Hooked to vitamin B12 since 1955: a historical perspective.

In our pioneering work in 1956, two binders of vitamin B12 (B12) alias cobalamin (Cbl) were identified in gastric juice, S with slow electrophoretic mobility, a 70 kD protein with intrinsic factor (IF) activity and another rapid (R), not IF active but probable digestion product. Numerous sources contained a protein immunologically identical to R (haptocorrin, Hc). Another IF-active component (I) was found. Isoelectric focusing showed that S, I and R were assemblies of "isoproteins" with different pI's due to varying glycosidation. Isolation of S, I and R in microquantities was achieved in 1962 using a series of ion exchange chromatographies and gel filtration. Ponderable products were obtained in 1965-1966. The B12-IF complex was a dimer, contained 13% carbohydrate and showed a different absorption spectrum than B12. Using the Schilling test, B12 absorption was shown to require Ca(++), bound in vitro to the ileal receptor and IF, but most of Ca(++) could be removed with sialidase. The receptor-substrate complex contained Ca(++) and carbohydrate. The purified receptor was shown to contain two main subunits. The Imerslund-Gräsbeck syndrome was discovered 1958-1960; it is caused by mutations in either of two genes, cubilin or amnionless, which form the multiligand receptor cubam. Testicular biopsies during and after B12-treated deficiency showed remarkable improvement after therapy. Studies of the turnover of radioactive B12 revealed biliary and fecal excretion, enterohepatic circulation and allowed calculation of biological half-life and daily need. The B12 coenzymes largely behaved like B12. To study whether radiocobalt in B12 was representative of the rest of the B12 molecule, (32)P and (57)Co labeled hydroxocobalamins were biosynthesized and shown to behave identically when given simultaneously to rats. The complex metabolism of B12 explains the pathogenesis of B12 deficiencies. Some of its mechanisms are not restricted to B12, e.g. the endocytosis of B12-IF also applies to other macromolecules.

The theory of reference values: an unfinished symphony.

The history of the theory of reference values can be written as an unfinished symphony. The first movement, allegro con fuoco, played from 1960 to 1980: a mix of themes devoted to the study of biological variability (intra-, inter-individual, short- and long-term), preanalytical conditions, standardization of analytical methods, quality control, statistical tools for deriving reference limits, all of them complex variations developed on a central melody: the new concept of reference values that would replace the notion of normality whose definition was unclear. Additional contributions (multivariate reference values, use of reference limits from broad sets of patient data, drug interferences) conclude the movement on the variability of laboratory tests. The second movement, adagio, from 1980 to 2000, slowly develops and implements initial works. International and national recommendations were published by the IFCC-LM (International Federation of Clinical Chemistry and Laboratory Medicine) and scientific societies [French (SFBC), Spanish (SEQC), Scandinavian societies…]. Reference values are now topics of many textbooks and of several congresses, workshops, and round tables that are organized all over the world. Nowadays, reference values are part of current practice in all clinical laboratories, but not without difficulties, particularly for some laboratories to produce their own reference values and the unsuitability of the concept with respect to new technologies such as HPLC, GCMS, and PCR assays. Clinicians through consensus groups and practice guidelines have introduced their own tools, the decision limits, likelihood ratios and Reference Change Value (RCV), creating confusion among laboratorians and clinicians in substituting reference values and decision limits in laboratory reports. The rapid development of personalized medicine will eventually call for the use of individual reference values. The beginning of the second millennium is played allegro ma non-troppo from 2000 to 2012: the theory of reference values is back into fashion. The need to revise the concept is emerging. The manufacturers make a friendly pressure to facilitate the integration of Reference Intervals (RIs) in their technical documentation. Laboratorians are anxiously awaiting the solutions for what to do. The IFCC-LM creates Reference Intervals and Decision Limits Committee (C-RIDL) in 2005. Simultaneously, a joint working group IFCC-CLSI is created on the same topic. In 2008 the initial recommendations of IFCC-LM are revised and new guidelines are published by the Clinical and Laboratory Standards Institute (CLSI C28-A3). Fundamentals of the theory of reference values are not changed, but new avenues are explored: RIs transference, multicenter reference intervals, and a robust method for deriving RIs from small number of subjects. Concomitantly, other statistical methods are published such as bootstraps calculation and partitioning procedures. An alternative to recruiting healthy subjects proposes the use of biobanks conditional to the availability of controlled preanalytical conditions and of bioclinical data. The scope is also widening to include veterinary biology! During the early 2000s, several groups proposed the concept of 'Universal RIs' or 'Global RIs'. Still controversial, their applications await further investigations. The fourth movement, finale: beyond the methodological issues (statistical and analytical essentially), important questions remain unanswered. Do RIs intervene appropriately in medical decision-making? Are RIs really useful to the clinicians? Are evidence-based decision limits more appropriate? It should be appreciated that many laboratory tests represent a continuum that weakens the relevance of RIs. In addition, the boundaries between healthy and pathological states are shady areas influenced by many biological factors. In such a case the use of a single threshold is questionable. Wherever it will apply, individual reference values and reference change values have their place. A variation on an old theme! It is strange that in the period of personalized medicine (that is more stratified medicine), the concept of reference values which is based on stratification of homogeneous subgroups of healthy people could not be discussed and developed in conjunction with the stratification of sick patients. That is our message for the celebration of the 50th anniversary of Clinical Chemistry and Laboratory Medicine. Prospects are broad, enthusiasm is not lacking: much remains to be done, good luck for the new generations!

