ORIGINAL ARTICLE Egypt. J. Med. Hum. Genet. Vol. 10, No. 1, May, 2009 Clinical, anthropometric, radiological and molecular characteristics of Egyptian patients

Mona S. Aglan1, Alice K. Abdel-Aleem2, Ahmed IS. EL-Katoury1, Mona H. Hafez3, Sahar N. Saleem4, Ghada A. Otaify1 and Samia A. Temtamy1

1Department of Clinical , 2Medical Molecular Genetics Department, and Stem Cell Unit, Genetics & Research Division, National Research Centre, Departments of 3Pediatrics and 4Radiology, Faculty of , Cairo University ABSTRACT

Background: Achondroplasia is the most common form of non lethal skeletal dysplasia. It is a fully penetrant autosomal dominant disorder and the majority of cases are sporadic resulting from de novo associated with ad- vanced paternal age. The of achondroplasia is related to disturbance in endochondral bone formation due to mutations in the fi broblast growth fac- tor -3 (FGFR3) . Aim of the Work: Evaluation of the cardinal phenotypic features in achondro- plasia, the body physique using anthropometric measurements, the character- istic radiological signs in the patients as a main tool for diagnosis and detec- tion of the most common mutations in achondroplasia patients in the studied sample. Subjects and Methods: From 42 cases referred to us as achondroplasia, we se- lected 20 cases where clinical manifestations were consistent with achondropla- sia. Cases were subjected to full clinical examination, detailed anthropometric measurements, whole body skeletal survey and molecular studies of the most common mutations of the FGFR3 gene using PCR amplifi cation technique. Results: Nineteen cases were sporadic (95%) and one case had an affected father (5%). A paternal age above 35 years at the time of child’s birth was present in 7 cases (35%). Paternal exposure to occupational heat was noted in 6 cases (30%) and parental exposure to chemicals in 3 cases (15%). All cases showed typical clinical and radiological manifestations of achondroplasia. An- thropometric measurements quantitatively confi rmed the body physique in the studied cases. G380R common mutations of the FGFR3 gene were detected in 15/18 cases (83%) with the G to A transition at nucleotide 1138 in 14 cases (77%). Agenesis of corpus callosum, not previously reported in association with achondroplasia, was present in the only case with the G-C transversion at nucleotide 1138 (5%). Conclusions: Awareness of the cardinal features of achondroplasia, proper anthropometric measurements and detailed skeletal survey are the key for accurate diagnosis, and avoidance of over diagnosis. The majority of studied Egyptian achondroplasia patients have the same common mutation that has been most often defi ned in patients with achondroplasia from other countries. Copyright: All rights reserved for The Egyptian Journal of Medical 13 Characteristics of Egyptian achondroplasia patients

Key Words: Corresponding Author: Achondroplasia, fi broblast growth factor recep- Mona S. Aglan tor3, skeletal dysplasia, paternal heat exposure. E-mail: [email protected]

