J Med Genet 1996;33:957-961 957 Syndrome of the month J Med Genet: first published as 10.1136/jmg.33.11.957 on 1 November 1996. Downloaded from type 1B

Andrea Superti-Furga

Historical notes which tried to provide a quantitative basis (the In 1952, the name achondrogenesis (Greek for "femoral cylinder index") for qualitative "not producing ") was given by Marco changes, did not prove helpful and was later Fraccaro, a young Italian pathologist (later to abandoned. In the late 1980s, it was shown become a well known cytogeneticist), to the that achondrogenesis type II was caused by condition he observed in a stillborn female structural in II and thus with severe micromelia and marked histological constituted the severe end of the spectrum of changes of cartilage.' Fraccaro noted a similar the collagen II chondrodysplasias.l'" case published by Parenti in 1936.2 Fraccaro's Borochowitz et al'4 provided convincing report (written in Italian) came to the know- histological criteria for the further subdivision ledge ofHans Grebe, who in 1939 had observed of achondrogenesis type I into IA (with ap- sisters (aged 7 and 11 years, born to con- parently normal cartilage matrix but inclusions sanguineous parents from the Black Forest re- in chondrocytes) and IB (with abnormal car- gion of Germany) with markedly short limbs tilage matrix; see below). These findings were and digits but normal trunk. Grebe became confirmed by another group shortly there- convinced that his patients were affected by after.'5 Using these criteria, some older pub- the same achondrogenesis as Fraccaro's short lished cases can be unequivocally diagnosed as limbed stillborn patient, and he had his report type IB,'617 others as type IA,'8 while Parenti's (written in German) published in the same case probably should be classified as type II.2 14 journal.' However, the condition described by The classification of achondrogenesis type IB Grebe was different (although superficially sim- as a separate group has recently been confirmed ilar to Fraccaro's achondrogenesis because of by biochemical and molecular findings (see limb shortening) and has later become known below). The history of achondrogenesis shows as Grebe chondrodysplasia or Grebe syndrome. how subjective clinical and radiological clas- The name achondrogenesis was then used sifications can be. Histology and ultrastructure to characterise the most severe forms of chon- have allowed a more solid classification and

drodysplasia in humans, invariably lethal before provided the basis for biochemical and mo- http://jmg.bmj.com/ or shortly after birth. Precise figures on the lecular studies. incidence of achondrogenesis are not available, The present classification ofachondrogenesis but it is not exceedingly rare: reviews with large is shown in table 1. Recessive inheritance of numbers of patients have been published.45 achondrogenesis type IA is not formally proven, In the 1970s, the heterogeneity of achon- and this type may still contain different sub- drogenesis was recognised. Using a com- groups. To avoid misunderstandings in the era bination ofradiological and histological criteria, of gene symbols and computerised databases, achondrogenesis type I (then also called Frac- it is advisable (though regrettable) to convert on October 1, 2021 by guest. Protected copyright. caro-Houston-Harris type) and type II (called Roman numerals to Arabic numerals. Ac- Langer-Saldino type) were distinguished.67 In cordingly, the following abbreviations are pro- 1976, it was found that chondrocytes of some posed: achondrogenesis type IA (ACGGA), (though not all) type I patients contained cyto- achondrogenesis type lB (ACGIB), and plasmic inclusions.8 In 1983, a new clas- (ACG2). Division of Metabolic and Molecular sification of achondrogenesis (types I to IV) Diseases, Department was proposed9 and adopted in the McKusick of Pediatrics, catalogue. This radiological classification, University Children's Clinical features of achondrogenesis type Hospital, lB Steinwiesstrasse 75, (JrMed Genet 1996;33:957-961) CH-8032 Zurich, Newborns with ACGGB frequently present Switzerland Key words: ; cartilage; differ- in breech position. After delivery, their ap- A Superti-Furga ential diagnosis. pearance is immediately perceived as abnormal

