Autoinflammatory diseases and osteoclasts mediated osteoimmunology.

       

TNF family growth factors

. Lymphotoxin alfa/TNF alfa

. lymphotoxin beta/TNF beta

. FAS Ligand

. Nerve Growth Factor

. CD40 ligand or CD154

. CD27 ligand

. CD30 ligand

. OX-40 ligand

. 4-IBB ligand

. RANK/ODF/SOFA (Receptor Activator of NF-kB/ Osteoclasts differentiation Factor)

. RANK Ligand

. OPG (Osteoprotegerin)

 

Receptors for TNFalfa

TNFR1: p55 - CD120a Tumor necrosis factor receptor I (Chr. 12)

TNFR2: p75 - CD120b Tumor necrosis factor receptor II

CD95 FAS

TNFRSF4: OX40, OX40 antigen

TNFRSF5: CD40 B cell associated molecule

TNFRSF9: 4-IBB homologue of mouse 4-IBB

TNFRSF11A: RANK, Receptor Activator of Nuclear factor kB (Chr.18q21-22)

TNFRSF11B: OPG, Osteoprotegerin (Chr. 8q24.2 )

TNFRSF16: Nerve Growth Factor Receptor

 

Recently a classification of these receptors have been reported all containing

1. FADD domain: a protein sequence thought to be Fas-Associated Death Domain: a bipartite bridge that directy binds to CD95 Ligand and to pro-caspase 8.

2. TRADD: an adapter protein with liker function with FADD sequence

The molecular structure of this receptor has been defined as formed by a 55 kDa protein with:

1. four cysteine –rich extracellular domains (the first two are involved in most mutations founded in syndromic complex)

2. Intracellular FADD domain involved in signal transduction through protein-protein interaction.

The receptors at present know are:

- TNF-R1: the true receptor for TNF alpha

- TRAIL-R1: using FADD sequense and linking to pro-caspase 8

- TRAIL-R2: using FADD sequence.

Soluble forms of these last two recptors are present in extracellular fluids, with an inhibitor action on apoptotic signals:

- TRAIL-R3 also know such as TRID

- TRAIL-R4 also know such as TRUNDD

We can consider OPG-Ligand and RANK-Ligand such as TRAIL like molecules ie TNF Related Apoptosis Inducing Ligands.

OPG (Osteoprotegerin) on the contrary is a TRAIL-Receptor soluble form ie with inhiting action on TRAIL-R induced apoptosis.

On the osteoclasts instead of linking to true TRAIL-Rs the ligands can link to RANK a specific receptor able to activate NF-kB.

 

Autoinflammatory diseases

The receptor activation of TNF alfa have been clearly demonstrated to be the masterplayers in regulation those we begin to call “autoinflammatory syndromes”.

These are represented by:

- Familial Mediterranean Fever - pyrin

- Familial Hibernian Fever – TNF receptor type 1

- Muckle-Wells syndrome - cryopyrin

- Familial cold urticaria - cryopyrin

- Chronic infantile neurological cutaneous and articular syndrome - cryopyrin

- Neonatal onset multisystem inflammatory disease - cryopyrin

- Hyperimmunoglobulinaemia D and periodic Fever syndrome – mevalonate kinase

First of all we have to remember that these syndromic complexes are very rare diseases and may be only a model of more large diffuse arthritic diseases. Anyway considering the genetic selectivity pressure involving the genes areas mutated in these subjects, we can suppose that the selectivity pressure has favored the selection of heterozygous people. People affected are from Mediterranean ancestral origin such as Sepharditic Jwes, Armenian, Turkish, Greek, Italian people and these diseases affect humans during early year of life. We can suppose that people heterozygous for such mutations react with a very intense systemic way leading to great survival chances.

The molecular pathway involves the cleavage of precursor of IL-1 beta before its secretion affecting a group of proteins showing the same molecular structure now called NALPs proteins able to activate the caspase-1 complex involved in degradation of pro-IL1 ( pyrin ) such in the case of Familial Mediterranean Fever.

The defect of mevalonate kinase involved into the genesis of hyperglobulinemia D is involved in an increased secretion of IL-1 beta that is linked to the production of isopreoid molecules, normally linking IL-1 to the plasmamembrane.

The defect of TNF receptor type 2 lead to an increase stimulation of TNF alfa, due to defect in cleavage site of this receptor normally acting such as scavenger receptor for circulating TNF alfa, such as the case of so called Familial Hibernian fever. The autosomal dominant inheritance of this syndrome has been reported in many ethnic groups but in particular in Irish and Scottish family. So far more than 20 families have been described in Australia, United States and Europe too. Linkage analysis mapped the susceptibility gene for two separate families to the short arm of chromosome 12. Identifying several missense mutations, at least 16, in the gene for the type 1 TNF receptor in the exons 2,3 and 4 of genomic sequence has lead to hypothesis that receptor activation usually lead to cleavage and shedding of its intracellular portion into the circulation, where it acts as an inhibitor of TNF alpha. Activation of receptor can leads to activation of a protease that shed the TNF receptor from cell surface. It is postulated that in affected patients the TNF receptor cannot be shed by proteases leading to persistent inflammatory response.

Familial meditterranean fever is defined such as disease linked to an alteration on TNFalfa Receptor but due to a pyrin defect. The prolonged attacks, conjunctivitis, and localized myalgias differentiated the TNF-receptor associated periodic fever from other syndromic periodic fever syndromes.

Finally cryopyrin defect is associated at different level with the last three syndromic complexes including neonatal-onset multisystem inflammatory diseases.

Cryopyrin belong to NALP superfamily, and is able to activate caspase system at N terminal site, to link to nucletide sequence in its central portion (Leucine rich repeat), and at C terminal site it shows many similaryties with Toll like receptor family able to link to many bacterial and host molecules. Finally a particular attention deserve the presence in these hereditary periodic fever syndromes of amyloidosis. It’s interesting to note that many bone diseases are characterized by the presence in abnormal level of amyloidotic structures in extracellular space. In particular it would be interesting to evaluate the different arhtropatyes affecting adults for the presence of amyloidotic proteins in synovial flluids or anyway in extracellular degraded matrix. In considerations of recent evidences of involvement of matrix metalloproteinases and aggrecanases gene mutations in diseases affecting cartilagineous structures, it would be intersting to evaluate the hypothesis of an increased production and secretion at kidney level of amyloid like proteins. The pathways of production of amyloid include the gamma secretase enzymes involvement into degradations of HLA linked moleculed in particular of beta2 microglobulin. It has been suggested that after the apototitc processes due to same mechanisms involving the increased secretion of IL-1 beta would produces increased amount of plasmamebrane linked proteins containing strucural motif able to form agregates of fibrils in extracellualr matrix that can only be secreted by kidney with possible damage of renal filtration at glomerular level. Interestingly in the disease affecting older people the levels of amyloid like structures are increased for example at level of cerebral tissue in degenerativve diseases such as Alzheimer’s diseases. The same mechanism would be present also at bone tissue livel in particular at articular level. It si interesting to note that amyloidosis is preset in Armenian people living in Armenia wheres it is absent in Armenian living elsewere; it is possible that the presence of only one allele of this gene responsible of autosomal recessive diseases, previously described, account for an increased production of amyloid proteins, production that require also a predisposition of SAA1 genotype in the amyloid precursors.

Arthritic diseases and malignancies

Virchow suggested in the nineteenth century that chronic inflammation might give rise to malignancy, and the link between inflammation and cancer was not widely inderstood until recently. Clinical evidences of this link is demonstrated by the relationships between chronic infections with hepatitis B virus (HBV) and hepatitis C virus with hepatocellular carcinoma; infections with Helicobacter pylori and association with most gastric cancers; chronic inflammatory bowel diseases, such as Ulceratice colitis, and colorectal cancer; chronic airway irritation and inflammation caused by airborne partocles and tobacco smoke and lung carcinomas. Thus epidemiological studies were an excellent source of new working hypothetis concerning the pathogenesis of cancer. Hanahan and Weinberg summarized the processes underlying the emergence of neoplasia with the presence of self sufficiency in growth signals, insensitivity to growth inhibition signals, evasion from apoptosis, limitless replicative potential, tissue invasion and sustained angiogenesis.