INTRODUCTION

Achondroplasia (OMIM: 10800)1 is a is fl attened and the abdomen protrudes genetic disorder evident at birth. It oc- with thoraco-lumbar kyphosis, with age curs in all races and in both sexes. It exaggerated lumbar lordosis becomes is the most common non lethal type of more prominent. Sleep apnea, respira- skeletal dysplasia and the most com- tory diffi culties and otitis media are mon type of short-limb dwarfi sm. Its common complications. Spinal defor- depiction in ancient Egyptian art makes mities are potentially disabling prob- it one of the oldest recorded birth de- lems as the short thickened pedicle and fects2. Achondroplasia affects more reduced inter-pedicular distance reduce than 250,000 individuals worldwide3. It the size of the spinal canal, both antero- is a fully penetrant autosomal dominant posteriorly and transversely.8 disorder with an incidence rate rang- ing between 1/15,000 and 1/40,000 A skeletal survey is of utmost impor- live births4. The majority of cases are tance to confi rm the diagnosis of achon- sporadic resulting from de novo muta- droplasia. In children caudal narrowing tion associated with advanced paternal of the inter-pediculate distance rather age5. The phenotype of achondroplasia than the normal caudal widening and a is related to a disturbance in endochon- notch like sacro-iliac groove are typi- dral bone formation due to mutation in cal features in addition to the other ra- the fi broblast -3 diological fi ndings characteristic of the (FGFR3) gene and consequently, af- limbs, vertebrae and skull.9 fected individuals exhibit characteristic clinical and radiological features.6 Once the gene for achondroplasia was assigned to 4p16.3 by linkage analy- Phenotypically, achondroplasia is con- sis, causative mutations were identi- sidered a short-limb dwarfi ng condi- fi ed by the approach10. tion with the proximal segments more Achondroplasia is caused by missence severely affected than the distal seg- mutation in the fi broblast growth fac- ments7. The are short and broad tor receptor-3 gene (FGFR3), which is with fi ngers exhibiting a three-pronged located at 4p16.3. Two mutations in the (trident) appearance at birth due to an FGFR3 gene, a G-to-A transition at nu- inability to fully oppose the third and cleotide 1138 and a G-to-C transversion fourth digits. Other characteristic fea- at the same nucleotide, both resulting in tures include, a large head, frontal G380R amino acid substitutions, cause bossing, fl attening of the nasal bridge, over 99% of cases with achondropla- midface hypoplasia and relatively sia.11 prominent mandible. Macrocephaly is due to ventricular enlargement in many The purpose of our study was to evalu- cases; however, intracranial pressure ate the cardinal phenotypic features in is not elevated signifi cantly. The chest achondroplasia, the body physique us- 14 Aglan et al. ing anthropometric measurements, the • Detailed clinical examination with characteristic radiological signs in the special emphasis on dysmorphic patients as a main tool for diagnosis and features, skeletal system, limb seg- to detect the most common mutations in ments and examination of different achondroplasia patients in our sample. body systems. Clinical examina- tion was carried out for parents and sibs. SUBJECTS AND METHODS • Anthropometric measurements in- This study included 20 cases with cluding height (Ht), weight (Wt), achondroplasia. Cases were selected head circumference (HC) and sit- from 42 patients referred as achondro- ting height. Body mass index (BMI) plasia to the Limb Malformations and was calculated for each case (BMI Skeletal Dysplasia Clinic, Medical = Wt in kilograms/Ht2 in meters2). Services Unit at the National Research Measurements were compared to Centre (NRC), Cairo, Egypt in the last age and sex matched normal Egyp- two years. Cases were selected based tian control12,13. Statistical analysis upon the presence of the typical charac- including standard deviation scores teristic stigmata of achondroplasia. The (SDS) were calculated. Upper seg- study included 11 females and 9 males; ment (US)/ lower segment (LS) their mean age at presentation was 3.42 ratio was calculated. Total limb years, ranging from 9 months to 9 years. length, arm length, forearm length A full explanation of the study has been and length were measured and provided to the studied cases and their compared to normal standards14. parents and a written consent has been Absolute measurements for Ht and obtained. HC were plotted on achondroplasia curves.15 For each case the followings were conducted: • A skeletal survey including radio- graphs of skull, spine, pelvis, short and long bones. Neuroimaging • Three - generation pedigree con- of the brain including CT or MRI struction including consanguinity, scan. similar conditions and other affect- ed members in the family. • Molecular studies were conducted on 18 cases, who provided their • Complete history including parents, consent for withdrawal, us- occupation, pregnancy and delivery ing the following steps: histories, exposure to drug intake, fever, trauma, irradiation, or any ο DNA has been extracted from fresh maternal chronic illness. Parental peripheral blood leucocytes by ages at birth of the child, family standard salting out protocol.16 history and developmental mile- stones as well as the occurrence ο PCR amplifi cation across the trans- of complications as respiratory membrane domain, which contains problems, otitis media and history the common mutations G1138A and of orthopedic complications. G1138C of the FGFR3 gene. PCR 15 Characteristics of Egyptian achondroplasia patients amplifi cation was performed in a ent in 3 cases (15%); a mother had Da- 50 μl PCR reaction containing 50 fl on for treatment of varicose veins at ng of genomic DNA as a template, the 5th month of pregnancy, a father had 100 μM of each dNTPs, 10 pmol of lipidol injection 6 months before con- each primer, 1 unit Taq polymerase ception and another was working in the and its buffer (Fermentase). petrol and chemical industry for more than 10 years. ο Restriction endonuclease analysis was the method used to detect the presence or absence of the common mutation. SfcI was the restriction applied to detect the G to A transition at nucleotide 1138; the enzyme has been incubated with the completed PCR product at 37ºC. MspI restriction enzyme was used to detect the G to C transversion at the same nucleotide position 1138 of FGFR3.

ο Digested samples were loaded on 3% ethidium stained agarose gel, electrophoresed and visualized on UV-transilluminator.

