Biochemical Screening for MCAD Deficiency

Question 1

What is MCAD deficiency?

Answer 1

• MCAD deficiency is a disorder of fat oxidation.
• Generally speaking you maintain your glucose homeostasis via 3 mechanisms:
  1) . Gluconeogensis - you can make new glucose molecules. Generally speaking the starting substrate for this is alanine from muscle breakdown.
  2) . Glyconeolysis - the breakdown of glycogen which is a glucose polymer stored in the liver. Releases glucose from glycogen polymer.
  3) . Fat Oxidation - using an alternative energy source. You start with triglycerides and you produce ketone bodies. These ketone bodies provide an alternative energy source. Triglycerides have a glycerol 3 carbon backbone, usually shown diagrammatically with fatty acids sticking out at right angles. When you get fat oxidation you get cleavage of that glycerol and you end up with the fatty acid being transported into the mitochondria and then you successively chop off 2 carbon fragments, the ketone bodies, which you can use as an energy source. The fatty acids are cleaved by a process called beta oxidation - a process that involves 17 different enzymes.
• Young children are quite vulnerable from at least 2 of these mechanisms. Generally speaking muscle mass in a small child is very low compared with their head size. The brain has a relatively high demand for glucose and they have very little muscle mass. Therefore, if they don’t feed regularly the potential to produce new glucose molecules through gluconeogenesis is just not there. Similarly, the amount of glycogen stored in their liver compared with their head size is comparatively small so again they have very little reserve and they cannot go for long fasting. One thing they can do comparatively well is produce alternative energy sources from the oxidation of fat. Indeed shortly after birth they depend very heavily on fat as the lypolytic stress of birth causes lypolysis and they use ketone bodies as an important alternative energy substrate. If you’ve got a problem with fat oxidation then you’ve got a real difficulty.
• In fatty acid oxidation the fatty acids are cleaved by a process called beta oxidation - a process that involves 17 different enzymes. If this process is defective it means that when the child is fed they will be fine, but if you suspend that feeding for any reason then very quickly they will become hypoglycaemic if they have a block in fat oxidation.
• What happens in MCAD which is the commonest of the fat oxidation defects is that they become hypoglycaemic, but it is a particular form of hypoglycaemia where they have a less than usually high concentration of ketone bodies. Usually if your glucose goes down then you ketone bodies will go up - you become ketotic. However, children with MCAD cannot produce an alternative energy source and so they become hypoglycaemic and hypoketotic.

Question 2

Generally speaking, how do you maintain glucose homeostasis?

Answer 2

Generally speaking you maintain your glucose homeostasis via 3 mechanisms:

1) . Gluconeogensis - you can make new glucose molecules. Generally speaking the starting substrate for this is alanine from muscle breakdown.
2) . Glyconeolysis - the breakdown of glycogen which is a glucose polymer stored in the liver. Releases glucose from glycogen polymer.
3) . Fat Oxidation - using an alternative energy source. You start with triglycerides and you produce ketone bodies. These ketone bodies provide an alternative energy source. Triglycerides have a glycerol 3 carbon backbone, usually shown diagrammatically with fatty acids sticking out at right angles. When you get fat oxidation you get cleavage of that glycerol and you end up with the fatty acid being transported into the mitochondria and then you successively chop off 2 carbon fragments, the ketone bodies, which you can use as an energy source.

Question 3

What are the symptoms of MCAD? How can it be treated?

Answer 3

• Hypoglycaemia
• Hypoketosis
• Genereally speaking if you are an older person an become hypoketotic you go pale and become aggressive , may shake - is obvious. This is because you produce a counter andrenergic response that helps mediate a correction of that blood glucose. As a young child it will nit be as obvious.
• Young children don’t mount a strong counter adrenergic response, they just go floppy and pale and lethargic. It is actually quite different to tell whether a young child is hypoglycaemic/hypoketotic. Lethargy, coma, death.
• Typically would be following a mild intercurrent infection or viral illness, typical onset 1 year of age.
• If they are not eating they can become hypoglycaemic quite quickly because their stores are depleted and they can very often die - 30% do die at first presentation of ketotic hypoglycaemia.
• If you put them on to regular feeds then they are very easy to treat because you just need to keep feeding them. If they get an intercurrent infection then you need to make sure you have some other energy source such as putting them onto an IV drip.
• Clinically with MCAD you get a secondary accumulation of key metabolites - best analogy is having a block in a stream = high level at one side and low level at the other.
• In MCAD you get a high concentration of the precursors of fat oxidation which produce dicarboxylic acids and you get a low concentration of the ketone bodies = can demonstarte a dicarboxylic aciduria, particularly accumulation of the c8 compound octanoyl carnitine that occurs just before the metabolic block. You get an increased concentration of these free fatty acids (FFA) that would normally be converted to ketone bodies. Ketone bodies low and free fatty acids high.
• i.e. an increased FFA:3OHB ratio.

Question 4

Why is MCAD a good candidate for screening?

Answer 4

• It is difficult to detect clinically
• It is easily treatable
• It is severe
• What we were lacking originally was a test for MCAD. There was a lot of reason to do it but not a test.

Question 5

What metabolic indications are you likely to see in MCAD?

Answer 5

• Clinically with MCAD you get a secondary accumulation of key metabolites - best analogy is having a block in a stream = high level at one side and low level at the other.
• In MCAD you get a high concentration of the precursors of fat oxidation which produce dicarboxylic acids and you get a low concentration of the ketone bodies = can demonstarte a dicarboxylic aciduria, particularly accumulation of the c8 compound octanoyl carnitine that occurs just before the metabolic block. You get an increased concentration of these free fatty acids (FFA) that would normally be converted to ketone bodies. Ketone bodies low and free fatty acids high.
• i.e. an increased FFA:3OHB ratio.

