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 KM; 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 O2 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
stunted growth
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
acid-base and electrolyte disorders
polyuria with polydipsia
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.
abnormal blood cell count or hypogammaglobulinemia

      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, glycogen storage 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) Lysosomal storage disorders.
8) Mitochondial diseases
9) 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
e) immunodeficiencies
f) anemias

      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 non-diabetic mellituria.

Genetic defects of monosaccharide metabolism (non-diabetic melliturias)
Disease (enzyme/gene; pathway) inheritance symptoms laboratory findings
Abnormal variants of Glucose-6-phosphate dehydrogenase (pentose phosphate pathway) X-linked decreased erythrocyte lifespan; in some cases sensitivity to fava beans may cause acute crises anemia, usually of moderate degree; hemolytic crises with hemoglobinuria may be caused by contact with or ingestion of fava beans (favism)
Galactosemia (galactose 1 phosphate uridyl transferase; Leloir pathway for the isomerization of galactose to glucose) autosomal recessive stunted growth, possible mental retardation (prevented by galactose-free diet) presence of galactose and galactose 1 phosphate in the blood and urine
Galactokinase deficiency (Leloir pathway for the isomerization of galactose to glucose) autosomal recessive early cataract due to galactitol accumulation in the lens (prevented by galactose-free diet) presence of galactose in the blood and urine
UDP-galactose epimerase deficiency (Leloir pathway for the isomerization of galactose to glucose) autosomal recessive benign presence of galactose and galactose in the urine
Hereditary fructose intolerance (phosphofructo aldolase deficiency) autosomal recessive hypoglycemia, renal tubular acidosis, possible convulsions and coma presence of fructose in the urine
Fructosemia / Fructosuria (fructokinase) autosomal recessive benign presence of fructose in the blood and urine
Fructose 1,6 diphosphatase deficiency (gluconeogenesis) autosomal recessive hypoglycemia, acidosis presence of glucogenesis precursors in the blood and urine (e.g. ketoacids, lactic acid)
Pentosuria (L-xylulose dehydrogenase) autosomal recessive benign, characteristic of some populations (e.g. american Jews) presence of L-xylulose in the blood and urine

      Favism: a defect of Glucose-6-Phosphate Dehydrogenase (G6PDH). Glucose-6-Phosphate Dehydrogenase catalyzes the first reaction of the pentose phosphate pathway; its genetic defects are frequent, and at least 250 variants have been described, often with reduced activity. The gene of G6PDH is encoded on the X chromosome, thus its defects are inherited as a sex-linked recessive trait. The pentose phosphate pathway in some cells, notably the erythrocyte, may be the principal source of NADPH, and provides the reducing equivalents requiredby anabolic reactions and to counteract oxidative damage. The erythrocytes of affected individuals may have reduced lifespan and a mild anemia may be evident at laboratory tests. Several defects of G6PDH confere resistance to malaria because the infected erythrocyte ruptures before the parasite can complete its life cycle; this is the reason why defects of G6PDH attain high prevalence in populations originating from regions where malaria is or has been endemic. Hemolytic crises may be caused by several stressors. Some substances contained in fava beans (vicine, divicine, convicine) may cause oxidative stress and require an increased production of NADPH that the patient's erythrocytes cannot meet; thus the subjects suffering of these mutations may experience severe, and potentially lethal hemolytic crises when exposed to fava beans or its pollens (favism).
Laboratory findings: hemolytic crises; hemoglobinuria; pre-hepatic jaundice (increased unconjugated bilirubin); mild anemia; diagnosis is confirmed by gene sequencing.
Further readings
Luzzatto L. and Arese P. Favism and Glucose-6-Phosphate Dehydrogenase Deficiency N.E. J. Med. 2018, 378: 60-71.

      Disorders of the Leloir pathway for conversion of galactose to glucose. The conversion of galactose to glucose (Leloir pathway) is surprisingly complex, given that the two sugars are epimers, differing only because of the sterechemical configuration of C4. The Leloir pathway is as follows:
      1) αD galactose + ATP → galactose-1-P + ADP (enzyme: galactokinase)
      2) galactose-1-P + UDP-glucose → UDP-galactose + glucose (enzyme: galactose-1-P uridyl ransferase)
      3) UDP-galactose → UDP-glucose (enzyme: UDP-galactose epimerase)
      In addition to the above enzyme the cells also possess the enzyme galactose mutarotase to accelerate the conversion of βD galactose to αD galactose.
      Mutations are described for all the enzymes of the Leloir pathway and may lead to diseases inherited as mendelian autosomal recessive traits.