RESULTS

This study included 20 cases (11 fe- males & 9 males) and one father with the characteristic features of achon- droplasia. Parental consanguinity was noticed in only 2 cases (10%). The Fig. 1: A father (A) and his son (B) with the paternal ages at the time of the child’s characteristic features of achondroplasia. birth ranged from 22 to 41 years. A pa- ternal age above 35 years was present in Results of clinical fi ndings: 7 cases (35%) while a maternal age of All studied cases had the characteristic 35 was present in one case only (5%). features of achondroplasia as shown in Ninety fi ve percent of cases were spo- (Figures. 1, 2). The characteristic facial radic; only one case (5%) had a father phenotype in the form of frontal boss- with achondroplasia (Figure. 1). ing, mid face hypoplasia, depressed nasal bridge and short neck was pres- History of prolonged paternal exposure ent in all cases. Examination of upper to heat as a result of occupation was and lower limbs in the studied cases noted in 6 cases (30%), three of the ex- revealed that all cases had apparent posed fathers were at the mid twenties rhizomelic shortening, limited elbow and 3 were at the early thirties. Parental extension and generalized joint laxity exposure to chemical agents was pres- in addition to brachydactyly and trident 16 Aglan et al. hand confi guration. Bowing of legs or in all cases. Upper respiratory tract in- genu varum was noticed in all except 5 fection, recurrent snoring, sleep apnea cases (75%). Many cases had multiple and otitis media were common com- skin creases over the limbs (Michelin plications in most cases. Neurological tire creases). Examination of the trunk examination revealed in 70% revealed spinal deformities in the form of cases. Brisk refl exes were present in of lumbar kyphosis in 5 cases (25%) 6 cases (30%) with dilated ventricles while exaggerated lumbar lordosis was and cortical atrophic changes evident present in 75% of cases. Protuberant ab- by brain neuroimaging. Motor develop- domen was noticed in all cases. , mental milestones were delayed in 50% chest and abdomen were clinically free of cases.

Fig. 2: The characteristic clinical features of achondroplasia (A), exaggerated lumbar lordosis and short limbs (B), kyphosis (C), brachydactyly and trident hand confi guration (D).

Results of Anthropometric measure- centile in all cases denoting short limbs. ments: Mean SDS for weight was -1.41 ± 1.7, Using available Egyptian standards12,13, only 3 cases showed a SDS below -2.0 height was below -2.0 SD in 95% of SD (15%). Using BMI as an estimate of cases with a mean SDS of -4.06 ± 2.73. showed that 12 cases were rela- Only one case who was 9 months old tively obese (60%) with a mean SDS had normal height of -0.76 SD. It was of 2.07 ± 0.98 while the rest of cases noted that short stature becomes more had normal scores. Macrocephaly (HC apparent with age. Mean SDS for sitting above 2.0 SD) was present in 11 cases height was -1.06 ± 1.7. Sitting height (55%) with a mean SDS of 2.06 ± 1.97. was below -2.0 SD in only 5 cases Measurements of total limb length, arm, (25%). US/LS ratio was above the 97th forearm and hand were all below the 17 Characteristics of Egyptian achondroplasia patients 3rd centile for age compared to normal curves15 for Ht and HC except one case standards14, with more affection of the with height just below -2.0 SD and 3 arm length. All cases were within the males who had a HC less than -2.0 SD normal range using the achondroplasia (Figures. 3, 4, 5).

Fig. 3: Absolute height measurements of the Fig. 4: Absolute height measurements of the studied male cases plotted on achondroplasia studied female cases plotted on achondroplasia height chart for males.15 height chart for females.15

Fig. 5: Absolute head circumference measurements of the studied cases plotted on achondroplasia head circumference charts for both males & females.15 18 Aglan et al. Results of radiological fi ndings: with age. Exaggerated lumbar lordosis Examples of the characteristic radio- was noticed in most cases (75%). logical features in the studied cas- es of achondroplasia are shown in The pelvis had small square iliac wings, (Figure. 6). small short sacrosciatic notch, fl at hori- zontal acetabular roof and short femoral Frontal bossing, relative small skull neck with elongated trochanters. base and narrow foramen magnum were common radiological fi ndings in Long bones were short and thick with the lateral view of skull X-ray. metaphyseal fl aring. The humerus was markedly shortened. The distal femo- Anteroposterior (AP) view of the spine ral physes had an inverted-V shaped revealed caudal narrowing of the inter- confi guration best noted with age. The pediculate distance in all cases. Lateral fi bula was typically longer than the view of the spine revealed shortening of tibia. The hands were broad with short the pedicles with signifi cant posterior metacarpals and phalanges and a trident scalloping that became more apparent confi guration.

