine will accumulate ate in the blood and urine.

References:

Shevell MI, Matias--uk N, Ledley FD and Rosenblatt DS; Varying neurological phenotypes among muto and mut- patients with methylmalonyl coA mutase deficiency.  Am J. Med.  Genet. 45 "616-624, 1993

Ledley FD and Rosenblatt DS, Mutations in mut methylmalonic academia. clinical and enzymatic correlations, Hum.  Mut. 9: 1-6, 1997.

  6   December, 1997

Non ketotic Hyperglycinaemia

Introduction

In the most common form of non-ketotic hyperglycinaemia acute neuro- logical problems may begin only a few hours after birth and generally in the first week of life. Somnolence, lethargy and lack of spontaneous movement progress to deep coma with apnoeic spells. Exaggerated reflexes may be present and there may be myoclonic seizures. The EEG shows a pattern of very low activity interspersed with occasional high-amplitude synchronous bursts. This abnormal pattern may be shown 30 min after birth in spite of apparent clinical normality at this stage. Milder forms of non-ketotic hyperglycinaemia may show non-specific mental retardation though some later- onset patients experience rapid neurological deterioration. As yet we cannot correlate the clinical variability with the biochemical heterogeneity.

Diagnosis.

This is based on the finding of hyperglycinaemia and hyper- glycinuria in the absence of an organic acid disorder. These abnormalities may be obvious but in a minority of cases the diagnosis must be actively sought as the plasma glycine levels may be only marginally increased and the glycinuria modest. If a diagnosis of non-ketotic hyperglycinaemia is suspected on clinical grounds, a quantitative amino acid analysis of plasma is mandatory. Spurious hyperglycinaemia can develop in infants fed on very low protein diets. Determination of CSF glycine will provide supporting evidence of non-ketotic hyperglycinaemia since, at least in the more severe forms of the disease, the CSF/plasma glycine ratio (0.2-0.33 compared with normal 0.02-0.03) as well as the absolute CSF glycine concentration (0.09-0.36 mmol/l, normal 0.004-0.010 mmol/l) are abnormally high. A transient neonatal form with the biochemical and clinical features of classical non-ketotic hyperglycinaemia but spontaneous normalisation has been described.

Glycine is an important inhibitory neurotransmitter in the central nervous system where the glycine cleavage system may well play a role in terminating its action. In patients with ketotic hyperglycinaemias (secondary to defects in organic acid metabolism) the CSF/plasma glycine ratio is similar to that in normal children, suggesting that in these disorders the liver is the main site of inhibition. Some such patients have experienced high plasma and CSF glycine concentrations for many years without neurological symptoms.

The glycine cleavage system is not expressed in cultured amniotic fluid cells. The glycine to serine ratio in amniotic fluid shows some discrimination between affected and unaffected foetuses but this is insufficient for the method to be reliable. Chorionic villus biopsy contains the glycine cleavage system and may be used for prenatal diagnosis though a very large sample is required. Diagnosis by foetal liver biopsy is a theoretical possibility.

Treatment.

Efforts aimed at reducing the overall glycine levels in the body by restricting supply or removal as conjugates have met with varied success. Of these, the administration of large doses of benzoate have been the most successful, as judged by reduction of CSF glycine concentration and of the frequency of seizures. However, such manoeuvres have been uniformly unsuccessful in halting the overall course of the disease and allowing normal intellectual development. Supplementing the supply of` various cofactors has been equally unsuccessful where it has been tried but there is no reason to suppose that cofactor responsive variants might not occur. In a few cases improvement in neurological function has been achieved by blocking glycine receptors with high doses of strychnine but results have mostly been disappointing. Diazepam in high dosage may have some benefit.

Carnitine Therapy for Fatty Acid Oxidation Defects and Organic Acidaemias

Susan C. Winter, MD

Medical Director, Medical Genetics/Metabolism

Valley Children’s Hospital

Madera, CA, USA

winter2571@aol.com

Carnitine is a natural substance important to the transport of fat into the mitochondria where it is “burnt” for energy Camitine is also important in removing the biochemical ‘ashes” remaining after the fat is metabolized to energy. it does this by binding to the biochemical ashes and carries them out of the mitochondna and then out of the body as carnitine bound “ashes” (acylcarnitine derivatives) dissolved in the urine. Carnitine is eaten in the diet in red meats and dairy products, including breast milk, and is also made in the body from breaking down muscle protein and converting it to carnitine.