Table 1 Classification of achondrogenesis Name Former eponyms Abbreviation Inheritance Molecular basis Achondrogenesis type IA Houston-Harris type ACGIA AR (?) Unknown Achondrogenesis type lB Fraccaro type ACGIB AR Mutations in sulphate transporter gene, DTDST, on 5q Achondrogenesis type 2 Langer-Saldino type ACG2 AD (de novo) Mutations in collagen II gene, COL2A1, on 12q 958 Superti-Furga

proven ACG1B,'41519 22 as well as in pub- lished cases who could be classified as ACGlB by their histological features.' 17 The following

should be noted: (1) there is a certain degree J Med Genet: first published as 10.1136/jmg.33.11.957 on 1 November 1996. Downloaded from of variability, and no single feature should be regarded as obligatory; (2) contrary to what has been reported,'4 distinction between ACGIB and ACGlA on radiographs is not always possible; (3) the degree of ossification is age dependent, and caution is needed in comparing radiographs from patients at differ- ent gestational ages. In ACGGB, the skull is only mildly affected: it is slightly less ossified than expected for gestational age, and the orbits may be extended laterally and superiorly. The disproportion be- tween the skull of almost normal size and the hypoplastic skeleton is noticeable. The clavicles are mildly affected; they are ossified, and, albeit somewhat short, of near normal shape. The scapulae are small and their contour is irregular. The vertebral bodies are usually not ossified; Figure I (Left) Clinical appearance of a patient with ACGIB, born at 34 weeks and at most, rudimentary calcification in the central died 25 minutes after birth. Note the flat face, the narrow thorax with protuberant part is seen. The vertebral lateral pedicles are abdomen, and particularly the severe nicronmelia with short stubby finigers anld toes. usually ossified, and part of the neural arches (Right) X ray of the same newborn. Following the resuscitation attempts, there is air in the stomach and intestine but not in the lungs. For details of the skeletal systeni, refer to may also be. The ribs are slightly thinner and the text. For radiographs of other newborns with ACGIB, see refs 14, 15, 16, 17, or 21. much shorter than normal; they are usually not (Reprintedfrom ref 19, with perniission of the publisher.) fractured (although occasionally, one or two fractures have been seen) and show slight cup- ping at their distal ends. The iliac are (fig 1, left). The abundance of soft tissue rel- smaller than usual and only their upper (cra- ative to the short skeleton gives these newborns nial) half is ossified, often in an irregular fash- a fat or hydropic appearance. There is a dis- ion; their shape can be compared to that of a proportion between the head, which is of nor- crescent or a paraglider. The ischium is not mal or near normal size, and the rest of the ossified or only minimally. body, which is much shorter than normal. The The tubular bones are those most markedly face is flat, the neck short, and the soft tissue affected (fig 1). Femora and humeri are usually of the neck may be thickened. The thorax shortened to a degree where no major axis can is narrow and the abdomen is protuberant. be recognised. As some metaphyseal spurring Umbilical or inguinal herniae are frequent. The occurs, these bones end up resembling cartoon most striking abnormality concerns the limbs. stars, thorn apples, or, for the haematological http://jmg.bmj.com/ They are severely shortened, sometimes re- expert, acanthocytes. The tibiae and fibulae sembling flippers, sometimes, when an articular are similarly misshapen and the fibulae are not crease at the hip or shoulder is present, re- ossified in more premature cases. In most cases, sembling sausages. The fingers and toes are the ulnae are amorphous, while the radii show similarly short and stubby. The feet and toes some tubulation and may have a peculiar distal are rotated inwards in a fashion reminiscent dichotomy thereby resembling a waterfork. The