[Homozygous mutation in the intrinsic factor gene in a child with severe vitamin B12 deficiency]. / Homozygot mutation i intrinsic factor-genet hos et barn med svær vitamin B 12-mangel.

A 28 month-old boy was hospitalized with pallor and weight stagnation. He had macrocytic anaemia and pancytopenia due to cobalamin deficiency and a rare homozygous mutation in the intrinsic factor gene. His sister showed similar symptoms at the age of 15 months. The heterozygous father had no symptoms, but did have a low cobalamin level. Gastroscopy with biopsies showed no pathology. All were given monthly cyanocobalamin injections which, however, caused leg cramps. Replacement with monthly hydroxocobalamin was successful.

Juvenile selective vitamin B12 malabsorption: 50 years after its description-10 years of genetic testing.

Fifty years have passed since the description of juvenile selective malabsorption of cobalamin (Cbl). Quality of life improvements have dramatically reduced the incidence of parasite-induced or nutritional Cbl deficiency. Consequently, inherited defects have become a leading cause of Cbl deficiency in children, which is not always expressed as anemia. Unfortunately, the gold standard for clinical diagnosis, the Schilling test, has increasingly become unavailable, and replacement tests are only in their infancy. Genetic testing is complicated by genetic heterogeneity and differential diagnosis. This review documents the history, research, and advances in genetics that have elucidated the causes of juvenile Cbl malabsorption. Genetic research has unearthed many cases in the past decade, mostly in Europe and North America, often among immigrants from the Middle East or North Africa. Lack of suitable clinical testing potentially leaves many patients inadequately diagnosed. The consequences of suboptimal Cbl levels for neurological development are well documented. By raising awareness, we wish to push for fast track development of better clinical tools and suitable genetic testing. Clinical awareness must include attention to ethnicity, a sensitive topic but effective for fast diagnosis. The treatment with monthly parenteral Cbl for life offers a simple and cost-effective solution once proper diagnosis is made.

Imerslund-Gräsbeck syndrome (selective vitamin B(12) malabsorption with proteinuria).

Imerslund-Gräsbeck syndrome (IGS) or selective vitamin B(12) (cobalamin) malabsorption with proteinuria is a rare autosomal recessive disorder characterized by vitamin B(12) deficiency commonly resulting in megaloblastic anemia, which is responsive to parenteral vitamin B(12) therapy and appears in childhood. Other manifestations include failure to thrive and grow, infections and neurological damage. Mild proteinuria (with no signs of kidney disease) is present in about half of the patients. Anatomical anomalies in the urinary tract were observed in some Norwegian patients. Vitamin B(12) absorption tests show low absorption, not corrected by administration of intrinsic factor. The symptoms appear from 4 months (not immediately after birth as in transcobalamin deficiency) up to several years after birth. The syndrome was first described in Finland and Norway where the prevalence is about 1:200,000. The cause is a defect in the receptor of the vitamin B(12)-intrinsic factor complex of the ileal enterocyte. In most cases, the molecular basis of the selective malabsorption and proteinuria involves a mutation in one of two genes, cubilin (CUBN) on chromosome 10 or amnionless (AMN) on chromosome 14. Both proteins are components of the intestinal receptor for the vitamin B(12)-intrinsic factor complex and the receptor mediating the tubular reabsorption of protein from the primary urine. Management includes life-long vitamin B(12) injections, and with this regimen, the patients stay healthy for decades. However, the proteinuria persists. In diagnosing this disease, it is important to be aware that cobalamin deficiency affects enterocyte function; therefore, all tests suggesting general and cobalamin malabsorption should be repeated after abolishment of the deficiency.

Hereditary juvenile cobalamin deficiency caused by mutations in the intrinsic factor gene.