Osteitis Deformans or Paget’s Disease of bone

Paget’s disease of bone is also called Osteitis Deformans and it is a more common metabolic disorder resulting from rapid bone remodelling. Interestingly on 1% of affected patients an osteosarcoma, fibrosarcomas or chondrosarcomas are present ; the more common site sarcoma transformed is the femur; higher risk patients are those with polyostotic form of the disease, and it has been linked to the presence of a single gene mutation tightly located on chromosome 18. So that Osteitis Deformans can be considered a precancerous lesion of bone.

Cardiovascular system is affected in polyostotic form with an increase in cardiac output during the active resorbing phase; cardiac failure is rarely present. The increase vascular flow rate is the first sing of Paget disease of bone when it stops after a correct antiresorptive therapy.

The hematic flow increase on skull bone can lead to a cerebral ischaemic attacks like those we see during vertebrobasilar insufficiency.

 

Alkaline phoshatase linked diseases

Inherited diseases are known to be associated with an alteration on ALP secretion:

. Hypophosphatasia: low levels of NTS-ALP due to chromosomal alteration on chromosome 1.

. Aphosphatasia: heritable deficit of NTS-ALP

. Inherited Hyperphosphatasia (Paget disease of bone)

Aphosphatasia

Is an heritable deficit of NTS-ALP where we have a typical biochemical characterization due to increased levels of:

- Phosphoetanolamine

- Pyridoxate phosphate (Vit. B6)

- Pyrophosphates

All molecules targets of ALP activity under normal conditions.

Recently, after the discovery of Osteoprotegerin as a “uncoupling factor” able to modulate bone remodelling, many studies have been devoted to unravel the gene location and transcription control of Osteoprotegerin-coding genes.

At present two chromosomal loci have been identified coding for two different kind of OPG-like proteins:

1. TNFRSFIIA on chromosome 18q21-22

2. TNFRSFIIB on chromosome 8q24.2

These gene locations have been identified thanks to the study of rare inborn errors of bone metabolism by White’s MP group, and in particular of

- Familial Expansile Osteolysis – MIM174810 - TNFRSFIIA

- Expansile skeletal Hyperphosphatasias – MIM 239000 - TNFRSFIIB

- Early onset Paget’s disease of bone in Japan

- Idiopathic Hyperphosphatasia (Juvenile Paget’s disease) MIM 239000

OPG, a soluble member of the superfamily of TNF receptors, is normally secreted in vivo into the marrow space by cells derived from embrional mesenchimal tissue. Physiological actions of OPG is to function as a decoy receptor free into extracellular space, necessary and sufficient for osteoclasts development.

KO mice for TNFRSFIIB develop osteoporosis with numerous osteoclasts and rapid remodelling bone tissue.

OPG serum levels have been described to be undetectable in patients affected by Hereditary Hyperphosphatasia (Juvenile Paget’s disease) according to the model described above.

TNFRSF11B: Osteoprotegerin (Chr. 8q24.2 ) mutations and osteoporosis

Interestingly, some recent data demonstrated the presence of a genetic polymorphism in the codon 3 at exon 1 of gene coding for OPG, causing an aminoacid substitution from lysine to asparagine at position 3. This gene mutation is presented in osteoporotic fractured older women. Several studies have reported an association between the Asn (C) allele presenting the mutation of Lys3Asn and increased lumbar spine BMD, as well’s lower risk of osteoporosis and osteopenia, with a reduced fracture risk.

Also the Study of Osteoporotic Fractures Research Group (SOF Research) studying 6695 women aged 65 years and older confirmed the previous studies demonstrating an association between high BMD level at diffeent skeletal sites and Ans3Asn (C/C) genotype. In particular C/C genotype is associated in SOF study women with high BMD values at calcaneal level, distal radious, intertrochanter, and lumbar spine. However it is important to note that also fracture risk was strictly related to OPG polymorphism. Interestingly, many of skeletal sites examined showed an association only with high BMD level but not with fracture risk, whereas the largest increase in fracture risk was seen at femural neck level, where there was no significant association with BMD values.

Studies evaluating serum concentration levels of OPG with BMD or fracture risk in humans have yielded conflicting results, partly because serum concentration levels of OPG, don’t reflect the OPG biologic activity within the bone microenvironment.

Genetically determined differences in OPG expression or function may be more reliable indicator of long term OPG activity. This possibility is supported by the association between the OPG Lys3Asn missense polymorphism and the risk of fracture over an average of 13.6 years of follow up in the SOF Research Group. Interestingly, the Lys3 Asn mutation occurs in a potential exonic splicing enhancer site. Changes in these sequences can have functional effect on the protein biologic activity. These findings for OPG Lys 3 Asn polymorphism are consistent with the common disease, common variant model inwhich high frequency alleles may contributes a modest relative risk but an appreciable proportion of disease burden in the population.

Aseptic losses of periprostetic bone

Inflammation reaction is strictly related to production and activation of membrane linked lipids mainly derivatives of arachidonic acid able to be transformed into their active metabolites: prostaglandins, leukotrienes, lipoxins and forming sfingolipids.

Between prostaglandins the main metabolic actions are exerted by PGE2 those receptors have been found in humans to be of 4 types EP1,2,3,and 4.

EP1 is linked to IP3 signaling and PLC with subsequent mobilization of intracellular calcium stores.

EP2 and EP4 stimulate formation of cAMP at intracellular level

EP3 activating Gi a G protein able to inhibits the enzyme adenyl cyclase.

Many data seems to demonstrate that the receptor responsible for induction of RANK-L expression by bone cells is represented by EP4.

PGE2 induction by RANKL seems to be mediated by EP4 receptor, so that double KO EP4 mice show few osteoblasts and showed a dramatic reduction in RANKL expression by osteoblastic cells.

Periprostetic osteolysis is a serious orthopedic problem often present at prosthesis / bone interface linked to activation of inflammatory prostaglandins: it is called “Aseptic Loosening of bone”. It leads to great limitation of many total joint replacement surgical interventions.

Wear debris production at tissue implant interfacce stimulates the activation of osteoclasts through RANKL secretion. Possible mediators of osteolytic action of wear debris are believed to be PGE2.

Interestingly sporadic reports of Bone Phenotype in “Darier Disease” suggested an involvment of SERCA 2b channels.

SERCA 2 b heterozygous mice -/+ showed a reduced frequency of calcium oscillation in bone cell membrane linked to loss of NFATc1 expression and loss of osteoclasts. SERCA 2b channels are present at cellular level at sarco-endoplasmic reticulum surface and they function such as Calcium ATPase type 2 channels involved in processes of osteoclasts differentiation.

These heterozygous mice showed a phenotype very similar to human bone phenotype of Darier disease showing bone tissue affected by bone cyst and fractures as well’s by periodontal gingival and mucosal inflammation.

 

References

Gafni J, Ravid M, Sohar E. The role of amyloidosis in familial Mediterranean Fever: a population study. Isr J Med Sci 1968;4:995-9.

Zemer D, Revach M, Pras M et al. A controlled trial of colchicine in preventing attacks of familial Mediterranean Fever. N Engl J Med 1974;291:932-4.

Dinarello CA, Wolff SM, Goldfinger SE et al. Colchicine therapy for familial Mediterranean Fever: double blind trial. N Engl J Med 1974;291:934-7.

Keat A. Reiter’s syndrome and reactive arthritis in perspective. N Engl J Med 1983;309:1606-15.

Goldenberg DL, Reed JI. Bacterial arthritis. N Engl J Med 1985;321:764-71.

Hoffmann G, Gibson KM, Brandt IK et al. Mevalonic aciduria – an inborn error of cholesterol and nonsterol isoprene biosynthesis. N Engl J Med 1986;314:1610-4.

Beutler B, Cerami A. Cachectin: more than a tumor necrosis factor. N Engl J Med 1987;316:379-85.

Steere AC. Lyme disease. N Engl J Med 1989;321:586-96.

Cover TL, Aber RC. Yersinia enterocolitica. N Engl J Med 1989;321:16-24.

Bisno AL, Group A streptococcal infections and acute rheumatic fever. N Engl J Med 1991;325:783-93.

Pras F, Aksentijevich I, Gruberg I et al. Mapping of a gene causing familial Mediterranean fever to the short arm of chromosome 16. N Engl J Med 1992;326:1509-13.

Pinals RS. Polyarthritis and fever. N Engl J Med 1994;330:769-74.

Bazzoni F, Beutler B. The tumor necrosis factor ligand and receptor families. N Engl J Med 1996;334:1717-25.

Case Records of the Massachussetts General Hospital (Case 25-1999). N Engl J Med 1999;341:593-9.