of . The external genital carpals and phalanges are usually very poorly on October 1, 2021 by guest. Protected copyright. organs are unremarkable. Death may occur ossified and can rarely be identified on whole before birth for causes which are not un- baby x rays. Sometimes bipartite ossification derstood. Even when heart action is present at of the medial phalanges can be seen. birth, respiratory insufficiency follows shortly. Fetuses with achondrogenesis may be as- certained by the observation of short femora Histology of cartilage on routine ultrasonography during pregnancy. In ACG1B, the cartilage matrix is rarefied and Other ultrasonographic signs may be nuchal partially replaced by a larger number of cells. oedema, reduced rump length, poor os- After haematoxylin-eosin staining, the matrix sification of the vertebral bodies and of the does not have the characteristic homogeneous, limb bones (leading to difficulties in de- ground glass appearance; instead, it shows termining their length), and polyhydramnios. coarsened collagen fibres. The fibres are often If pregnancy is interrupted, it is imperative to radially arranged around individual chon- obtain good clinical and radiographic docu- drocytes and bridge the space between chon- mentation as well as appropriate biological ma- drocytes. The fibres are particularly dense terial to allow a precise diagnosis and adequate around chondrocytes where they can form so- counselling (see below). called collagen rings'4 (fig 2). The coarsened fibres are seen well on silver stained sections. In normal cartilage, cationic dyes such as toluidine Radiological features blue or Alcian blue give a homogeneous, deep Radiological features have been reviewed in blue, or violet staining of the matrix owing to six cases with biochemically and molecularly the abundance of polyanionic sulphated pro- Achondrogenesis type IB 959 dysplasia. The structure of II, IX, and XI was shown to be normal in cartilage from the patient shown in fig 1 (B Steinmann,

unpublished data, 1988), suggesting to me a J Med Genet: first published as 10.1136/jmg.33.11.957 on 1 November 1996. Downloaded from defect in some other structural component. The total sulphate content of ACG1B car- tilage is less than one-fifth that of control car- tilage.20 When cartilage extracts are separated by polyacrylamide or agarose gel electro- phoresis and the gels stained with cationic dyes, the sulphated proteoglycans appear to be re- duced in concentration'9 and to migrate more slowly because of reduced negative charge."2 Chondrocytes and skin fibroblasts cultured from ACG 1 B patients are unable to incorporate exogenous sulphate'9 22; this is best shown by a double labelling test with 3H-glycine and 35S- sodium sulphate.20 In both cell types, a defect in the uptake of extracellular sulphate can be . shown.2022 Figure 2 (A) Section through normal fetal cartilage. The cartilage matrix is homogeneous and no structures can be recognised. (B) Section through ACGGB cartilage (case shown in fig 1, see ref 19). The number of cells is increased. The matnrx is devoid of Molecular basis of ACGIB ground substance and fibrillar structures can be recognised, which tend to forni rings The discovery of the sulphate incorporation around chondrocytes. (Both parts: haematoxylin-eosi n staining.) defect in ACG1B came at the same time as identification