Laboratory Medicine - Sapienza University of Rome
Inborn errors of metabolism
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Genetic diseases constitute a major diagnostic challenge. The number of known genetic diseases is huge, several thousands of them having been identified. If we consider them one by one, they are relatively uncommon unless some special selective condition applies, as in the case of genetic defects of hemoglobin that offer (partial) protection against malaria in areas where malaria is endemic. However, as a group they are extremely frequent, their summed incidence being in the order of one case every 100 births.
Broadly speaking, inherited genetic defects can be classified in four major groups:
1) Monogenic diseases (e.g. thalassemias)
2) Chromosomal abnormalities (e.g. Down syndrome)
3) Multifactorial diseases in which affected genes and environmental factors co-operate
4) Mitochondrial genetic diseases.
This lecture is concerned only with the laboratory diagnosis of monogenic diseases affecting enzymes of common metabolic pathways. Other monogenic diseases have been considere elsewhere in this and other courses (e.g. hemoglobinopathies, thalassemias, coagulation defects, porphyrias, ereditary forms of gout, jaundice, etc.) and will be occasionally mentioned but not described here.
As to the mechanism of inheritance, monogenic defects can be:
1) Autosomal and dominant (heteroxygotes and homozygotes are both affected. One of the parents is affected, transmission can be traced vertically in the ancestry).
2) Autosomal and recessive (Only homozygotes are affected. Parents are usually heterozygotes and not affected, but brothers and sisters may be affected; the disease cannot be traced vertically but may have occurred in an apparently ranndom fashion in the family). This is the most frequent type of inheritance.
3) Sex-linked and dominant (the gene is located on the X chromosome; both sexes may be affected)
4) Sex-linked and recessive (males are affected, female cases are extremely uncommon).
Gene mutations preventing the production of the enzyme or causing loss of function are usually inherited as recessive traits, because the healthy gene can at least partially compensate the defect. Gene mutations causing gain of function are often dominant, because the healthy gene cannot compensate.
Examples of genes located on the X chromosome, and thus leading to sex-linked inheritance include those coding for coagulation enzymes (whose defect can result in hemophylias); hypoxantine-guanine phosphorybosyl transferase (whose defect can cause the Lesch-Nyhan syndrome) and Phosphoribosyl Pirophosphate Synthetase; red-green color blindness; Duchenne musular distrophy.
Biochemistry of mendelian enzyme deficiencies
In a typical autosomal recessive genetic enzyme defect, the gene presents mutations in the promoter region or in the coding region, and these cause the protein to be expressed to a reduced extent or not at all. In some cases a mutated protein is produced that has reduced stability or impaired function. The net result of this condition is that the heterozygous patient produces a lower than normal amount of the affected enzyme. However, the usual physiological concentration of substrates is around the enzyme's K
; as a consequence a defect of enzyme concentration causes an increase of substrate concentration and this, in turn, allows the available enzyme to increase its activity, thus partially compensating the defect (this is an example of a negative feedback control mechanism).
If the described compensation occurs, a heterozygous individual may appear perfectly healthy, but in some cases the clinical laboratory may reveal that the concentration of the substrate of the affected enzyme is increased (this however occurs rarely, because substrates are usually present in the cytoplasm and thus are not accessible to analysis).
In some cases the affected enzyme (or protein) is produced and is stable but has abnormal functional properties, that cannot be compensated by the normal enzyme present in the cell. When this condition occurs, the disease is either dominant or clinically manifest even though recessive. For example in methemoglobinopathies the O
carrying capacity of blood is reduced even in the heterozygous state, and the heterozygous β-thalassemia causes a mild anemia. A more dramatic case occurs in mutations of membrane pumps or channels that, due to a mutation, may be permanently in the closed or open conformation: the first condition is usually recessive, the second is usually dominant.
When the defect is recessive, but the patient is homozygous, the affected enzyme is produced to a very little extent or not at all (at least in a functional state), and no compensation is possible. The same occurs in male subjects for recessive genetic defects of genes located on the X chromosome.