Drewe E, McDermott EM, Powell RJ. Treatment of nephrotic syndrome with etanercept in patients with the tumor necrosis factor receptor-associated periodic syndrome. N Engl J Med 2000;343:1044-5.

Drenth JP, van der Meer JWM. Hereditary periodic fever. N Engl J Med 2001;345:1748-57.

Hawkins PN, Lachmann HJ, McDermott MF. Interleukin-1 receptor antagonist in the Muckle-Wells syndrome. N Engl J Med 2003;248:2583-4.

Cundy T, Davidson J, Rutland MD et al. Recombinant osteoprotegerin for Juvenile Paget’s Disease. N Engl J Med 2005;353:918-23.

Deftos LJ. Treatment of Paget disease – Taming the wild osteoclast. N Engl J Med 2005;353:872-75.

Reid IR, Miller P, Lyles K et al. Comparison of a single infusion zolendronic acid with risendronate for Paget’s Disease. N Engl J Med 2005;353:898-908.

Kim K, Fisher MI, Xu SQ et al. Molecular determinants of esponse to TRAIL: in killing normal and cancer cells. Clin Cancer Res 2000;6:335-346.

Hugher EA, Ralston SH, Whyte MP et al. Mutations in TNFRSFIIA, affecting the signal peptide of RANK, cause Familial Expansile Osteolysis. Nat. Genet 2000;24:45-8.

White MP, Hughes AE. Expansile skeletal hyperphosphatasia is caused by a 15 base pair tandem duplication in TNFRSFIIA encoding RANK and is allelic to Familial Expansile Osteolysis. J Bone Min Res 2002;17:26-9.

White MP, Obrecht SE, Finnegan PM et al. Osteoprotegerin deficiency and Juvenile Paget’s disease. N Engl J Med 2002;347:175-84.

Langdahl BL, Carstens M, Stenjaer L et al. Polymorphisms in the osteoprotegerin gene are associated with osteoporotic fractures. J Bone Miner Res 2002;17:1245-55.

Arko B, Prezelj J, Kocijancic A et al. Association of the osteoprotegerin gene polymorphisms with bone mineral density in postmenopausal women. Maturitas 2005;51:270-9.

Zhao HY, Liu JM, Ning G et al. The influence of Lys3Asn polymorphism in the osteoprotegerin gene on bone mineral density in Chinese postmenopausal women. Osteoporosis Int. 2005;16:1519-24.

Wynne F, Drummond F, O’Sullivan K et al. Investigation of the genetic influence of the OPG, VDR (Fok1), and COL1A1 Sp1 polymorphisms on BMD in the Irish population. Calcif Tissue Int 2002;71:26-35.

Vidal C, Brincat M, Xuereb Anastasi A. TNFRSF11B gene variants and bone mineral density in postmenopausal women in Malta. Maturitas 2006;53:386-95.

Moffett SP, Oakley JI, Cauley JA et al. Osteoprotegerin Lys3Asn polymorphism and the risk of fracture in older women. J Clin Endocrinol Metab 2008;93:2002-8.

Samelson EJ, Broe KE, Demisse S et al. Increased plasma protegerin concentrations are associated with indices og bone strength of the hip. I Clin Endocrinol Metabol 2008;93:1789-95.

Darier disease and periprostetic aseptic bone losses:

Kariya Y, Homma M, Aoki S et al. Vps33a mediates RANKL storage in secretory lysosomes in osteoblastic cells. J Bone Miner Res 2009;24:1741-52.

Tsutsumi R, Xie C, Wei X et al. PGE2 signaling through the EP4 Receptor on fibroblasts upregulates RANKL and stimulates osteolysis. J Bone Miner Res 2009;24:1753-62.

Yang Y-M,Kim MS, Son A et al. Alteration of RANK-L induced osteoclastogenesis in primary cultured osteoclasts from SRCA2-/+ mice. J Bone Mineral Res 2009;24:1763-9.

Frezzini C, Cedro M, Leao JC et al. Darier disease affecting the gingival and oral mucosal surfaces. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006;102:e29-e33.

Menne T, Nielsen AO. Bone cysts and spontaneous fractures in two siblings with dyskeratosis follicularis Darier. Acta Derm Venereol 1978;58:366-7.

Ahn W, Lee MG, Kim KH et al. Multiple effects of SERCA2b mutations associated with Darier’s disease, J Biol Chem 2003;278:20795-801.

Sheridan AT, Hollowood K, Sakuntabhai A et al. Expression of sarco/endoplasmic reticulum Ca2 ATP ase type 2 isoforms (SERCA2) in normal human skin and mucosa , and in Darier’s disease skin. Br J Dermatol 2002;147:670-4.

Identification of vertebral fractures

 

    

        

                                                                                         

 

 Placebo effect has been know from time of Plato cited by Socrates about the cure of headache “ was a kind of leaf, which required to be accompanied by a charm, and if a person would repeat the charm at the same time that he used the cure, he would be made whole; but that without the charm leaf would be of no avail.” However a therapy to be true placebo, ethically speaking, must be safe to use, without any adverse effects and possible less invasive we can. That’s a problem of some “minimally invasive” techniques that in the developed countries during the last decade are increasing exponentially. Future of surgeons would be “endoscopically and fluoroscopically” followed tecniques, allowing shorter hospitalization times, prompt delivery and fast recovery of patients. Radiologists, if not involved into Nuclear Medicine, are better called Interventional Radiologists, a way to identify a Medical Doctor directly involved into the therapeutic measures adopted for increasingly diseases. The old radiologists, reading and comparing X-ray exams, are disappearing and substituted by Neuroradiologist. The problems is a correct use and approach to new available tecniques, too fast approaching on the free market without the due time required to completely understand their potential utility-damage ratio.

A clear example of a wrong use of these tecniques is clearly demonstrated by two article published on The Journal on the isssue of August 6, 2009 concerning vertebroplasty use in osteoporotic patients. If we don’t know the pathophysiology of osteoporotic verbral fractures, but also only what is osteoporosis, clearly we could be charmed and easly attracted by these technologically revolutionary tecniques.

Under fluoroscopic guidance, the neuroradiologist first infiltrated the skin underlying pedicles of fracture site with a 25 Gauge needle with 1 % lidocaine reacing the subcutaneous tissue. Sorthly after, using a 23 Gauge needle with 0.25% of bupivacaine infiltrated the periostium of the posterior verterbral lamina. At this point aan incision is made on the skin, and a 11-13 Gauge needle is placed postero-laterally relative to the eye of vertebral pedicle. Gently the operator mouves the needle through the pedicle into the anterior two third of the fractured vertebral body following the needle progression with fluoroscopic images antero-posteriorly and laterally. Barium opacified Polymethylmetacrylate (PMMA) is now infused under fluoroscopic lateral control. A Unipedicular approach can be used infusing a total of approximately 3 ml of PMMA injected directly into a single side. However a Bipedicular approach can be used if there is inadequate instillation of cement into vertebral body. The PMMA is stopped when a substantial resistance is met or when the PMMA reached the posterior quarter of vertebral body or if the PMMA leaked into extraosseous structures or veins. After the vertebroplasty the patients are followed in the supine position for 1 to 2 hours before discarge administering intravenously immediately after PMMA injection a cephalotin antibiotic treatment.

In the two studies cited above the Authors demonstrated quite inequivocably that vertebroplasty is equal to placebo treatment in back pain relief particularly in short term. The anesthetic effect is probably important in explaining the shorter pain relief and easy manipulation by operators of vertebral bodies; however it cannot explaine the beneficial effect the Authors founded also after 4 or 6 weeks in most patients.

As the Editor outlines, President Barack Obama recently called for more comparative-effectiveness research in order to establish true safety and effectiveness of a given treatment compared to other one as part of American Recovery and Reinvestement Act (ARRA). People of developed countries probably pay the prize of great advances in technologies, so that in great medical challenges, such as acute pain of vertebral fracture, informed patient take also an less invasive choiche, as vertebroplasty, in order to have best and faster pain relief. However we have to pay attention to correctly inform the patients in order to obtain a true informed choiche, and the demonstration of equal affectiveness of placebo anestetic injection and PMMA vertebral reconstruction help us in doing that.

 

Also the presence of intravertebral edema during MRI, used in the studies under discussion, is not universally accepted target of vertebral osteoporotic fracture. Such as CT scan, also MRI, are usually only required in the presence of localized pain, focal neurological signs, or symptoms suggesting cord compression, disc erniation, a radiculopathy, or the clinical suspicion of primary or metastatic lesions, but not in osteoporotic patients.