of the gene for a sulphate trans- teoglycans. In ACGGB cartilage, the intensity porter, DTDST,23 on chromosome 5q as the of staining with these dyes is very much re- gene locus responsible for the non-lethal dis- duced, and the matrix between the coarsened order, diastrophic dysplasia (DTD),24 for collagen fibres may not stain at all. These which no biochemical clues had existed. This features, probably the result of a deficiency made DTDST a plausible candidate gene for in sulphated proteoglycans,' 1'9 distinguish ACGIB. Indeed, a series of mutations in the ACGGB both from ACGGA (where the matrix DTDST gene have been identified in patients is close to normal and inclusions can be seen in with ACG1B, DTD, and a disorder of inter- chondrocytes) and from ACG2 (where cationic mediate severity called atelosteogenesis type 2 dyes give a normal staining pattern and coar- (A02). Mutations include point mutations and sening of the fibres and collagen rings is not deletions leading to premature stop codons, seen). substitutions or deletions of amino acids within transmembrane domains, substitutions of amino acids in intra- or extracellular Physical and biochemical findings in domains, and a presumed lying out- http://jmg.bmj.com/ cartilage side the coding region but causing low mRNA ACGGB cartilage is brownish and translucent levels.2023 2526 Genotype-phenotype cor- rather than white like normal cartilage, and relations indicate that the amount of residual friable rather than firm and elastic. Its con- activity of the sulphate transporter modulates sistency is similar to that of a cooked apple. It the phenotype from lethal ACGGB to non- can be easily cut through with a scalpel. In lethal DTD.26 Homozygosity or compound het- this respect, it resembles cartilage from severe erozygosity for mutations predicting stop co- on October 1, 2021 by guest. Protected copyright. collagen II disorders such as ACG2 or Kniest dons or structural mutations in transmembrane domains are associated with ACG1B, while mutations in extracellular loops or the cyto- plasmic tail or the presumed regulatory muta- tion giving low mRNA usually result in the less severe phenotypes, A02 and DTD. Thus, there is a chondrodysplasia family caused by allelic mutations in the DTDST gene.26 Hetero- zygotes are ofnormal stature and do not appear to have degenerative joint disease, confirming recessive inheritance. Impaired activity of the sulphate transporter in chondrocytes and fibroblasts results in the synthesis of proteoglycans which are not sulph- ated or only insufficiently,'922 probably by de- C2171t, A715V pletion of intracellular sulphate. Proteoglycan Figure 3 Schematic representation of the diastrophic undersulphation has a pronounced effect on dysplasia sulphate transporter (niodified from ref 23) with localisation of the known pathogenic mutations. Several of the composition of the extracellular matrix these mutations have been found in ACG1B, A02, and of cartilage, as shown by the physical, bio- DTD; the conmbination of niutations is responsible for chemical, and histological findings.'9 22 How modulating the phenotype. Further details concerning individual mutations can be found in ref 26; nucleotide this ultimately leads to the chondrodysplasia and amino acid nunibering follow ref 23. is not known. Interestingly, there is no clear 960 Superti-Furga