As with all natural substances, deficiency can occur. Carnitine deficiency is nearly always secondary to other problems and may often be due to more than one factor. In infants and small children with small muscle masses, camitine deficiency can develop easily due to poor muscle protein supplies for synthesis. These small children are very dependent on dietary carnitine for their supply If the diet is inadequate from generalized malnutrition, or due to a special formula not supplemented with carnitine or Total Parenteral Nutrition (TPN) that is unsupplemented, deficiency can develop within weeks. Children and adults with gastrointestinal malabsorption, such as those with cystic fibrosis or chronic diarrhoea, can develop deficiency Increased loss of carnitine from the blood or urine can occur with hemo- or peritoneal dialysis as it is a small chemical and comes out in the dialysis fluids. Camitine deficiency is also seen in children with kidney disorders affecting the reabsorption of needed chemicals from the filtered urine, renal Fanconi syndrome. In children with genetic metabolic disorders affecting fat oxidation or organic acidaemias, camitine deficiency occurs due to a massive excretion of carnitine in the urine bound to the unbumt “ashes” of fat metabolism. These unburnt fats attached to carnitine can be detected in the urine of these patients in high levels and this is the basis of the acylcarnitine derivative testing being used for newborn screening using the tandem MS-MS method.

Carnitine deficiency is associated with many symptoms. Since the deficiency is nearly always secondary to another disease process, the symptoms are often those of the primary disease plus additional problems that can be reversed with carnitine replacement therapy Deficiency of carnitine results in decreased energy available to muscle and muscle weakness and low muscle tone. Growth of muscle, and thus weight gain, also requires energy and the child with carnitine deficiency usually has failure to thrive. Carnitine deficiency can affect the cardiac muscle and result in poor cardiac contractions (cardiomyopathy). Energy is important to brain function and abnormalities of brain function can be seen including convulsions, lethargy irritability, and even coma. These children are very susceptible to infections and with the frequent infections they often show signs of deterioration of mental and physical status. Liver function may worsen and liver failure may occur. In children with genetic metabolic diseases, carnitine deficiency can be life threatening due to the inability to excrete the unburnt “ashes” left over from incomplete fuel burning. These accumulating ashes are toxic and poison the individual. Without carnitine to take these toxins out, the individual may die or suffer irreversible damage.

Camitine is available as a medication and is approved by the FDA for treating secondary deficiency due to metabolic diseases. In the USA, only one company, Sigma-tau Pharmaceuticals, Inc., sells pharmaceutical grade L-carnitine (Carnitor®) that is available through prescription. Oral L-­camitine is available as a liquid with 100 milligrams of camitine in each millilitre and as a tablet with 330 milligrams of carnitine per tablet. Intravenous L-carnitine is also available in vials each containing 1 gram in 5 millilitres of solution. Oral carnitine is poorly absorbed and only about 1/4 of what is swallowed is taken into the body The rest is excreted in the stool. This can result in diarrhoea, stomach upsets and in about 5% of people, a very fishy odour caused by certain bacteria in the bowel of some people converting camitine to trimethylamines. Intravenous carnitine is fully available for body use as it bypasses the bowel absorption problems and for this reason is the preferred route of administration in children in life threatening crisis. Doses of carnitine used are variable and range from 50 to 600 milligrams/kg/day with oral carnitine and 25 to 300 milligrams/Kg/day with IV camitine. Higher doses are usually used in children and adults with serious metabolic disorders during times of metabolic stress and decompensations.

Complications of long term or short-term carnitine treatment reported have been few and not serious. The body odour due to trimethylamines can be treated by taking a low dose of an antibacterial substance such as metronidazole to kill off the bacteria making the trimethylamines. The gastrointestinal upset and diarrhoea is often short lived and usually improves if the dose is lowered or given with food or more frequently. With 1V carnitine, the medication may burn if infused too quickly and may cause reversible pain and irritation if it gets under the skin (interstitial).

Treatment of fatty acid oxidation defects and organic acidurias with L-carnitine has been shown to be safe and, especially during the times of metabolic stress, life saving. Theoretical concerns regarding cardiac arrythmias in children with long chain fat metabolism defects have never been substantiated and no ill effects have been reported in this group of patients. Many children with long chain defects have been shown to reverse serious complications such as cardiomyopathy on carnitine therapy. In general, carnitine therapy has markedly improved the health and life style of children with fatty acid oxidation defects and organic acidurias.

 This article first appeared in the OAA Newsletter - Fall 2004 and is reproduced by permission