      Metabolism of fructose occurs in the liver and is relevant for glyconeogenesis; moreover it provides a different access to the intermediates of glycolisis. The essential steps are as follows:
      1) fructose + ATP → fructose-1-P + ADP (enzyme: fructokinase)
      2) fructose-1-P → dihydroxy acetone phosphate + glyceraldheyde (enzyme: phosphofructose aldolase)
      3) glyceraldehyde + ATP → glyceraldehyde-3-P + ADP (enzyme: glyceraldehyde kinase)

      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 severe hypoglycemia, which becomes manifest very early in life.

Genetic defects of glycogen metabolism (glycogenoses)
Disease (enzyme/gene) inheritance symptoms Organs affected
0: UDP-Glucose glycogen transferase (glycogen biosynthesis) autosomal recessive Liver steatosis, fasting hypoglycemia liver, muscle
Ia: Von Gierke disease (glucose-6 phosphatase, glycogen degradation) autosomal recessive Liver steatosis, severe hypoglycemia, stunted growth, acidosis liver, kidney
Ib: deficiency of glucose-6 phosphate translocase, glycogen degradation) autosomal recessive very similar to Von Gierke disease liver, leukocytes
II: Pompe disease (lysosomal glycosidase, glycogen degradation) autosomal recessive Liver steatosis, cardiomegaly all organs, especially liver and heart
III: Forbes disease (debranching enzymatic system, glycogen degradation) autosomal recessive Liver steatosis, severe hypoglycemia, muscular weakness liver, muscle, heart, leukocytes
IV: Andersen disease (branching enzymatic system, glycogen biosynthesis) autosomal recessive liver cirrhosis; myopathy and heart failure liver, muscle, heart
V: McArdle disease (muscle glycogen phosphorylase, glycogen degradation) autosomal recessive cramps on exercise muscle
VI: Hers disease (liver glycogen phosphorylase, glycogen degradation) autosomal recessive Liver steatosis, hypoglycemia of variable severity liver
VII: Tarui disease (phosphofructokinase, glycolysis) autosomal recessive similar to McArdle disease muscle, erythrocytes

      Inherited defects of lipid metabolism
      Inherited defects of lipid metabolism include hyperlipoproteinemias, which have been discussed in a preceding lecture, and lipidoses. 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 filipin (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) inheritance symptoms and laboratory findings organs affected
Gaucher's disase (glucocerebrosidase) autosomal recessive 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) autosomal recessive defect of hexosaminidase A, causes accumulation of gangliosides in the brain; usually lethal within the first years of life brain
Niemann-Pick disease (sphingomyelinase) autosomal recessive 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 mevalonate kinase. Two different diseases are associated to defects of MVK, depending on the type of mutation and the extent of loss of enzymatic activity. Mevalonic aciduria is the more severe form and is associated to mental retardation, hepatosplenomegaly, metabolic acidosis and various dismorfisms; hyperimmunoglobulinema D 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 hormone biosynthesis manifest themselves as congenital endocrine insufficiencies, and are usually transmitted as autosomal recessive traits. Laboratory diagnosis is as described in the lecture of endocrine diseases, except that these diseases appear in the newborn and require gene sequencing of the possibly responsible gene(s). The most frequent case is that of congenital thyroid insufficiency, wihch may be due to several genetic causes, the most important of which are defects in the transport and utilization of iodine; for a more detailed description see this link.

      Other inherited defects of metabolism have been presented elsewhere in this course: e.g. heme biosytnthesis (porphyrias), nucleotide metabolism, coagulation disorderd, hemoglobinopathies, etc.