indication that other organs besides the skeletal Prenatal diagnosis or exclusion of ACG1B system are affected by the disorder, in spite (and ofA02 or DTD) can be done by mutation of the ubiquitous expression of the sulphate analysis of chorionic villus DNA around weeks

transporter.23 Possibly, other tissues have a 10 to 11, provided that both alleles have been J Med Genet: first published as 10.1136/jmg.33.11.957 on 1 November 1996. Downloaded from lower requirement for sulphate which can be characterised beforehand and each has been met either by transport through other anion shown to be inherited from one of the parents. carriers or by endocellular synthesis from sul- Mutation analysis can also be used to ascertain phur containing amino acids. carriers, particularly in consanguineous famil- ies. Biochemical analysis of fibroblast cultures has not yet enabled distinction between hetero- Differential diagnosis and diagnostic zygotes and normal homozygotes. Analysis of procedures sulphate incorporation in chorionic villi might The differential diagnosis of achondrogenesis theoretically be used for prenatal diagnosis, is that of lethal , a long but experience is lacking. Finally, the available list of conditions.27 The most frequent among experience indicates that all three types of these disorders are lethal osteogenesis im- achondrogenesis can be recognised as early as perfecta, , and the short in gestational week 13 or 14 by experienced rib-polydactyly syndromes. In osteogenesis im- sonographers,28-30 making ultrasound detection perfecta, the skull is soft, the sclerae blue, and an acceptable option when molecular studies the bones bowed but clearly not as short as in are unavailable or not feasible. achondrogenesis. In thanatophoric dysplasia, I am indebted to J Bonaventure, J Briner, D Cohn, G Eich, A the limbs are longer and the shape of the Giedion, J Hastbacka, D L Rimoin, A Rossi, Th Stallmach, W Wilcox, B Steinmann, and R Gitzelmann for patient material, thorax is narrow but elongated. The short rib- laboratory data, financial support, or help in the preparation of polydactyly syndromes resemble thanatophoric the manuscript. This work was supported by the Swiss National Science Foundation (32-45401.95) and the Julius Klaus Stif- dysplasia and are usually associated with hexa- tung of the University of Zurich. dactyly. Marked limb shortening may suggest Roberts syndrome, in which the axial skeleton is 1 Fraccaro M. Contributo allo studio del mesenchima os- teopoietico - l'acondrogenesi. Folia Hered Pathol (Milanio) not affected or only mildly. Distinction between 19521:190-207. ACG1A, ACG1B, and ACG2 on clinical 2 Parenti GC. La anosteogenesi. Pathologica (Genova) 1936; 28:447-62. grounds is difficult. Almost normal hands are 3 Grebe H. Die Achondrogenesis: ein einfachrezessives seen in ACG2, whereas in ACG1A and Erbmerkmal. Folia Hered Pathol (Milano) 1952;2:23-8. 4 Wiedemann HR, Remagen W, Hienz HA, Gorlin RJ, Ma- ACG1B the hands are evidently shortened; roteaux P. Achondrogenesis within the scope of connately radiological evidence of rib fractures may sug- manifested generalized skeletal dysplasias. Z Kinderheilkd 1974;116:223-51. gest ACG1A. 5 Schulte MJ, Lenz W, Vogel M. Letale Achondrogenesis: Most cases with ACGIB occur eine Ubersicht iuber 56 Falle. Klin Padiatr 1978;191: sporadically 327-40. and therefore the ultrasonographic or clinical 6 Spranger J, Langer LO, Wiedemann HR. Bonie dysplasias - finding of severe short limbed chondrodysplasia an atlas of constitutional disorders of skeletal developtent. Stuttgart: Gustav Fischer Verlag, 1974. arises unexpectedly. Experience shows that in 7 Yang SS, Brough AJ, Garewal GS, Bernstein J. Two types such a setting it is rarely possible to make of heritable lethal achondrogenesis. _7 Pediatr 1974;85: the correct any between 796-801. diagnosis; diagnosis 8 Yang SS, Heidelberger KP, Bernstein J. Intracytoplasmic http://jmg.bmj.com/ , thanatophoric dys- inclusion bodies in the chondrocytes oftype I lethal achon- drogenesis. Hum Pathol 1976,7:667-73. plasia, and achondrogenesis is a good attempt. 9 Whitley CB, Gorlin RJ. Achondrogenesis: new nosology it is to secure a later with evidence of genetic heterogeneity. Radiology 1983; However, important diag- 148:693-8. nosis by obtaining good radiographs, EDTA 10 Eyre DR, Upton MP, Shapiro FD, Wilkinson RH, Vawter blood for DNA analysis, a skin biopsy under GF Nonexpression of cartilage type II collagen in a case of Langer-Saldino achondrogenesis. Anti . Hunt1 Geniet sterile conditions for fibroblast culture, and 1986;39:52-67. and cartilage tissue for histology and bio- 11 Godfrey M, Hollister DW. Type II achondrogenesis-