Question 6

What metabolic characteristics of MCAD can be used for screening? How is this performed?

Answer 6

• Clinically with MCAD you get a secondary accumulation of key metabolites - best analogy is having a block in a stream = high level at one side and low level at the other.
• In MCAD you get a high concentration of the precursors of fat oxidation which produce dicarboxylic acids and you get a low concentration of the ketone bodies = can demonstarte a dicarboxylic aciduria, particularly accumulation of the c8 compound octanoyl carnitine that occurs just before the metabolic block. You get an increased concentration of these free fatty acids (FFA) that would normally be converted to ketone bodies. Ketone bodies low and free fatty acids high.
• i.e. an increased FFA:3OHB ratio.
• We can use Octanoyl Carnitine as a marker for screening. This is performed using a rapid process called tandem mass spectrometry

Question 7

Describe the study led to MCAD screening being introduced nationally.

Answer 7

• trial was eventually carried out in 6 centres screening 375,000 cases per year for 2 years (slightly more than 50% of the population). Studied the screened and unscreened group. Was looking to ascertain the frequency, outcome, cost and overall impact in both groups.
• For the trial used the dried bloodspot card and looked for Octanoyl Carnitine after eluting out the bloodspot into 96 well plate.
• Cut-off was set at 0.5mM/L - anything >0.5 was investigated further and reported as a presumptive positive. Anything less than 0.5 was reported as normal.
• Over 2 years screened over 800k patients and identified 121 presumptive positive cases out of which 90 we MCAD - had some carriers and another disorder. Had some false positives possibly 1 false negative. The test was very specific and quite sensitive (only missed 1 case). PPV was well over 80%. In 2007 on the basis of those results it was recommended that this screening was rolled out nationally.
• An announcement made by ministers in Feb 2007 that screening for MCAD deficiency would be adopted throughout England in 2009.
• Needed to work together to agree a screening protocol, a diagnostic protocol, treatment guidelines, a useful quality assurance system.

Question 8

What does the future hold for MCAD screening? What has been done to examine whether screening should be rolled out for more inherited metabolic disorders? What conditions were revealed as the most important candidates for screening expansion?

Answer 8

• Could screen for may other conditions using the same technique that is used for MCAD screening.
• Currently USA screen for 29 core conditions and 24 secondary conditions. In total the USA screen for 53 inherited metabolic disorders whereas we are currently screening for 2 - MCAD and PKU.
• In the rest of Europe they are beginning to use the technology in the same way - in mainland Europe 10 countries screen for more than 10 conditions.
• Felt needed to look carefully at which other conditions to screen for as it is so easy to do it - may generate false positives and alarm families for no reason.
• Carried out a careful literature search of literature between the last HTA done between 2002 and 2009. A lit search revealed 1,817 publications on the clinical validity of MS/MS screening between 2002 and 2009. There 8 HTAs for this period. Looked at other centre’s published experience also. Came up with a list of likely candidates to screen for in the UK.
• The candidates included:
  1) . GA1
  2) . IVA
  3) . LCHAD
  4) . MSUD
  5) . Homocystinuria
• Often in these conditions it is the proximal accumulation of intermediates causing a kind of endogeneous poisoning that causes issues, in some cases it can also be the lack of products that is the problem.

Question 9

Describe Glutaric Acidurea Type 1 (GA1).

Answer 9

• GA1 is a deficiency of Glutaryl-CoA-Dehydrogenase (GCDH).
• GCDH is on the lysine degradation pathway. If you have the missing enzyme then you are fine if you are feeding normally. However, if you stop feeding for any reason then proteinolysis kicks in and you tip the balance towards catabolism. You begin to break down protein and begin to break down amino acids.
• When you try and metabolise lysine you get the accumulation proximal to that block of various intermediate, particularly 3-hydroxyglutarate.
• 3-hydroxyglutarate is neurotoxic and it particularly causes a severe type of dystonia and once that has happened you’ve got it. You have what is described as a metabolic decompensation. You are essentially endogenously poisoning yourself.
• At birth nothing is usually detectable, may have a slightly large head. They appear quite well. Typically at 10 or 11 months of age they have a period of intercurrent infection, they have a metabolic decompensation and they are left with sever dystonic posturing and in a wheelchair. Their intellect is preserved. They have around a normal life expectancy.
• If you screen for it as they have been in Germany of the 37 cases that they have identified that have been appropriately treated, 35 are totally asymptomatic and 2 have mild dystonia.
• Just need to screen and then treat with a low lysine diet and carnitine supplementation.
• Prevalence of GA1 is 1 in 109,000 - would identify about 6 or 7 patients a year.

Question 10

What does the screening of inherited metabolic diseased via tandem mass spectroscopy mean in practice?

Answer 10

• Tandem mass spec is a gateway tech and is being widely adopted to detect IEMs.
• The laboratory cost of adding an additional test is small, maybe 1.5 Euro per sample once the equipment is in place and cost effective.
• The overall cost of the programme is proportionate, approximately 3 Euro per baby tested.
• Quality standards need to be agreed and monitored including EQA.
• Sensitivity is not a great problem.
• The screening protocol needs to be developed to reduce false positive results.
• Confirmatory testing need to be well organised and available quickly (less than a week).
• We need to be clear about what constitutes diseases, i.e. disease definitions need to be agreed within the programme.
• Information and support for parents needs to be in place before testing and while confirmatory tests are underway.
• Agreed approaches to treatment would be very helpful.
• Monitoring where appropriate should be available.
• Evidence indicates that stress is reduced and treatment is more effective when organised at specialist centres.