The pioneer of the study of inherited defects of metabolism is Archibald Garrod, whose essay
Inborn errors of metabolism
published in 1909 makes a very interesting reading even today. The student may be perplexed by the observation that the enzymes affected rarely belong to the major metabolic pathways, those he or she studied with most attention: the glycolisis, beta-oxidation of fatty acids, the Krebs cycle, and when this occurs, the mutations do not completely inactivate the enzyme. The reason is that an embrio presenting severe defects of these pathways would never develop and would encounter a very early miscarriage: diseases may be observed and diagnosed only for those defects that are compatible at least with intrauterine life. An important example of this rule is that of genetic defects of the urea cycle, a major metabolic pathway whose disruption is incompatible with extrauterine life, but compatible with the development of the fetus, given that during intrauterine life ammonia may be disposed of by the mother via placental exchange.
Laboratory diagnosis of genetic enzyme deficiencies
A genetic enzyme deficiency is suspected very early in life, because of stunted growth, or mental retardation, or other symptoms (e.g. immune deficiencies, dysfunctions of the acid-base balance often with metabolic acidosis, etc.). Even though each defect is rare or very rare, with a frequency of one in several thousands births, the sum of all genetic defects yields a frequency of over 1% of live births. If the physician suspects the disease (the suspicion will usually be a generic one) he/she may look for gross abnormalities (e.g. metabolic acidosis or aciduria; symptoms aggravated by specific foods; etc.). A systematic search for ammonia, the principal metabolytes of aminoacids, abnormal blood or urinary concentrations of metals and other substances is usually the next step. Once the possible diagnoses have been restricted to a reasonable number, gene sequencing will provide the final proof.
Audio: Genetic defects of metabolism
Possible reasons to suspect an inherited defect of metabolism in a baby
positive familiar anamnesis
neurological symptoms (coma, seizures, unresponsivity, mental retardation etc.)
repeated infections (possibly due to immunodeficiencies)
abnormal pigmentation of the skin and mucosae (e.g. albinism, jaundice, etc.)
abnormal smell or color of the urine
Generic laboratory tests that may point to an inherited defect of metabolism
hyper- or hypo-glycemia
hyper-bilirubinemia and jandice
metabolic acidosis with increased anion gap
abnormal density or color of the urine; excess loss of ions, bicarbonate, glucose, etc.
For the purposes of this lecture we shall classify the genetic defects of metabolism according to the class of metabolytes affected:
1) Sugars, including favism, glycogenstorage diseases and mucopolysaccaridoses
2) Lipids, including gangliosidoses
3) Aminoacids and the urea cycle
4) Nucleotides biosynthesis, salvage and degradation
5) Heme biosynthesis (porphyrias) and degradation (genetic joundice)
6) Metal accumulation diseases.
7) Defects of membrane channels and pumps
Diseases classified in groups 4 and 5 were treated under separate headings and will not esplicitly considered here. It is important to stress that this classification is not extensive, e.g. it does not include the defects of structural proteins (Marfan syndrome, Ehler Danlos syndrome, achondroplasia, etc.). This is because in this course we focus our attention on diseases that cause alterations in the clinical laboratory results.
The reason of the above classification is that from the laboratory diagnostic view-point the metabolic pathway affected is indicative of the biochemical alterations one may find in the analysis: e.g. defects of the heme biosynthesis pathway cause the accumulation and excretion of pyrrole and tetrapyrrole ring derivatives. However, a classification according to the metabolic pathway affected may not be clinically indicative, because diferent defects of the same metabolic pathway may lead to very different clinical syndromes. Thus the above classification hould be cross referenced to a clinical one, that takes into account at least the most frequent syndromes, that include:
a) mental retardation
b) morphological abnormalities (e.g. dwarfism, cardiac defects, etc.)
c) stunted growth
d) metabolic acidoses
Inherited defects of carbohydrate metabolism
Several genetic defects of carbohydrate metabolism have been considered in the lecture on diabetes; thus in this lecture these defects will be treated quite schematically. The inherited enzymatic defects of monosaccharide metabolism usually cause an increased concentration of the affected sugar in the blood and urine causing a
Genetic defects of monosaccharide metabolism (non-diabetic melliturias)
Disease (enzyme/gene; pathway)
Galactosemia (galactose 1 phosphate uridyl transferase)
stunted growth, possible mental retardation (prevented by galactose-free diet)
presence of galactose and galactose 1 phosphate in the blood and urine
early cataract due to galactitol accumulation in the lens (prevented by galactose-free diet)
presence of galactose in the blood and urine
Galactose epimerase deficiency
presence of galactose and galactose in the urine
Hereditary fructose intolerance (phosphofructo aldolase deficiency)
hypoglycemia, renal tubular acidosis, possible convulsions and coma
presence of fructose in the urine
Fructosemia / Fructosuria (fructokinase)
presence of fructose in the blood and urine
Fructose 1,6 diphosphatase deficiency (gluconeogenesis)
presence of glucogenesis precursors in the blood and urine (e.g. ketoacids, lactic acid)
Pentosuria (L-xylulose dehydrogenase)
benign, characteristic of some populations (e.g. american Jews)
presence of L-xylulose in the blood and urine
Glycogen storage disorders (glycogenoses)
Defects of glycogen biosynthesis or degradation are called glycogenoses and are classified using roman numerals from 0 to XI. They are inherited as autosomal recessive traits, except for type IX, which is sex-linked. Interpreting the clinical and laboratory findings requires some familiarity with glycogen metabolism, as schematically illustrated below.