What is the correct approach to a vertebral fracture?

 

The problem is besides all the definition of vertebral fracture, at present there’s no Consensus Giudelines on osteoporotic vertebral fracture definition. Identification of vertebral fracture can be very difficult, because the shape of normal vertebral bodies varies widely between individuals. Vertebral bodies can be present abnormal features because non-osteoporotic deformities and errors in radiological projection can induce a misdiagnosis of fractured body. We have to remember that about 50% of vertebral fractures are asymptomatic and therefore are only casually identified. They are not the source of pain !Even when chest radiographs or vertebral images are correctly obtained only 35 to 50% of all radiographic vertebral fractures are correctly reported. It has been estimated that only 19% of these fractures reach clinical attention and can be correctly treated with a antiosteoporotic treatment. In view of high radiation exposure routine chest and lumbar radiographs are not recommended, but the availability of vertebral imaging using DEXA take the advantage of utilize an image of near radiographic quality available with a low fraction of the radiation dose.

Imaging vertebral fractures using DXA is called Vertebral Fracture Assessment (VFA).

The disadvantage of VFA is poor image resolution compared to conventional radiography, CT or MRI and the increased difficulties in imaging the thoracic spine, expecially above T7. Between 5 to 15% of thoracic vertebrae can be visualized only by conventioonal radiography.The sensitivity and specicificty of this approach compared with conventional radiography varies with the kind of approach used to definy a vertebral fracture:

- Morphometric

- Semiquantitative (SQ)

- Visual identification

One advantage of DXA imaging is that the scan are not subjected to the same degree of projection distortion as conventional radiography because the X-ray beam is always orthogonal to the spine. Reducing the X ray diffraction effect. Moreover DXA reduces the frequency that soft tissue obscure the endplates compared to single energy mode. Side-by-side viewing facilitates the identification of incidental vertebral fractures.

Morphometrical analysis

It uses the measurement of vertebral height to define vertebral fractures. A normative daatabase is established against which the vertebrae are compared, There are a number of different morphometric approaches that vary with the criteria by which they define a vertebral fracture and in the reference data used, The most widely used approaches to identify prevalent and incidental vertebral fractures are the two different algorithms proposed by McCloskey et al (3) and Eastell R et al (4). Morphometric analysis has a high sensitivity and moderate high specificity in discriminating between normal vertebrae and fractured vertebrae. Moreover, all the morphometric approaches for defining prevalent and incident vertebral fractures are correlated with clinical risk factors for vertebral fractures. A loss of vertebral height of 20 to 25 % is usually used to define an incident vertebral fracture; using this definition comparable ability to identify any vertebral frcture is present irrespective of aproach used to define a baseline fracture. As mentioned above VFA is more effective in identifying moderate to severe deformities with a sensitivity of 81.6% for grade 2 deformities, whereas mild grade 1 deformities identifiation has a sensitivity as low as 22%. Finally , the precision error is small if compared with the reduction in vertebral height of 20 to 25% threshold used to define vertebral fractures and it is less using conventional radiology than using VFA.

Semiquantitative analysis

SQ analysis combines measurements of vertebral height with subsequent evaluation of all vertebrae with a short vertebral height by an expert reader. This combined approach enables the identification of non-osteoporotic fracture vertebral deformities, which are not identified using morphometric analysis alone. As a consequence, SQ analysis is able to reduces false positive results.The most widely used SQ analysis is that of Genant HK (6). Baseline or prevalent vertebral fractures are graded from “0” equal to normal to “3” equal to severe fracture, and incident fractures are defined as an increase of more than or equal than 1 grade on follow-up radiographs.

Genant grade 1 corresponds to an 20 to 25% reduction in anterior, middle or posterior height

Genant grade 2 corresponds to a 25 to 40% reduction in any height

Genant grade 3 corresponds to more than 40% reduction in any vertebral height

Mild grade 1 SQ vertebral deformities are frequently not associated with low BMD values.The interobserver agreement for conventional radiographs or DXA images is similar with a K score of 0.53 and 0.51 respectively.This approach is currently those recommended by International Society of Clinical Densitometry for diagnosing vertebral fractures with VFA.

Algorithm Based Qualitative Approach

ABQ approach differs from SQ analysis because the last one is based only on variations of vertebral height; not considering variations on endplates cracks or breaks as the primary event with a subsequent evaluation of vertebral height. ABQ focus more attention on the vertebral endplate alterations rather than on short vertebral height. Using ABQ we have a greater association with low BMD and interobserver agreement for radiography and DXA images of 0.74 and 0.65 respectively. So that mild vertebral fractures identified with ABQ are more strngly associated with osteoporosis than when this mild fractures are identified with SQ method. The definition of vertebral fractures includes the presence of breaks in the cortex of vertebral body; these breaks always occurs in the center or either the superior or inferior endplates that are the weakest area of endplate because it is more distant from the strong outer vertebral ring. As a consequence, the endplate buckles or collapses under pressure because of interventebral disc and it results in a concave appearance to the superior and/or inferior endplate. If the concavity extend beyond the inner border of the vertebral ring , it is unlikely to represent an osteoporotic fracture. A vertebral fracture initially involves a crack of the superior or inferior endplate with or without the simultaneous loss of vertebral height. As severity of the fracture progresses, the vertebral ring fractures resulting in loss of height and buckling of the anterior, lateral and occasionally posterior cortex. It is important to outlined these aspects because there is considerable variation in vertebral shape resulting in osteoporotic and non osteoporotic deformities that can result in considerable intraobserver error even among expert readers.

Commonly we can see wedge deformity fracture associated with endplate fracture where is present a fracture of the anterior cortex of vertebral body.

A true compression fracture associated with endplate fracture is an osteoporotic compression fracture of superior endplate associated with fracture of anterior and posterior cortex of vertebral body.

Clinical recommendations for screening for vertebral fractures

The current recommendations for using fracture assessment through DXA imaging (VFA) by the International Society of Clinical Densitometry are:

1. When the results may influence clinical management
2. If BMD is indicated then consider performing VTA if clinically indicated in:

- Documented height loss greater than 2 cm

- Historical height loss greater than 4 cm since young adult

- History of fracture after 50 years old

- Commitment to long term oral or parental glucocorticoid therapy

- History or findings suggestive of vertebral fracture not     documented by previous radiographic imaging

Therefore, it is reasonable to screen all patients with osteopenia using VFA, if it will alter the management of the patient. In a study at Mayo Clinic 16% of patients 60 to 69 years old and 45% of those older than 70 years had a previously undiagnosed vertebral fracture on VFA.

References

Kallmes DF, Comstock BA, Heagerty PJ et al. A randomized trial of vertebroplasty for osteoporotic spinal fractures. N Engl J Med 2009;361:569-79.

Buchbinder R, Osborne RH, Ebeling PR et al. A randomized trial of vertebroplasty for painful osteoporotic vertebral fractures. N Engl J Med 2009;361;557-68.

Weinstein JN. Balancing science and informed choice in decision about vertebroplasty. N Engl J med 2009;361:619-21.

McCloskey EV, Spector TD, Eyres KS et al. The assessment of vertebral deformity: A method for use in population studies and clinical trials. Osteoporosis Int 1993;3:138-47.

Eastell R, Cedel SL, Wahner HW et al. Classification of vertebral fractures. J Bone Miner Res 1991;6:207-15.

Genant HK, Jergas M, Palermo L et al. Comparison of semiquantitative visual and quantitative morphometric assessment of prevalent and incident vertebral fractures in osteopororsis. The Study of Osteoporotic Fractures Research Group. J Bone Miner Res 1996;11:984-96.

Rea JA, Li J, Blake GM et al. Visual assessment of vertebral deformity by X-ray absorptiometry: A highly predictive method to exclude vertebral deformity. Osteoporosis Int 2000;11:660-8.

Siminoski K, Jiang G, Adachi JD et al. Accuracy of height loss during prospective monitoring for detection of incident vertebral fractures. Osteoporosis Int 2005;16:403-10.

Siminoski K, Warshawski RS, Jen H et al. The acuracy of historical height loss for the detection of vertebral fractures in postmenopausal women. Osteoporosis Int 2006;17:290-6.

Schousboe JT, Ensrud KE, Nyman JA et al. Cost-effectiveness of vertebral fracture assessment to detect prevalent vertebral deformity and select postmenopausal women with a femoral neck T-score lower than 2.5 SD for alendronate therapy: A modeling Study. J Clin Densitom 2006;9:133-43.