Fig. 6: Examples of the characteristic radiological features of achondroplasia in the studied cases: A - Plain X ray, lateral view of the skull showing frontal bossing, thick calvaria and relatively small skull base. B - Plain X ray, AP view of spine showing caudal narrowing of interpediculate distance. C - Plain X ray, lateral view of vertebrae showing platyspondyly with posterior scalloping and exaggerated lumbar lordosis. D - Plain X ray, AP view of the pelvis showing square iliac wings, fl at acetabular roof and narrow sacrosciatic notch. E - Plain X ray of long bones of right upper limb showing short broad long bones mainly in the humerus. F - Plain X ray of long bones of lower limbs showing short broad long bones, metaphyseal fl aring of the distal femoral ends taking the form of inverted-V (chevron) shaped metaphyseal notch. G - Plain X ray of both hands showing brachydactyly & trident confi guration. 19 Characteristics of Egyptian achondroplasia patients Neuroimaging of the brain revealed Results of Molecular study: abnormal fi ndings in 8 cases (40.0 %). The G380R common mutations were Dilated ventricles and cortical atrophic detected in 15 patients out of the 18 changes were present in 3 cases (15%). ascertained cases on clinical and radio- Cortical atrophy was noted in 2 cases logical bases (83%). The observed mu- (10%) while dilated ventricles only tation in 14 of the studied cases (77%) were seen in 1 case (5%). CT scan brain was the G to A transition at nucleotide of one case showed agenesis of corpus 1138, located in the transmembrane do- callosum (Figure. 7 A). MRI of cranio- main of FGFR3 as shown in (Figure. 8). cervical region in another case revealed The detected G to A point mutation cre- narrow foramen magnum without spi- ates a Scf I enzyme produced two frag- nal cord compression (Figure. 7 B). ments of 109 bp and 55 bp.

On the other hand digestion of the same 164bp PCR fragment with MspI edo- nuclease (indicating G to C transver- sion) produced two fragments of 107bp and 57bp. This MspI restriction site was evident in only one patient (5%) (Figure. 8, lane 10). The two common mutations were not detected in three pa- tients (16%), who were consistent with the clinical and radiological criteria of achondroplasia.

Fig. 7: CT scan brain of achondroplasia case with the G to C transversion at nucleotide 1138 of FGFR3 showing: colpocephaly, anterior dis- placement of 4th ventricle & dilated 3rd ventricle denoting agenesis of corpus callosum (A).

Sagittal section in MRI of craniocervical re- gion in an achondroplasia case showing nar- Fig. 8: FGFR-3 mutations in some of the studied row foramen magnum without spinal cord cases with achondroplasia. compression (B).