Hereditary juvenile megaloblastic anemia due to vitamin B12 (cobalamin) deficiency is caused by intestinal malabsorption of cobalamin. In Imerslund-Grasbeck syndrome (IGS), cobalamin absorption is completely abolished and not corrected by the administration of intrinsic factor (IF); if untreated, the disease is fatal. Biallelic mutations either in the cubilin (CUBN) or amnionless (AMN) gene cause IGS. In a series of families clinically diagnosed with likely IGS, at least six displayed no evidence of mutations in CUBN or AMN. A genome-wide search for linkage followed by mutational analysis of candidate genes was performed in five of these families. A region in chromosome 11 showed evidence of linkage in four families. The gastric IF (GIF) gene located in this region harbored homozygous nonsense and missense mutations in these four families and in three additional families. The disease in these cases therefore should be classified as hereditary IF deficiency. Clinically, these patients resembled those with typical IGS; radiocobalamin absorption tests had been inconclusive regarding the nature of the defect. In the diagnosis of juvenile cobalamin deficiency, mutational analysis of the CUBN, AMN, and GIF genes provides a molecular characterization of the underlying defect and may be the diagnostic method of choice.

The evolution of the reference value concept.

This manuscript reviews the introduction of the concept of reference values, the corresponding philosophy, and subsequent recommendations reflecting the different subtopics of the field. The generally unrecognised phenomenon that laboratory results of the population tended to be lognormal instead of Gaussian attracted the attention of the author, who became sceptical of the concept of normal values because of its ambiguity. Together with N.-E. Saris a new concept of reference values was launched at a congress in 1969. Briefly, clinical measurements should be interpreted against values from proper control subjects. Subsequently international, regional and national societies established expert panels which produced recommendations covering the general principles and terminology of reference values, the concept of health, standardised specimen collection and preanalytical factors, statistical treatment of collected values, stratification of data, relating observed (patient) values to reference values, etc. The importance of using correct terminology, taking into consideration the aim of ordering the laboratory test and some neglected procedures (e.g., survival values) are emphasised. New developments such as reference changes are mentioned. The field has evolved largely as the result of constructive international teamwork.

Genetically heterogeneous selective intestinal malabsorption of vitamin B12: founder effects, consanguinity, and high clinical awareness explain aggregations in Scandinavia and the Middle East.

Selective intestinal malabsorption of vitamin B(12) causing juvenile megaloblastic anemia (MGA; MIM# 261100) is a recessively inherited disorder that is believed to be rare except for notable clusters of cases in Finland, Norway, and the Eastern Mediterranean region. The disease can be caused by mutations in either the cubilin (CUBN; MGA1; MIM# 602997) or the amnionless (AMN; MIM# 605799) gene. To explain the peculiar geographical distribution, we hypothesized that mutations in one of the genes would mainly be responsible for the disease in Scandinavia, and mutations in the other gene in the Mediterranean region. We studied 42 sibships and found all cases in Finland to be due to CUBN (three different mutations) and all cases in Norway to be due to AMN (two different mutations), while in Turkey, Israel, and Saudi Arabia, there were two different AMN mutations and three different CUBN mutations. Haplotype evidence excluded both CUBN and AMN conclusively in five families and tentatively in three families, suggesting the presence of at least one more gene locus that can cause MGA. We conclude that the Scandinavian cases are typical examples of enrichment by founder effects, while in the Mediterranean region high degrees of consanguinity expose rare mutations in both genes. We suggest that in both regions, physician awareness of this disease causes it to be more readily diagnosed than elsewhere; thus, it may well be more common worldwide than previously thought.

Proteinuria in cubilin-deficient patients with selective vitamin B12 malabsorption.

Selective vitamin B(12) malabsorption or Gräsbeck-Imerslund disease (megaloblastic anemia 1) is frequently accompanied by proteinuria. The malabsorption-proteinuric syndrome of Finnish patients is caused by a defect in the multiligand receptor cubilin. We studied the urinary proteins of control subjects and 13 adult patients with three defined cubilin mutations (FM1, FM2, FM3), all diagnosed during childhood and subsequently observed. The overall kidney function was unimpaired and did not deteriorate with time. The excretion of total protein and albumin, and to lesser extent of transferrin, immunoglobulin light chains, and alpha(1)- and beta(2)-microglobulins, was clearly elevated in 3 patients, mildly elevated in 3, and hardly or not at all increased in the rest. The urinary cobalamin-intrinsic factor receptor was low in 5 patients studied and lowest in the group with clear-cut proteinuria. The proteinuria was not of the classical glomerular or tubular type, but apparently due to the lack of cubilin function needed for tubular reabsorption of some, but not all, proteins of the primary urine.