: identification of abnormal type II on October 1, 2021 by guest. Protected copyright. chemistry. The combination ofradiological and collagen. Ant _7 Hunt Genet 1988;43:904-13. histological findings will give a provisional diag- 12 Vissing H, D'Alessio M, Lee B, Ramirez F, Godfrey M, Hollister DW. Glycine to serine substitution in the triple- nosis which can then be confirmed by selected helical domain of proil1(II) collagen results in a lethal biochemical or molecular or perinatal form of short-limbed . _7 Biol Chent investigations 1989;264: 18265-7. both. For ACG1B, these investigations would 13 Spranger J, Winterpacht A, Zabel B. The type II col- include the of lagenopathies: a spectrum of chondrodysplasias. EurJ study sulphate incorporation Pediatr 1994;153:56-65. in cultured fibroblasts or chondrocytes and 14 Borochowitz Z, Lachman R, Adornian GE, Spear G, Jones mutation analysis of the DTDST gene. K, Rimoin DL. Achondrogenesis type I: delineation of further heterogeneity and identification of two distinct subgroups. _7 Pediatr 1988;112:23-31. 15 van der Harten HJ, Brons JTJ, Dijkstra PF, et al. Achon- drogenesis-hypochondrogenesis: the spectrum of chon- Genetic and drogenesis imperfecta. Pediatr Pathol 1988;8:571-97. counselling prenatal 16 Urso FP, Urso MJ. Achondrogenesis in two sibs. Birth diagnosis Defects 1974;1O:10-17. 17 Nardi F, Gerlini G, Bonucci E. Achondrogenesis: report on ACG1B and the related disorders, A02 and a case, with particular reference to ultrastructure and DTD, are inherited as autosomal recessive histochemistry. Virchows Arch A Pathol Anat Histol 1974; a recurrence 363:311-22. traits. For given couple, the risk 18 Molz G, Spycher MA. Achondrogenesis type I: light and is 1 in 4, or 25%, and thus markedly higher electron-microscopic study. Eur_7Pediatr 1980; 134:69-74. for 19 Superti-Furga A. A defect in the metabolic activation of than that ACG2 which is usually caused sulphate in a patient with achondrogenesis type IB. Ant _7 by a new dominant mutation. Asymptomatic Hum Genet 1994;55:1137-45. 20 Superti-Furga A, Hastbacka J, Wilcox WR, et al. Achon- carriers may be present in the families of affec- drogenesis type IB is caused by mutations in the di- ted patients. Therefore, genetic counselling astrophic dysplasia sulphate transporter gene. Nat Genet 1996;12:100-2. must rely on differentiation between ACG1B 21 Freisinger P, Stanescu V, Jacob B, Cohen-Solal L, and ACG2. This can be done as outlined above. Maroteaux J, Bonaventure J. Achondrogenesis type IB Achondrogenesis type lB 961

(Fraccaro): study of collagen in the tissue and in chon- porter gene (DTDST): evidence for a phenotypic series drocytes cultured in agarose. Anm Med Genet 1994;49: involving three chondrodysplasias. Am _J Hum Genet 1996; 439-46. 58:255-62. 22 Rossi A, Bonaventure J, Delezoide AL, Cetta G, Superti- 26 Superti-Furga A, Rossi A, Steinmann B, Gitzelmann R. A Furga A. Undersulphation of proteoglycans synthesized chondrodysplasia family produced by mutations in the gene: by chondrocytes from a patient with achondrogenesis diastrophic dysplasia sulphate transporter genotype/ J Med Genet: first published as 10.1136/jmg.33.11.957 on 1 November 1996. Downloaded from type lB homozygous for a Leu483Pro substitution in the phenotype correlations. Am Med Genet 1996;63:144-7. diastrophic dysplasia sulphate transporter. Biol Chem 27 Spranger J, Maroteaux P. The lethal osteochondro- 1996;271: 18456-64. dysplasias. Adv Hum Genet 1990;19:1-103. 23 Hastbacka J, de la Chapelle A, Mahtani MM, et al. The 28 Sharony R, Browne C, Lachman RS, Rimoin DL. Prenatal diastrophic dysplasia gene encodes a novel sulphate trans- diagnosis of the skeletal dysplasias. Anz _7 Obstet Gynecol porter: positional cloning by fine-structure linkage dis- 1993;169:668-75. equilibrium mapping. Cell 1994;78:1073-87. 29 Soothill PW, Vuthiwong C, Rees H. Achondrogenesis type 24 Lamy M, Maroteaux P. Le nanisme diastrophique. Presse 2 diagnosed by transvaginal ultrasound at 12 weeks' gest- Med 1960;68:977-1980. ation. Prenat Diagn 1993;13:523-8. 25 Hastbacka J, Superti-Furga A, Wilcox WR, Rimoin DL, 30 Meizner I, Barhard Y. Achondrogenesis type I diagnosed Cohn DH, Lander ES. Atelosteogenesis type II is caused by transvaginal ultrasonography at 13 weeks gestation. by mutations in the diastrophic dysplasia sulphate trans- Ant _T Obstet Gynecol 1995;173:1620-2. http://jmg.bmj.com/ on October 1, 2021 by guest. Protected copyright.