      Lysosomal storage disorders
      Lysosomes are intracellular organelles produced by the Golgi apparatus and containing digestive enzymes. The main function of lysosomes is the fusion with endocytic vesicles (formation of secondary endosomes) and digestion of their content. Mutations can affect virtually every lysosomal enzyme, and may cause the inability of the secondary endosome to digest a specific content, which accumulates to a great extent. Many lysosomal storage disorders affect the liver and cause its enlargement and, on the long run, its functional failure; thus the disease may not be manifest at birth and the onset of symptoms mayoccur in late infancy or even later . Since lysosomal storage disorders are defined by the intracellular location of the defect, rather than the metabolyte, this group includes some diseases that have been already considered under separate headings: e.g. Pompe glycogenosis, considered under the glycogen metabolism disorders is a lysosomal storage disease. The lysosomes are the principal seat of degradation of sphingolipids and mucopolysaccharides; thus shingomyelinoses and mucopolysaccharidoses figure preminently among lysosomal storage disorders. Diagnosis is suspected on clinical grounds and confirmed by means of gene sequencing. Symptoms are variable; mental retardation and marked enlargement of the liver and the spleen are very common. Inheritance is almost always autosomal and recessive. Lysosomal storage disorders cause scarce or absent laboratory findings because the accumulated metabolytes remain trapped in the secondary endosomes and are not released in the blood nor do they reach the urine. Liver and spleen biopsy, with appropriate histochemical staining are strongly indicative and suggest the appropriate genetic analyses.
diseaseenzyme affectednotes
GM1 gangliosidosisβ-galactosidase 
GM2 gangliosidosis (Tay-Sachs disease and its variants)hexosaminidase A 
Galactosyl-ceramide lipidosis (Krabbe disease)galactosyl-ceramide β-galactosidase 
Metachromatic leukodystrophy (and its variants)arylsulfatase A 
Sphingomyelin lipidosis (Niemann-Pick disease)sphingomyelinase 
Glucosyl-ceramine lipidosis (Gaucher disease)β-glucocerebrosidase 
Triexhosyl ceramidosis (Fabry disease)α-galactosidase AX-linked dominant
Acid lipase deficiencyacid lipase 
Ceramide accumulation (Farber disease)ceramidase  
Glycogenosis type II (Pompe disease)α-glicosidase (acid maltase/isomaltase) 
Acid phosphatase deficiencyacid phosphatase 
AspartylglucosaminuriaAspartylglucosamine glucosidaseaspartylglucosamine peptides released
in the blood and urine
Mucopolysaccaridosis I (Hurler disease)α-iduronidase 
Mucopolysaccaridosis II (Hunter disease)iduronosulfate sulfatase 
Mucopolysaccaridosis III (Sanfilippo disease; several variants)heparansulfate hydrolases 
Mucopolysaccaridosis IV (Morquio disease)N-actylgalactosamine sulfate sulfatase 
Mucopolysaccaridosis VI (Moroteaux-Lamy disease)N-acetyl hexosamine sulfate sulfatase (arylsulfatase B) 
Mucopolysaccaridosis VIIβ-glucuronidase  
Multiple sulfatase deficiencyarylsulfatases A, B, and C 
Sialidosissialidase (glycoprotein neuroaminidase) 
Mucolipidosis I, II, IIIUDP-N-acetylglucosamine transferase  
Mucolipidosis VIganglioside neuroaminidase  

      Mitochondrial diseases
      Inherited diseases of mitochondria may be due to mutations of cellular genes, coding for mitochondrial enzymes, or to mutations of mitochondrial genes (in which case are inherited via maternal transmission). The principal symptoms is muscular weakness, and the clinical picture may be severe, even lethal. At least 350 mitochondrial genetic diseases are described, some affecting spcific organs (e.g. the eye), others affecting all organs. The clinical laboratory findings are usually scarce; diagnosis is suggested by the symptoms and the familial anamnesis and confirmed by gene sequencing (caution: mitochondrial genes must be sequenced, together with nuclear genes coding for mitochondrial enzymes).
      A special, and very interesting case is that of genetic defects of mitochondrial transporters. Mitochondrial transporters are encoded by the nuclear DNA and their mutations are inherited as recessive autosomal traits. The peculiarity of these diseases is that the biochemical abnormality is not confined to the mitochondrion, but causes accumulation of typical mitochondrial metabolytes in the cell cytoplasm, from which these molecule leak to the blood and urine, where they can be demonstrated by the usual laboratory analyses. Typical examples are the inherited defects of carnitine/acylcarnithine transporter or glutamate/aspartate transporter. For an extended list of inherited defect of mitochondrial transporters see this link

      Inherited defects of the metabolism of oligoelements (metal accumulation diseases; thesaurismoses)
      Primary hemochromatosis 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
      Cystic fibrosis is a relatively frequent 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.
      Laboratory tests for cystic fibrosis. The sweat test is a measure of the concentration of chloride in the sweat (increased if the disease is present). It is reliable and non-invasive. The diagnosis is confirmed by gene sequencing.

      Further readings
      The World Health Organization web-page on genetic diseases.
      The NIH web-page on genetic diseases.
      HR Waterham Defects of cholesterol biosynthesis FEBS Letters 200; 580: 5442-9.
      Two interesting articles on Cystic fibrosis; cystic fibrosis.