Collagenopathies and Marfan's Syndrome.

 

Here you can found some one-line material concerning my letter to New England about the Dietz's article on Marfan's sindrome and losartan therapy, published on the June 26, 2008 issue of The Journal. As usually all writed material is based on personal reading of scientific articles and possible are apparently unrelated each other. However they represent the course of my scientific actual knowledge concerning collagenopathies and TGFbeta related disease starting from Marfan's syndrome study.

Since more than a century ago the first professor in pediatrics in Paris, Antoine Marfan, described a young girl with long, spider-like fingers and other curious skeletal anomalies, understanding of the syndrome that now bears his name.

The Marfan syndrome is an inherited disorder of connective tissue characterized by pleiotropic manifestations of many organs, including the eyes, heart, aorta, skeleton, skin, and lung.

The cardinal ocular manifestation, ectopia lentis, was not recognized as being associated with the skeletal changes for some decades.

The cardiovascular system was found to be involved at about the same time, when severe mitral regurgitation was observed; until 1943 was identified the involvement of aortic root, and it was remained for McKusick to show in 1955 that the disease of the aorta accounted for most deaths. Life expectancy is reduced by one thrid, on average because of emergent cardiovascular complications.

By the 1930s, it was recognized that the disease was transmitted by mendelian dominant phenotype; not until 1949 the study of large pedigrees convinced skeptics that the condition was due to a single mutant gene, which needed to be present in only one copy (heterozygosity) to cause the disease.

Half a century was required to to identify the effect of the mutation of the classic triad of Marfan disease:

1. ectopia lentis
2. cardiovascular disease: aortic aneurism and mitral valve prolapse
3. skeletal disease: joint laxity and bone overgrowth

Microfibrillar fibers make up a discrete, widely distributed, and pleiomorphic fiber system in human tissues. When visualized by electron microscopy, the fibers apper as linear bundles containing many individual microfibrils with a tubular cross-section and an average diameter of 10 to 12 nm, showing characteristic like collagen fibers type 3.

Microfibrillar fibers are considered integral components of elastic elements, but such fibers are much more widely distributed than elastin. They have been visualized by immunolocalization studies in skin, tendon, cartilage, muscle, kidney, perichondrium, periosteum, blood vessels, pleura, dura mater, and ciliary zonules of the ocular lens.

In particular McKusick has suggested that understanding the common factor in aortic media lamellar structure and the ciliary zonules causing “ectopia lentis” often present in Marfan syndrome may reveal the basic defect of this disease.

The consistent finding of stretched and occasionally broken zonular fibers in the ectopia lentis of patients with Marfan syndrome argues that these microfibrillar fibers are functionally incompetent to resist to normal stress and elongate progressively over time.

The progressive dilatation of aortic root with the fragmentation of elastic lamellae of the tunica media, the striae atrophicae in the skin, pulmonary bullae, dural ectasia as well’s seletal overgrowth can be linked to functional alteration of the lamellar structure. In particual skeltal overgrowth may be linked to diminished forces generated by periosteal and perochondrial membranes that oppose bone growth.

The absence of reticular meshworks on epidermal sections and in dermal fibroblasts culture of patients affected by Marfan syndrome shows that an alterations is present in these extracellular structures.

However other common clinical pictures can present the same alterations in fiber disposition such as in patients affected by:

- Homocystinuria: due to cystationina beta synthse defect

- Ectodermal dysplasia

- Epidermolysis bullosa-like syndrome

- Coronary artery dissection

- Paudoxantoma elasticum

- Cutis laxa: due to elastin gene mutation

The first major advance came in 1991 when missense mutations in the fibrillin-1 gene (FBN1) were discovered in two unrelated patients with the syndrome by Dietz HC et collegues. Three works on the same issue of Nature outlined the presence of such a mutation as a cause of syndromic complex. These findings were the culminations of biochemical studies that identified “fibrillin” as an extracellular matrix component and specifically such as the principal component of microfibrils associated with elastin fibers.

Whereas the work of Hollister DW in 1990 demonstrated the fibrillin deficiency in patients affected by Marfan syndrome by immunohystochemical studies, on 1994 Shores demonstrated with genetic investigations that the region of chromosome 15 was linked to marfanoid syndrome and it was shown to contain the gene coding for fibrillin.

The gene FBN1 is located on chromosome 15 q21.1, codes for fibrillin, the 350 kd protein that is the main component of extracellular microfibrils. The gene was demonstrated to be affected by mutations that resulted in a spectrum of connective tissue disorders, including but not limited to Marfan syndrome, involving structural fibrillin protein domains distributed uniformly over 10 kb FBN1 DNA sequence. So that such as in neurofibromatosis type 1 we have a relatively common dominant disorder with a high rate of new mutations, so that no easy screening test is possible. So that the demonstration of FBN1 mutation or abnormal fibrillin metabolism don’t permit or confirm the diagnosis of Marfan syndrome.

It has been estimated that the frequecy of fibrillin disorders is considerably greater than the estimated incidence of Marfan syndrome of 1 in 10.000 subjects.

Concerning Marfan syndrome four distinct phenotypically groups have been defined:

- alterations of FBN1 gene disrupting the second disulphidrile bridge in 1 of the 44 domains containing six cysteine residues shown to be related to reduced secretion of fibrillin

- nonsense mutations leading to premature termination of polypeptide synthesis shown to be related to a sinthesis of half the normal amount of fibrillin

- rapidly progressive with aortic dilatation, severe scoliosis, ectopia lentis, variable skeletal abnormalities and negligible cardiac involvement or with mitral valve prolapse with skeltal features (with absent fibrillin)

- adult type with dominant form of slowly progressive aortic aneurism without typical ocular and skeletal findings (with locally absent fibrillin)

The basic paradigm of Marfanoid habitus is that fibrillin gene mutations resulted in the production of abnormal fibrillin protein that, when incorporated into microfibrils along with normal fibrillin, resulted in structurally inferior connective tissue. This adverse effect of mutant proteins on normal ones, which genetists term “dominant negative”, appeared to explain many of the cardianal feaures of Marfan syndrome.

This explanantion was reinforced by the contemporaneous discovery of a second fibrillin gene, FNB2 located on chromososme 5, which is associated with a related connective tissue disorder:

. congenital contractural arachnodactyly (Beals’ syndrome)

On late ’90 some researchers developed imbred animal models for Marfan syndrome study; the introduction of mutations into the mouse fibrillin-1 gene, FBN1, recapitulated the disorder. Interestingly lungs were more affected in mice bearing the fibrillin gene mutations showing emphysematous changes in alveolar tissue.

However Dietz HC showed that, instead of damages due to increasing breakdown due to repeated stretching of connective tissue, affected lungs presented an abnormal septation of the distal alveoli in newborn mice pups; a finding more consistent with a developmental defects than a decreased elasticyty.

On the same year, on 2003, it was demonstrated that fibrillin has an homologous structure with Latent TGF beta binding-protein (LTBPs), which serve to hold TGF beta in an inactive complex in various tissues in extracellular matrix. It was showed tha fibrillin can bind TGF beta and LTBP. Dietz HC group hypothesized that abnormal fibrillin, or reduced levels of fibrillin, in connective tissue might result in an excess of active TGF beta. They demonstrated that blocking TGF beta with neutralizing antibodies, inbred mice strains showed a normalization of lung development.

In 2005 Loeys and Dietz showed that some patients can be classified such a separated clinical entity overlapping marfan syndrome, presenting:

- aortic aneurysm

- arachnodactyly

- dural ectasia

this syndromic complex is now called Loeys-Dietz syndrome and it was due to mutations affecting genes coding for TGF beta receptor type 1 and TGF beta receptor type 2 (TGFBR1 and FGFBR2); one affected patient was found to have an increase TGF beta activity in the aortic tissue.

Interestingly studying a large cohort of patients with TGFBR1 and TGFBR2 mutations, it was demonstrated that some had the classic Loeys-Dietz syndrome with better outcome after aortic surgery, whereas others resembling patients with Ehlers-Danlos syndrome known to be linked to defects in collagen type III gene.

Taken together, the genetic findings from these studies help us to describe a group of connective tissue diseases due to inhborn error of extracellular matrix proteins with a larger incidence on populations than previously known, we can call “fibrillinopathies”.