Glycogen metabolism. In black the glycogen synthesis pathway; in blue the cytoplasmic glycogen degradation pathway; in green the lysosomal degradation reaction. In red the diseases associated with each step, according to the standard classification (see table). Glycogenoses may affect (enzymes characteristic of) the liver, the skeletal muscle or the lysosomes. A very important laboratory finding in liver glycogenoses is
, which becomes manifest very early in life.
Genetic defects of glycogen metabolism (glycogenoses)
0: UDP-Glucose glycogen transferase (glycogen biosynthesis)
Liver steatosis, fasting hypoglycemia
Ia: Von Gierke disease (glucose-6 phosphatase, glycogen degradation)
Liver steatosis, severe hypoglycemia, stunted growth, acidosis
Ib: deficiency of glucose-6 phosphate translocase, glycogen degradation)
very similar to Von Gierke disease
II: Pompe disease (lysosomal glycosidase, glycogen degradation)
Liver steatosis, cardiomegaly
all organs, especially liver and heart
III: Forbes disease (debranching enzymatic system, glycogen degradation)
Liver steatosis, severe hypoglycemia, muscular weakness
liver, muscle, heart, leukocytes
IV: Andersen disease (branching enzymatic system, glycogen biosynthesis)
liver cirrhosis; myopathy and heart failure
liver, muscle, heart
V: McArdle disease (muscle glycogen phosphorylase, glycogen degradation)
cramps on exercise
VI: Hers disease (liver glycogen phosphorylase, glycogen degradation)
Liver steatosis, hypoglycemia of variable severity
VII: Tarui disease (phosphofructokinase, glycolysis)
similar to McArdle disease
Inherited defects of lipid metabolism
Inherited defects of lipid metabolism include hyperlipoproteinemias, which have been discussed in a preceding lecture, and lipidoses.
are genetic defects of the degradation of phospholypids, sfingomyelin, or gangliosides. These substance accumulate mainly in the endosomes/lysosomes of macrophages incapable of digesting them, causing huge hepato- and spleno-megaly in addition to other symptoms. The diagnosis of these diseases is difficult. Biopsy followed with specific staining with
(a compound that binds specifically to cholesterol) reveals that the enlarged macrophages are filled with undigested cholesterol. Gene sequencing confirms the inherited enzymatic defect.
Disease (genetic defect)
symptoms and laboratory findings
Gaucher's disase (glucocerebrosidase)
type I manifests itself in the adult, pancytopenia but no neurological involvement;
type II, infantile with severe neurological symptoms
type III, juvenile with neurological symptoms and pancytopenia
spleen, liver, bone marrow, nervous system
Tay Sachs disease (GM2 gangliosidosis)
defect of hexosaminidase A, causes accumulation of gangliosides in the brain; usually lethal within the first years of life
Niemann-Pick disease (sphingomyelinase)
several variants are known; all cause demyelinization of the nervous system and mental retardation. Hepato- and spleno-megaly, and pancytopenia are present.
nervous system, liver, spleen, bone marrow
Inherited defects of cholesterol biosynthesis
Several inheritable enzymatic defects of cholesterol biosynthesis are known. Since cholesterol is necessary for embrionic and fetal organogenesis these defects may be associated to multiple malformations (e.g. microcephaly) and hypocholesterolemia. The most important genetic defect of cholesterol biosynthesis is due to mutation of gene MVK encoding for the enzyme
. Two different diseases are associated to defects of MVK, depending on the type of mutation and the extent of loss of enzymatic activity.
is the more severe form and is associated to mental retardation, hepatosplenomegaly, metabolic acidosis and various dismorfisms;
is the less severe form and is associated to recurrent fever. Mevalonic acid accumulates in the serum and in the urine and is easily detected by the clinical laboratory.