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 :
lipid metabolism
aminoacid metabolism
carbohydrate metabolism

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:
Galactose metabolism
Tyrosine metabolism
Sphyngosine metabolism

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
uroporphyrinogen synthase.
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).

is it possible to take gulonolactone oxidase to synthesize vitamin C
instead of vitamin C supplement?
Bellelli: no, this approach does not work. The main reason is that
the biosynthesis of vitamin C, as almost all other metabolic processes, occurs intracellularly.
If you administer the enzyme it will at most reach the extracellular fluid but will not be
transported inside the cells to any significant extent. Besides, there are other problems
in this type of therapy (e.g. the enzyme if administered orally, may be degraded by digestive
proteases; if administered parenterally, may cause the immune system to react against a
non-self protein). In theory one could think of a genetic modification of the inactive human
gene of gulonolactone oxidase, but the risk and cost of this intervention would not be
justified. In addition to these considerations, except for cases of shipwreckage or
other catastrophes, a proper diet or administration of tablets of vitamin C is effective,
risk-free and unexpensive, thus no alternative therapy is reasonable. However, I express my
congratulations for your search on the biosynthesis pathway of ascorbic acid.

Resorption and not reabsorption would lead to hypercalcemia ie bone matrix being broken down.
Bellelli: I am not sure to interpret your question correctly. Resorption indicates destruction of the bone matrix and release of calcium and
phosphate in the blood, thus it causes an increase of calcemia. Reabsorption usually means active transport of calcium from the renal tubuli to the blood, thus
it prevents calcium loss. It prevents hypocalcemia, and thus complement bone resorption. To avoid confusion it is better use the terms "bone resorption" and "
renal reabsorption of calcium". If you have a defect in renal reabsorption, parthyroid hormone will be released to maintain a normal calcium level by means of
bone resorption; the drawback is osteoporosis.

In Reed and Frost model: I haven't understood what is the relationship
between K and R reproductive index. Thank you Professor!
Bellelli: in the Reed and Frost model K is the theoretical upper limit of
R0. R the reproductive index is the ratio (new cases)/(old cases) measured after
one serial generation time. R0 is the value of R one measures at the beginning
of the epidemics, when in principle all the population is susceptible.

What is the link between nucleotide metabolism and immunodeficiencies and mental retardation?
Bellelli: the links may be quite complex, but the principal ones are as follows:
1) the immune response requires a replication burst of granulocytes and lymphocytes, which in turn requires
a sudden increase of nucleotide production, necessary for DNA replication. Defects of nucleotide metabolism
impair this phase of the immune defense. Notice that the mechanism is similar to the one responsible of
anemia which requires a sustained biosynthesis of nucleotides at a constant rate, rather than in a burst.
2) Mental retardation is mainly due to the accumulation of nulceotide precursors in the brain of the
newborn, due to the incompletely competent blood-brain barrier.

How can ornithine transaminase defects cause hyperammonemia? Is it due to the accumulation
of ornithine that blocks the urea cycle or for other reasons?
Bellelli: ornithine transaminase is required for the reversible interconversion of ornithine
and proline, and thus participates to both the biosynthesis and degradation of ornithine. The enzyme is
synthesized in the cytoplasm and imported in the mitochondrion. Depending on the metabolic conditions
the deficiency of this enzyme may cause both excess (when degradation would be necessary) or defect
(when biosynthesis would be necessary) of ornithine; in the latter case, the urea cycle slows down. Thus
there is the paradoxical condition in which alternation may occur between episodes of hyperammonemia
and of hyperornithinemia.

When we use the Berthelot's reaction to measure BUN do we also have to
measure the concentration of free ammonia before adding urease?
Bellelli: yes, in principle you should. Berthelot's reaction detects ammonia,
thus one should take two identical volumes of serum, use one to measure free ammonia,
the other to add urease and measure free ammonia plus ammonia released by urea. BUN is
obtained by difference. However, free ammonia in our blood is so much lower than urea that
you may omit the first sample, if you only want to measure BUN.

Why do we have abnormal electrolytes in hematological neoplasia e.g.
Bellelli: I do not have a good explanation for this effect, which may have
multiple causes. However, you should consider two factors: (i) acute leukemias cause a massive
proliferation of leukocytes (or lymphocytes depending on the cell type affected) with a very
shortened lifetime; thus you observe an excess death rate of the neoplastic cells. The dying
cells release in the bloodstream their content, which has an electrolyte composition different
from that of plasma: the cell cytoplasm is rich in K and poor in Na, thus causing hyperkalemia.
(ii) the kidney may be affected by the accumulation of neoplastic white cells or their lytic products.