Inhborn error of genes coding for different types of collagens give rise to “collagenopaties”:

- Osteogenesis Imperfecta: due to collagen type 1 defect

- Spondyloepyphiseo dysplasia: due to collagen type 2 defect

- Ehlers-Danlos disease: due to collagen type 3 or type 5 defect

- Mutiple Epiphyseal Dysplasia: due to collagen 9 defect

- Methapyseal Dysplasia Schmid type: due to collagen type 10 defect

- Marshall syndrome: due to collagen type 11 defect

Its’ becoming clear that collagenopathies and fibrillinopathies show sometimes the same clinical picture in affected patients; and the more common manifestation of more subtle biochemical pathways involved into signal transduction share same proteins present on extracellular matrix space for their final actions.

It’s also likely that understanding the role of these proteins present in large amount on extracellular space may explain the physical properties of tissues such as the skin and bone where connective tissue components play a relevant role.

In particular in view of recent result on marphanoid habits, involving elastin associated microfibrils in TGF beta signal transmission, it is easy to imagine a role of Bone Morphogenetic Proteins action also on mechanical transduction of physical forces applied on bone and on regulation of bone stiffness.

References

Peyritz RE, McKusick VA. The Marfan syndrome: diagnosis and management. N Engl J Med 1979;300:772-7.

Pyeritz RE, McKusick VA. Basic defects in the Marfan syndrome. N Engl J Med 1981;305:1011-2.

Boucek RJ, Noble NL, Gunja-Smith Z et al. The Marfan syndrome: a deficiency in chemically stable collagen-cross-links. N Engl J Med 1981;305:988-91.

Gott VL, Pyeritz RE, Magovern GJ Jr et al. Surgical treatment of aneurysms of the ascending aorta in the Marfan syndrome: results of composite-graft repair in 50 patients. N Engl J Med 1986;314:1070-4.

Sakai LY, Keene DR, Engvall E. Fibrillin, a new 350 kD glycoprotein, is a component of extracellular microfibrils. J Cell Biol 1986;103:2499-509.

Hollister DW, Godfrey M, Sakai LY et al. Immunologic abnormalities of the microfibrillar-fiber system in the Marfan Syndrome. N Engl J Med 1990;323:152-9.

Kainulainen K, Pulkkinen L, Savolainen A et al. Location on chromosome 15 of the gene defect causing Marfan syndrome. N Engl J Med 1990;323:935-9.

Pyeritz RE. Marfan syndrome. N Engl J Med 1990;323:987-9.

Lee B, Godfrey M, Vitale E et al. Linkage of Marfan syndrome and a phenotypically related disorder to two different fibrillin genes. Nature 1991;352:330-4.

Maslen CL, Corson GM, Maddox BK et al. Partial sequence of a candidate gene for the Marfan syndrome. Nature 1991;352:334-7.

Dietz HC, Cutting GR, Pyeritz RE et al. Marfan syndrome caused by a recurrent de novo missense mutation in the fibrillin gene. Nature 1991;352:337-9.

Tsipouras P, Del Mastro R, Sarfarazi M et al. Genetic linkage of the Marfan syndrome, ectopia lentis, and congenital contractural arachnodactyly to the fibrillin genes on chromosomes 15 and 5. N Engl J Med 1992;326:905-9.

Shores J, Berger KR, Murphy EA et al. Progression of aortic dilatation and the benefit of long term beta adrenergic blockade in Marfan syndrome. N Engl J Med 1994;330:1335-41.

Francke U, Furthmayr H. Marfan’s syndrome and other disorders of fibrillin. N Engl J Med 1994;330:1384-5.

Loeys BL, Chen J, Neptune ER et al. A syndrome of altered cardiovascular, craniofacial, neurocognitive and skeletal development caused by mutations in TGFBR1 or TGFBR2. Nat Genet 2005;37:275-81.

Habashi JP, Judge DP, Holm TM et al. Losartan, an AT1 antagonist, prevents aortic aneurysm in a mouse model of Marfan syndrome. Science 2006;312:117-21.

Loeys BL, Schwarze U, Holm T et al. Aneurysm syndromes caused by mutations in the TGF beta receptor. N Engl J Med 2006;355:788-98.

Gelb BD. Marfan’s syndrome and related disorders – more than tightly connected than we thought. N Engl J Med 2006;355;841-4.

Brooke BS, Habashi JP, Judge DP et al. Angiotensin II blockade and aortic-root dilatation in Marfan's syndrome. N Engl J Med 2008;358:2787-95.

Pyeritz RE. A small molecule for a large disease. N Engl J Med 2008;358:2829-31.

Hypoparathyroidism and Hypercalciuria

 

           

Calcium homeostasis

The present discussion is about the recent articles published on NEJM, concerning Hypoparathyroidism and Hypercalciuria. They represent on line material of my studies on these topics, as I’ve done a summary on my recent letter to the Editor on Brief Report on NHERF1 gene mutations and parathyroid hormone by Prié Dominique working in Paris, France at Hopithal Necker – Enfants Malades.

Calcium crystallization process

Excessively high concentration of calcium ions in the urine is one identifiable and correctable factor in stone formation. Calcium stone formation is a process of mineral crystallization in body tissue or fluid.

Inorganic crystal are shaped to become an integral part of organic tissue to provide strength and hardness. Thsese inorganic substances are capable of reversible interactions with biomolecules so that the crystalline structures can be remodeled for physiological needs.

Calcium salts have an highly adaptable coordination geometry, that greatly facilitates the protein binding, in its solid state or solution, adapting theirself to irregular geometry of proteins.

The physical properties of bone and teeth result from the activities of proteins that functions as the organic-inorganic interface.

Proteins share specific domains that specifically are able to interact with calcium crystals. The sequence below seems to be specific in these extracellular matrix proteins:

Aspartate-phosphoserine-phosphoserine-glutamate-glutamate

(DpSpSEE)

The motif described in the saliva protein “statherin” is also found in other calcium crystal-interacting proteins, such as osteopontin.

Interestingly unlike EF domain hand that binds ionic calcium, this structure specifically binds to solid phase calcium phosphate crystals and it is conserved in all phylogenetically evolved forms of life in the heart from invertebrates, such as crustaceans, to higher vertebrates, such as humans.

Physiologic crystallization includes formation of exoskeleton, pearl, endoskeleton, and dentition, whereas pathophysiologic crystallization includes pyroposphate arhtropathy, pigmented gallstones, vascular calcifications, and urolithiasis.

Serum calcium concentration is tightly regulated in humans, so that also small decrement in its concentration can led to clinical manifestations of “tetania”, as we have in hypoparathyroidism where Trousseau and Kwostek signs are present as clinical maifestations of altered muscle cells contraction regulations.

On other side increments of calcium levels can lead to increased urinary calcium secretions with kidney function alterations, hypergastrinemia with gastric ulcer formation, hypertension, bone reabsorption with osteoporomalacia and bone fractures, parodontopathies with alterations in theet adherence to bone stucture in particular of mandible (inferior dental arc), as mention the more important clinical manifestations.

The calcium homeostasis is under control of four main organs, bowel and intestinal system, parathyroid glands, bone tissues, and kidneys. Interestingly small changes in calcium levels can be evident such as alterations in acid-base equilibrium (i.e blood PH), due to important action exerted on these physicochemical equilibrium by renal cells activity of secretion and reabsorption. So that small changes in blood PH have to be resetted by intervention of kidney system ( metabolic acidosis or alkalosis) and only after by lung gas exchanges ( respiratory acidosis or alkalosis).

The secretion and reabsorption of calcium ions by kidney, in that view, is essential to human life. When these equilibrium is altered the first alteration we see is an excessive excretion or loss of calcium ions in urine, so that we call hypercalciuria.

Isolated hypercalciuria per se is not detrimental, but clinician interest in hypercalciuria concerns the complications that include mainly nephrolithiasis and nephrocalcinosis.

Stones formation

It has been suggested that the importance of hypercalciuria versus hyperoxaluria in calcium oxalate stones formers is equal; so that both urinary concentration of calcium and oxalate are important contributing factors in formation of kidney stones.

Kidney stones have a lifetime incidence of up to 13% in USA; in at least 70% of cases the stones are formed by calcium oxalate crystals, often with calcium phosphate or sodium urate.

For a stone to form there must be “supersaturation” a chemical condition dependent from PH, Ionic strength and Ionic concentration; in the presence of a “nidus” the nucleation process occurs, where the “nidus” is formed by extracellular matrix components or cellular debris. The subsequent step is the formation of of a true stone by crystal growth and aggregation.