Inherited defects of the metabolism of oligoelements (metal accumulation diseases; thesaurismoses)
is inherited as a mendelian, autosomal recessive trait. It is relatively common (in the order of 5/1,000 births) and causes excess uptake and accumulation of iron. Humans do not have mechanisms to eliminate iron, whose poor water solubility causes urinary excretion to be limited. Indeed iron is protein-bound in the serum (to transferrin) and in cellular deposits (to ferritin; to hemosiderin). Iron is lost only in hemorrages, unless chelating therapy is instituted. In primary hemochromatosis the regulation of iron absorption is disturbed and the patient absorbs excess iron. However iron is so low in tissues that the disease takes many years to produce organ damage. Symptoms usually arise in the adult age (this is one of the reasons why the disease is frequent: counter selection cannot operate because the patient has already generated children before the disease becomes evident). Iron accumulates in various tissues; the liver is particularly affected (with cirrhosis). Panhypopituitarism and/or diabetes mellitus may occur. The skin has a dark colour due to iron accumulation in the subcutaneous tissues. The laboratory analyses reveal strongly increased plasma iron (> 200 μg/dL) and increased saturation of transferrin (> 70%). Urinary iron is > 2 μg/die.
Differential diagnosis is with secondary forms of hemochromatosis, especially in diseases requiring repeated transfusions (e.g. thalassemias), or in cases of especially iron-rich diets. Therapy takes advantage of iron chelators, that increase urinary excretion, but some organ lesions (e.g. cirrhosis) are irreversible.
Wilson's disease (hepatolenticular degeneration)
is an autosomal recessive inherited defect of copper etabolism leading to accumulation of the metal in the tissues.The disease takes several years to become clinically evident.Hemolytic anemia and liver cirrhosis are common signs; dementia may follow. Paradoxically, serum Cu is reduced (< 70 μg/dL; ceruloplasmin < 20 mg/dL), because the cells are unable to release Cu to ceruloplasmin. Cu in the urine is increased (> 50 μg/die).
Inherited defects of membrane transporters and pumps
is an autosomal recessive genetic defect of the membrane chloride channel CFTR. Since some 2,000 mutations of the CFTR gene have been described, 150 of which cause cystic fibrosis, the disease is heterogeneous and its severity is variable depending on the specific mutation. The life expectancy, on average, is reduced to 20 to 60 years, depending on the severity of the case. The disease is quite frequent and in Europe its prevalence approches 1/2,000. The mucous secretion of patients are thick and viscous; this causes obstruction of the ducts and airways. The lungs, pancreas, and liver may be more severely affected. Laboratory fundungs depend on which organs are most affected (e.g. recurring episodes of pancreatitis). The duodenal fluid, sweat and sputum contain increased concentration of chloride and decreased bicarbonate.
World Health Organization web-page on
NIH web-page on
Defects of cholesterol biosynthesis
FEBS Letters 200; 580: 5442-9.
Two interesting articles on
Questions and exercises:
1) An infant has an episode of confusion and coma; the laboratory analysis reveals severe hypoglycemia. You suspect:
a liver glycogenosys (e.g. Von Gierke's disease)
a defect of the metabolism of gangliosides (e.g. Gaucher's disease)
type 1 diabetes mellitus
2) Methylmalonic aciduria is a chronic metabolic acidosis resulting from a genetic defect of :
3) A laboratory analysis of the urine of a baby reveals the presence of phenylpiruvic acid. This finding is characteristic of phenylketonuria, a genetic defect of:
4) Primary hemochromatosis and Wilson disease are inherited as:
autosomal recessive traits
autosomal recessive and autosomal dominant traits, respectively
autosomal dominant and autosomal recessive traits, respectively
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Thank you Professor (lecture on bilirubin and jaundice).
The fourth recorded part, the one on hyper and hypoglycemias is not working.
Bellelli: I checked and in my computer it seems to work. Can you better specify
the problem you observe?
This Presentation (electrolytes and blood pH) feels longer than previous lectures
Bellelli: it is indeed. Some subjects require more information than others. I was
thinking of splitting it in two nest year.