20 Aglan et al. DISCUSSION noted, full extension and rotation of the elbow was restricted in most cases. In our study, all cases were sporadic ex- Kitoh and associates24 found limited el- cept one case with autosomal dominant bow extension in 68% of their studied inheritance. Seven cases were associ- cases with achondroplasia. In our study ated with advanced paternal age denot- thoracolumbar kyphosis was present in ing dominant mutations, a fi nding pre- only 5 cases with ages ranging from 9 viously confi rmed by Stoll et al.17 who months to 18 months. Cases above this noticed that de novo gene mutations age had exaggerated lumbar lordosis were associated with advanced paternal due to improvement of hypotonia with age, often defi ned as over 35 years. age. Motor milestones of development were delayed in 50% of our cases due Prolonged heat exposure because of the to associated early hypotonia. father’s occupation was noted in 6 of the studied cases starting at the age of In our study all cases were dispropor- twenties, a fi nding that was not specifi - tionately short with short limbs mainly cally reported with achondroplasia but proximal and relatively long narrow of signifi cant importance as exposure to trunk. Quantitatively confi rmed by heat is known to increase the exposure anthropometric measurements, it was noted that short stature becomes more of spermatozoa to mutagenic metabo- 18 distinctive with age. US/LS ratio was lites. Tarin et al. suggested that heat above the 97th centile in all cases in- exposure in the period before concep- dicating that disproportionate short tion could theoretically increase de stature was caused primarily as a re- novo mutations or alter the epigenetic sult of short limbs. Although total up- modifi cation of imprinted , par- per limb length was below normal in ticularly because late spermatids and all our studied cases, arm lengths were mature spermatozoa do not have repair the mostly affected thus, quantitatively . supporting the rhizomelic shortening in achondroplasia. All the clinical and Parental exposure to chemical agents in anthropometric data in our cases were consistent with the fi ndings reported by this study was present in 3 cases. Some Horton et al.3 of the most common exposures that have been studied for increased risk of Although the clinical features of achon- achondroplasia, but for which no asso- droplasia are so distinctive and can be ciation had been found included: che- easily identifi ed at birth in most cases, motherapy19, anticonvulsant drugs20, Nicoletti et al.25 said that about 20% of antihistaminics21, corticosteroids, cal- affected individuals are not recognized cium channel blockers22 and thalido- at birth. This was evident in case 1 as mide.23 short stature was not present at the age of 9 months. Achondroplasia curves Diagnosis of achondroplasia is based should be used whenever possible for upon clinical fi ndings and confi rmed affected cases. In our study the few by radiological and molecular studies. cases lying below -2.0 SD on achon- All included cases had the typical facial droplasia curves can be attributed to the features described in achondroplasia. patient’s genetic potential, possibility Although, hyperextensibility of knee, of associated growth hormone defi cien- wrist and interphalangeal joints was cy and the use of standards from other 21 Characteristics of Egyptian achondroplasia patients countries due to the unavailability of achondroplasia from other countries. To achondroplasia curves for Egyptians. our knowledge, the only available mo- lecular study on Egyptian patients with Skeletal survey was carried out for all achondroplasia was carried out by Ab- the studied cases to explore the charac- del-Aleem et al.28. The authors detected teristic radiological features of achon- G380R common mutation with the G to droplasia. Our results were consistent A transition at nucleotide 1138 in 8 out with the radiological criteria for diag- of 11 cases ascertained on clinical basis nosis of achondroplasia reported by as achondroplasia. Same results were Spranger et al.26 found in all of the 16 studied Sweden patients with achondroplasia29, ten Chi- Although clinical manifestations and nese achondroplastic patients30, eleven radiological investigations are crucial of 12 Turkish patients31 and three Thai for the diagnosis of achondroplasia, patients.5 defi nitive diagnosis and prenatal con- fi rmation in high risk pregnancies are The two common mutations were not carried out by molecular analysis. In detected in three of the studied patients the present study 14 out of 18 cases had who had the clinical and radiological the G-A transition and 1 case had the criteria of achondroplasia. This sug- G-to-C transversion at nucleotide 1138 gests the existence of other less fre- of the FGFR3 gene. Agenesis of corpus quent mutations in the FGFR3 gene. callosum was noticed in our case with Further molecular studies for rare muta- the G-C transversion mutation. To our tions in achondroplasia were not avail- knowledge this fi nding has not been de- able for us. Reports of achondroplasia scribed before in the medical literature cases with mutations other than the neither with achondroplasia in general common G380R alterations were de- or with this mutation in particular. scribed by Ikegawa et al.32, Superti- Furga et al.33, Nishimura et al.34 and The common mutations detected in our Prinos et al.35 cases were in the heterozygous state, no homozygous gene mutation was identi- Care should be taken when consider- fi ed in our sample of achondroplasia pa- ing the diagnosis of achondroplasia tients. Homozygosity in achondroplasia as it must be confi rmed by radiologi- gene results in a severe lethal skeletal cal studies showing the defi nite fi nd- dysplasia with progressive hydrocepha- ings of achondroplasia and confi rmed lus, brain abnormalities and respiratory by molecular study if possible. In this failure due to narrow chest and pulmo- study less than 50% of referred cases as nary hypoplasia. Survival beyond in- achondroplasia (20 out of 42 referred fancy is rare.27 cases) were consistent with the clini- cal and radiological manifestations of Molecular results of common mutations achondroplasia and were included in at nucleotide 1138 of the FGFR3 gene this study. of our achondroplasia patients coincide with other studies concluding that the Discovery of the genetic basis of achon- majority of Egyptian achondroplasia droplasia opened the door for explora- patients had the same mutation that has tion of the biochemical mechanisms been most often defi ned in patients with that cause the underlying clinical fea- 22 Aglan et al. tures of the disorder. Studies exploring 5. Shotelersuk V, Ittiwut C, Srivuthana these biochemical mechanisms as well S, Wacharasindhu S, Aroonparkmon- as targeting the effects of the gene mu- gkol S, Mutirangura A, et al. 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