Gaussian curve: If it is bimodal is it more likely to be a "certain diagnosis" than if it is
unimodal or does it only show the distinguishment from health?
Bellelli an obviously bimodal Gaussian curve indicates that the disease is clearly
separated from health: usually it is a matter of how precise and clear-cut is the definition of the disease.
For example tuberculosis is the disease caused by M. tuberculosis, thus if the culture of the sputum is
positive for this bacterium you have a "certain" diagnosis (caution: the patient may suffer of two diseases,
e.g. tuberculosis and COPD diagnosis of the first does not exclude the second). However, in order to have
a "certain" diagnosis it is not enough that distribution of the parameter is bimodal, it is also required that the
patient's parameter is out of the range of the healthy condition: this is because a distribution can be
bimodal even though it is composed by two Gaussians that present a large overlap, and the patient's
parameter may fall in the overlapping region. Thus, in order to obtain a "certain" diagnosis you need to
consider not only the distribution of the parameter(s) but also the patient's values and the extent of the
overlapping region.

Prof can you please elaborate a bit more on the interhuman variability and its difference
with the interpopulation variability please?
Bellelli: every individual is a unique combination of different alleles of the same genes;
this is the source of interindividual variability. Every population is a group of individuals who intermarry and
share the same gene pool (better: allele pool). Every allele in a population has its own frequency. Two
population may differ because of the diffferent frequencies of the same alleles; in some cases one
population may completely lack some alleles. The number and frequencies of alleles of each gene
determine the variance. If you take two populations and calculate the cumulative interindividual variance
of the population the number you obtain is the sum of two contributions: the interindividual variance within each population, plus the interpopulation variance
between the means of the allele frequencies. For example, there are human population in which the frequency of blood group B is close to 0% and other populati
ons in which it is 30% or more.

Prof can you please explain again the graph you have showed us in class about thromboplastin?
(Y axis=abs X axis= time)
Bellelli: the graph that I crudely sketched in class represented the signal
of the instrument (an absorbance spectrophotometer) used to record the turbidity of the
sample (turbidimetry). The plasma is more or less transparent, before coagulation starts.
When calcium and the tissue factor (or collagen) are added. thrombin is activated and begins
digesting fibrinogen to fibrin; then fibrin aggregates. The macroscopic fibrin aggregates cause
the sample to become turbid, which means it scatters the incident light. The instrument reads
this as a decrease of transmitted light ( an increase of the apparent absorbance) and the
time profile of the signal presents an initial lag phase, which is called the protrombin or
thromboplastin time depending on the component which was added to start coagulation
(tissue factor or collagen).

Prof can you please explain the concept you have described in class about
the simultaneous hypercoagulation and hemorrhagic syndrome? How can this occur?
Bellelli: The condition you describe is observed only in the Disseminated
Intravascular Coagulation syndrome. Suppose that the patient experiences an episode of
acute pancreatitis: tripsin and chymotripsin are reabsorbed in the blood and proteolytically
activate coagulation causing an extensive consumption of fibrinogen and other coagulation
factors. Tripsin and chymotripsin also damage the vessel walls and may cause internal
hemorrages, but at that point the consumption of fibrinogen may have been so massive that
not enough is left to form the clot where the vessel has been damaged, causing an internal
hemorrage. Pancreatitis is a very severe, potentially lethal condition, and DIC is only one of
the reasons of its severity.

You said that certain drugs (ethanol, cocaine, cannabis, opiates...) cause a
necessity of higher and higher dosage, for two reasons: the enzyme in the liver is inducible and
the receptors in the brain are expressed less and less. So, first, I am not sure I got it right, and
second I did not understand how expressing less receptors leads to a necessity of higher
Bellelli: You got it correctly, but the detailed mechanism of resistance may
vary among different substances, and not all drugs cause adaptation.
The reason why reducing the number of receptors may require an increased dosage of the drug
is as follows: suppose that a certain cell has 10,000 receptors for a drug. When bound to its
agonist/effector, each receptor produces an intracellular second messenger. Suppose that in
order for the cell to respond 1,000 receptors must be activated. The concentration of the
effector required is thus the concentration that produces 10% saturation. You can easily
calculate that this concentration is approximately 1/10 of the equilibrium dissociation constant
of the receptor-effector complex (its Kd), the law being
Fraction bound = [X] / ([X]+Kd)
where [X] is the concentration of the free drug.
After repeated administration, the subject becomes adapted to the drug, and his/her cells
express less receptors, say 5,000. The cell response will in any case require that 1,000
receptors are bound to the effector and activated, but this now represents 20% of the total
receptors, instead of 10%. The drug concentration required is now 1/4 of the Kd.
Continuing administration of the drug further reduces the cell receptors, but the absolute
number of activated receptors required to start the response is constant; thus the fewer
receptors on the cell membrane, the higher the fraction of activated receptors required.

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