The molecular mechanisms underlying the stone formation are described as:

. heterogeneous nucleation: in which the initial ion complex is attached to a foreign surface

. homogeneous nucleation: in which stones are formed independently from a nucleating surface

The heterogeneous nucleation occurs more often, requiring less energy and so at low level of supersaturation.

Some “factors” control the nucleation and crystal growth processes such as lowering supersaturation energy required and the presence of chemical inhibitors of crystal growth such as:

. pyrophosphate inorganic

. citrate

. glycoproteins

The majority of stone formers are defined such as affected by ”idiopathic hypercalciuria” . Any analysis of hypercalciuria should take into account a Pak pioneering work of 1975 introducing a tripartite classification of hypercalciuria:

1. Absorptive hypercalciuria
2. Reabsorptive hypercalciuria
3. Renal hypercalciuria

From pathophysiological point of view and also by genetic view, this classification may seem to be very important and today useful.

Accordingly the extracellular fluid compartment can be regulated by the exchanges with three systems:

1. Intestinal system
2. Bone system
3. Kidney system

Where the action of main hormones secreted and regulated by parathyroid glands is exerted : parathormone, 1,25 dihydroxy vitamin D3. Interestingly the physiological action exerted by the third hormone “calcitonin” in humans is not relevant, whereas in fish living in water mabient rich in calcium salts, this hormene is very important.

A possible classification of clinical parameters avaible if we consider renal hypercalciuria is the present:

- Parathyroid hormone and 1,25 vitmian D3 are higher than expected

- Hypercalciuria is inappropriate for the slightly elevated parathyroid homone, normal serum calcium, and normal filtered calcium.

- Persistent hypercalciuria is present even during fasting

- Increased bone resorption markers and/or reduced bone mineral density are present.

Interestingly half of patients labeled as havng idipatic hypercalciuria shown a family history of kidney stones. However the genetic rules observed by hypercalciuric patients don’t follow the Mendelian pattern of inheritance, but it seems likely a variable under the effect of

- Polygenic influence

- Polymorphism if a single gene locus (heterogeneity)

- Secondary and compensatory influences by three systems before described

- Under influece of external non genetic factors in particular dietary and lifestyle factors

So that also calciuria can be considered a continous variable with a polygenic determination and those phennotypic expression is modulated by non genetic environmental factors such as blodd pressure and body mass.

Parathyroid hormone hyposecretion or hypoactivity

Post-surgical

Radiation induced

Metastatic infiltration

Autoimmune (isolated or combined with polyglandular endocrine defects)

Autoimmune Polyglandular Syndrome type 1

It is linked to chromosome 21q22.3 coding for AIRE gene, inherited as autosomal recessive moitety. Loss-of-function mutation in AIRE, a zinc finger transcription factor present in thymus and lymph nodes, it is critical in mediating central tolerance by the thymus. NALP-5 is an intracellular signalling molecule strongly expressed in the parathyroid, and it can be target of specific parathyroid autoantigens in patients affected by APS-1. Autoantibodies to NALP5 were found in 49% of patients with APS-1 and hypoparathyroidism.

Clinical picture is variably present in people concentrated in Finnish, Iranian Jewis, and Sardinian populations, presenting more than 58 mutations. Classic triad is represented by:

1. Mucocutaneous candidiasis
2. Adrenal insufficiency
3. Hypoparathyroidism

(any of these two conditions are suffcient to formulate the diagnosis of APS-1). Other different features include hypogonadism, type 1 diabetes, hypothyroidism, vitiligo, alopecia, keratoconjuntivitis, hepatitis, pernicious anemia, and malabsorption. More than 80% of patients with APS-1 have hypoparathyroidism, as sole endocrinopathy. Typically the disease is presented in childhood or adolescence, but patients with only one disease manifestation is folowed long-term for the appearance of other signs of disease.

Deposition of heavy metals

Thalassemia for iron excess

Hemochromathosis

Wilson’s disease

Severe magnesium depletion

alchoolism, malnutrition, malabsorption, diabetes, metabolic acidosis, renal disorders leading to magnesium wasting (pyelonephritis, postostructive nephropathy, renal tubular acidosis, acute tubular necrosis, drugs toxicity ( diuretics, cisplatinum, aminoglycoside antibiotics, amphotericin B, cyclosporin)

Primary renal magnesium wasting or familial hypomagnesiemia with hypercalciuria and nephrocalcinosis (OMIM 248250) due to mutations in genes coding for parcellin-1 and claudin 16

Hypermagnesiemia

On patients receiving tocolytic therapy or in patients with chronic kidney disease receiving magnesium supplements, antiacids or laxatives

Genetic disorders of PTH biosynthesis and parathyroid gland development

PTH gene mutations

Familial Isolated hypoparathyroidism

It is linked to chromosomal alteration of gene coding for pre-pro-PTH located on chromosome 11p15, and it is inherited in an autosomal recessive fashion. Mutations in signal peptides, disrupting PTH secretion, or in a donor splice site of the PTH gene, leading to skipping of PTH exon-2, which contains the initiation codon and signal peptide, are the molecular gene derangements accounting for the clinical picture. Very low or undetectable levels of of PTH and symptomatic hypocalcemia are main features of this syndromic complex.

Instead of mutations in signal peptides, we can have on the same chromosomal locus point mutations in the signal sequence of the pre-pro PTH that prevents processing and translocation of PTH across endoplasmic reticulum and memebrane exocytosis. Mutant PTH is believed to be trapped into endoplasmic reticulum inside cells; resulting stress in endoplasmic reticulum is thought to predispose cells to undergo to apoptosis.

Large deletions in transcription factor for gene coding for PTH called Glial cell Missing B or 2 transcription factor coded on chromosome 6p23-p24 (GCMB or GCM2) are autosomal recessive transmitted gene mutations responsible for forms of familial hypoparathyroidism due to large deletions of these transcription factors with subsequent loss-of-function mutation or point-mutations in the DNA-binding domain of these transcription factors. Leading to loss of transactivating capacity. Interestingly the two transcription factors are highly expressed in parathyroid cells and they controls the embryologic development of parathyroid glands

X-linked Hypoparathyroidism

The X linked recessive mutations affecting the chromosome Xq26-27 involve deletions or insertions of genetic material from chromosome 2p25.3 to chromosome Xq27.1, causing a position effect on regulatory elements controlling SOX3 transcription factor. SOX3 transcription factoris believed to be expressed during developement of parathyroid glands and its mutations cause parahtyroid agenesis of these glands.

Hypoparathyroidism may is a part of complex genetic syndromes:

Familial hypocalcemia with hypercalciuria:

The gene locus responsible is located on chromosome 3q13 and it is coding for calcium sensing receptor, the mutation is transmitted as autosomal dominant form and the phenotypic appearance of affected patients is cause by a gain-of-function mutation in calcium sensing receptor leading to milf hypocaòcemia and hypomagnesemia with hypocalciuria. Mutant receptors caused a left shifted set point for PTH secretion , definied as extracellualr calcium level necessary for half maximal suppression of PTH secretion. Most than 40 mutations have been identified at present, some of them responsible for the Batter’s syndrome type 5 (OMIM 601199 ).

Constitutive active Calcium Sensing Receptors

Most commonly coused by mutations and rarely caused by acquired antibodies that stimulates the calcium sensing receptor; appears to be among the most common causes of hypoparathyroidism.

Syndrome of hypoparathyroidism, deafness, and renal anomalies.

This syndromic complex is linked to mutations on chromosome 10p14-10pter, coding for the transcription factor GATA3. This mutation is inherited through an autosomal dominant form and interfere with the ability of GATA3 to bind to DNA or other transcriptional complexes. GATA3 is a transcription factor known to be highly expressed in in parathyroid glands, kidney and otic-vescicles during organ development precesses. So that clinical alterations characterizing this syndromic complex are hypoparathyroidism, bilateral sensorineural deafness, and renal anomalies or disfunction.

Syndrome of hypoparathyroidism with growth retardation, mental diseases and dysmorphism.

- Kenny-Caffey syndrome

- Sanjad-Sakati syndrome

The syndromic complex is due to mutations affecting chromosome 1q42-q43 coding for transcription factor TBCE and transmitted as autosomal recessive trait. TBCE mutations cause loss of function and alter the assembly of microtubules in affected tissues. Kenny-Caffey syndrome is presented such as hypoparathyroidism probably due to agenesia of the glands, shorth stature, osteosclerosis, cortical bone thickening, calcifications of basal ganglia, ocular abnormalities; whereas Sanjad-Sakati syndrome is characterized by parathyroid aplasia, growth failure, ocular amalformations, microencephaly, and mental retardation.