Bellelli in response to a question raised by email: when we compare the blood pH
with the standard pH we do not mean to compare the "normal" blood pH (7.4)
with the standard pH. Rather we compare the actual blood pH of the patient, with
the pH of the same blood sample equilibrated under standard conditions.
Thus, if we say that standard pH is lower than pH we mean that equilibriation with
40 mmHg CO2 has caused absorption of CO2 and has lowered the pH with respect
to its value before equilibration.
(Lipoproteins) Is the production of leptin an indirect cause of type 2 diabetes since
it works as a stimulus to have more adipose tissue that produces hormones?
Bellelli: in a sense yes, sustained increase of leptin causes the hypothalamus to adapt
and to stop responding. Obesity ensues and this in turn may cause an increase in the
production of resistin and other insulin-suppressing protein hormones produced by the
adipose tissue. However, this is quite an indirect link, and most probably other factors
contribute as well.
(Urea cycle) what is the meaning of "dissimilatory pathway"?
Bellelli: a dissimilatory pathway is a catabolic pathway whose function is not to produce
energy, but to produce some terminal metabolyte that must be excreted. Dissimilatory
pathways are necessary for those metabolytes that cannot be excreted as such by the
kidney or the liver because they are toxic or poorly soluble. Examples of metabolytes
that require transformation before being eliminated are heme-bilirubin, ammonia,
sulfur and nitrogen oxides, etc.
Talking about IDDM linked neuropathy can be the C peptide absence considered a cause of it??
Bellelli: The C peptide released during the maturation of insulin, besides being an indicator
of the severity of diabetes, plays some incompletely understood physiological roles. For
example it has been hypothesized that it may play a role in the reparation of the
atherosclerotic damage of the small arteries. Thus said, I am not aware that it plays a direct
role in preventing diabetic polyneuropathy. Diabetic neuropathy has at least two causes: the
microvascular damage of the arteries of the nerve (the vasa nervorum), and a direct
effect of hyperglycemia and decreased and irregular insulin supply on the nerve metabolism.
Diabetic neuropathy is observed in both IDDM and NIDDM, and requires several years to
develop. Since the levels of the C peptide differ in IDDM and NIDDM, this would suggest
that the role of the C peptide in diabetic neuropathy is not a major one. If you do have
better information please share it on this site!
In acute intermitted porphyria and congenital erythropoietic porphyria why do the end product
of the affected enzymes accumulate instead of their substrate??
Bellelli: First of all, congratulations! This is an excellent question.
Remember that a condition is which the heme is not produced is lethal in the foetus; thus
the affected enzyme(s) must maintain some functionality for the patient
to be born and to come to medical attention. All known genetic defects of heme
biosynthesis derange but do not block this metabolic pathway.
Congenital Erythropoietc Porphyria (CEP) is a genetic defect of uroporphyrinogen
III cosynthase. This protein associates to uroporphyrinogen synthase (which is present
and functional in CEP) and guarantees that the appropriate uroporphyrinogen isomer is produced
(i.e. uroporphyrinogen III). In the absence of a functional uroporphyrinogen III
cosynthase other possible isomers of uroporphyrinogen are produced together with
uroporpyrinogen III, mostly uroporphyrinogen I. The isomers of uroporphyrinogen
that are produced differ because of the positions of propionate and acetate side chains,
and this in turn is due to the pseudo symmetric structure of porphobilinogen. Only
isomer III can be further used to produce protoporphyrin IX. Thus in the
case of CEP we observe accumulation of abnormal uroporphyrinogen derivatives, which, as
you correctly observed are the products of the enzymatic synthesis operated by
The case of Acute Intermittent Porphyria (AIP) is similar, although there may be variants
of this disease. What happens is that either the affected enzyme is a variant that does not
properly associate with uroporphyrinogen III cosynthase or presents active site mutations
that impair the proper alignement of the phoprphobilinogen substrates. In either case
abnormal isomers of uroporphyrinogen are produced, as in CEP.
Also remark that in both AIP and CEP we observe accumulation of the porphobilinogen
precursor: this is because the overall efficiency of the biosynthesis of uroporphyrinogens is
reduced. Thus: (i) less uroporphyrinogen is produced, and (ii) only a fraction of the
uroporphyrinogen that is produced is the correct isomer (uroporphyrinogen III).
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