DiGeorge Syndrome or VeloCardioFacial Syndrome

An heterozygous deletion of chromosome 22q11.2 coding for the transcription factor TBX1 is the known cause of this syndrome. Loss of function mutation of TBX1 is responsible for loss of adjuvating action by TBX1 on other transcription factors known to be involved into the development of thymus and parathyroid glands. Embriological alterations are demonstrated to occurs in these patients in the formation and development of thrid and fourth branchial pouches. Wide spectrum of phenotypc expression, may include conotruncal cardiac defects, parathyroid and thymic hypoplasia, neurocognitive problems, and palatal, renal, ocular, and skeletal abnormalities. Hypocalccemia (in 50% of patients) can be transient or permanent and can develop in adulthood. A screening test si available with confirmed dletion by FISH technique.

Mithocondrial disorders with hypoparathyroidism

- Kearns-Sayre syndrome

- MELAS syndrome

- MTPDS syndrome

Are known syndromic complexes due to deletions, mutations, rearrangments and duplications in the mitochondrial genome. These diseases are inherited uniquely by maternal cells (as all mithocondrial structures) and hypopararhytoidism can be present with various syndromic complexes:

In Kearns-Sayre syndrome with progressive external ophtalmoplegy, pigmentary retinopathy, hearth block or cardiomegaly, diabetes. In MELAS syndrome with diabetes only In MTPDS with fatty acids oxidation alterations, peripheral europathy, retinopathy, acute fatty liver in pregnancy.

Resistance to PTH action

Pseudo-Hypoparathyroisim Type 1a

The disease is due to inactivating mutation in the gene coding for the subunit alfa of G protein coupled with PTH Receptor (GNAS gene on chromosome 20q13.3).

GNAS gene is able to code for the a-subunit of the stimulatory G protein (G_Sa) and it is located on Chromosome 20q11, where 13 exons are present with differnt promoter regions. It is well demonstrated that this protein is linked to many transmembrane receptors such as Parathyroid hormone receptor, TSH Receptor, FSH and LH Receptors, GH Receptor.

During the past few years it became apparent that GNAS gene enchodes not only for for G_Sa but also for several splice variants:

1. XLas (paternal allele)

2. NESP55 neurosecretory protein (maternal allele)

3. A/B (1A) (paternal allele)

4. Antisense transcript

Later it was demosntrated that the alternative exons and their promoter regions are “methylated” on one parental allele, giving rise only to “non-methylated” allele transcription.

Moreover, in most tissues the transcripts encoding G_Sa are derived from both alleles; whereas in a few tissues such as

. proximal renal tubular cells

. adipocytes

. pituitary cells

G_Sa appears to be expressed only from maternal allele.

In the type 1a the mutation is an heterozygous inactivating mutation transmitted with autosomal dominant pattern with maternal transmission of the biochemical phenotype. Clinical features include those described first as Albright’s Hereditary Osteodystrophy such as round facies, mental retardation, frontal bossing, shorth stature, obesity, brachydactyly, ectopic ossification, hypocalcemia, hyperphopshatemia, evelated PTH levels, hypothyroidism, hypogonadism.

Pseudohypoparathyroidism Type 1b

The disease is due to a partenally imprinted defect in G protein due to methylation defect in exon A and exon B, this alterations lead to a selective resistance only to parathyroid hormone and not to other G coupled receptors linking other hormones. So the features are not those present in classic Albright Hereditary Osteodystrophy but hypoparathyroidism with elevalted PTH values, hypocacemia, hyperphosphatemia and elevated levels of urinary cAMP after administration of PTH.

Pseudohypoparathyroidism Type 2 or Pseudo-pseudo-hypoparathyroidism

It is due to GNAS inactivating mutation paternally inherited; however a resistance to PTH is present so that patients secrete normal urinary cAMP levels but not phosphaturic responses to PTH. It can have inheried or sporadic occurrence.

References

Calcium homeostasis

Starnes CW, Welsh JD. Intestinal sucrase-isomaltase deficiency and renal calculi. N Engl J Med 1970;282:1023-4.

Pak CY, Kaplan R, Bone H et al. A simple test for the diagnosis of absorptive, resorptive and renal hypercalciurias. N Engl J med 1975;292:497-500.

Coe FL, Parks JH, Moore ES. Familial idiopathic hypercalciuria. N Engl J Med 1979;300:337-40.

Tieder M, Modai D, Samuel R et al. Hereditary hypophosphatemic rickets with hypercalciuria. N Engl J Med 1985;312:611-7.

Charnas LR, Bernardini I, Rader D et al. Clinical and laboratory findings in the oculocerebrorenal syndrome of Lowe, with special reference to growth and renal function. N Engl J Med 1991;324:1318-25.

Coe FL, Parks JH, Asplin JR. The pathogenesis and treatment of kidney stones. N Engl J Med 1992;327:1141-52.

Curhan GC, Willet WC, Rimm EB et al. A prospective study of dietary calcium and other nutrients and the risk of symptomatic kidney stones. N Engl J Med 1993;328:833-8.

Lemann J Jr. Composition of diet and calcium kidney stones. N Engl J Med 1993;328:880-2.

Pearce SH, Williamson C, Kifor O et al. A familial syndrome of hypocalcemia with hypercalciuria due to mutations in the calcium-sensing receptor gene. N Engl J Med 1996;335:1115-22.

Simon DB, Lu Y, Choate KA et al. Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption. Science 1999;285:103-6.

Borghi L, Schianchi T, Meschi T et al. Comparison of two diets for the prevention of recurrent stones in idiopatic hypercalciuria. N Engl J Med 2002;346:77-84.

Bushinsky DA. Recurrent hypercalciuric nephrolithiasis – Does diet help? N Engl J Med 2002;346:124-5.

Bushinsky DA. Genetic hypercalciuric stone forming rats. Curr Opin Nephrol Hypertens 1999;8:479-488.

Lemann J Jr, Pleuss JA, Worcester EM et al. Urinary oxalate excretion increases with body size and decreases with increasing dietary calcium intake among healthy adults. Kidney Int 1996;49:200-8 [Erratum Kidney Int 1996;50:341]

Phillips MJ, Cooke JNC. Relation between urinary calcium and sodium in patients with idiopathic hypercalciuria. Lancet 1967;1:1354-7.

Silver J, Rubinger D, Friedlander MM et al. Sodium-dependent idiopatic hypercalciuria in renal-stone formers. Lancet 1983;2:484-6.

Parathyroid hormone hyposecretion or hypoactivity

Ahonen P, Myllamierni S, Sipila I et al. Clinical variation of autoimmune polyendocrinopathy-candidiasis ectodermal dystrophy (APECED) in a series of 68 patients. N Engl J Med 1990;322:1829-36.

Bilous RW, Murty G, Parkinson DB et al. Autosomal familial hypoparathyroidism, sensorineural deafness and renal dysplasia. N Engl J Med 1992;327:1069-84.

Brown EM, Pollak M, Seidman CE et al. Calcium-ion-sensing cell-surface receptors. N Engl J Med 1995;333:234-40.

Pearce SHS, Williamson C, Kifor O et al. A familial syndrome of hypocalcemia with hypercalciuria due to mutation in the calcium sensing receptor. N Engl J Med 1996;335:1115-22.

Shore EM, Ahn J, de Beur SJ et al. Paternally inherited inactivating mutations of the GNASI gene in progressive osseus heteroplasia. N Engl J Med 2002;346:99-106.

Juppner H. The genetic basis of progressive osseus heteroplasia. N Engl J Med 2002;346:128-130.

Juppner H, Schipani E, Bastepe M et al. The gene responsible for psuedohypoparathyroidism type 1b is paternally imprinted and maps in four unrelated kindreds to chromosome 20q13.3. Proc Nat Acad Sci USA 1998;95:11798-830.

Bastepe M, Pincus JE, Sugimoto T et al. Positional dissociation between the genetic mutations responsible for pseudohypoparathyroidism type 1b and the associated methylation defect at exon A/B: evidence for a long-range regulatory element within the imprinted GNASI locus. Hum Mol Genet 2001;10:1231-41.

Bastepe M, Lane AH, Juppner H et al. Paternal uniparenteral isodisomy of chromososme 20q – aand the resulting changes in GNASI methylation – as a plausible cause of pseudohypoparathyroism. Am J Hum Genet 2001;6:1283-9.