Fetal and Pediatric Pathology, 34:57–64, 2015 C Informa Healthcare USA, Inc. Copyright ISSN: 1551-3815 print / 1551-3823 online DOI: 10.3109/15513815.2014.962198
ORIGINAL ARTICLE
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Diagnosis of Fetal Osteogenesis imperfecta by Multidisciplinary Assessment: A Retrospective Study of 10 Cases Qichang Wu,1 Wenbo Wang,1 Lin Cao,2 Li Sun,1 Yasong Xu,1 and Xiaohong Zhong1 1
Prenatal Diagnosis Center of Xiamen’s Maternal & Child Health Care Hospital, Xiamen, Fujian, China; 2 Clinical laboratory Center of Beijing Genomics Institute at Shenzhen, Shenzhen, China
Objective: To describe our 2 year experience in diagnosing prenatal-onset osteogenesis imperfecta (OI) by multidisciplinary assessment. Methods: We retrospectively analyzed 10 cases of fetal OI by using prenatal ultrasound evaluation, postnatal radiographic diagnosis, and molecular genetic testing of COL1A1/2. Results: By postnatal radiographic examination, five patients were diagnosed with type II OI and five were diagnosed with type III OI. A causative variant in the COL1A1 gene was found in four cases of type II and one case of type III OI; a causative variant in the COL1A2 gene was found in two cases of type III OI. Conclusion: The definitive diagnosis of fetal OI should be accomplished using a multidisciplinary assessment, which is paramount for proper genetic counseling. With the discovery of COL1A1/2 gene variants as a cause of OI, sequence analysis of these genes will add to the diagnostic process. Keywords: osteogenesis imperfecta, ultrasound, radiograph, collagen type I, gene, COL1A1/2
INTRODUCTION Recent developments in prenatal ultrasonography have made it easy to detect fetal skeletal dysplasia, especially severe short-limb dwarfism. The differential diagnosis for fetal, severe, short-limbs should include achondrogenesis, thanatophoric dysplasia, osteogenesis imperfecta (OI), and other disorders. According to a recent study on prenatal sonographic diagnosis of fetal skeletal dysplasia, OI was the second most common disorder [1]. OI is an inherited bone fragility disorder with a wide range of clinical severities that are normally caused by mutations in COL1A1 or COL1A2, the genes that encode two type I collagen alpha chains [2]. With the discovery of COL1A1/2 gene variants as a cause of OI, we recognized that the sequence analysis of these genes should add to the diagnostic process. In order to specify a diagnosis, ultrasound, radiography, and molecular genetic examinations are often required. In this study, we describe our experience in diagnosing prenatal-onset OI by using a multidisciplinary assessment that included prenatal ultrasound evaluation, postnatal radiographic diagnosis, and molecular genetic testing of COL1A1/2. Received 28 January 2014; Revised 27 August 2014; accepted 2 September 2014. Address correspondence to Dr. Qichang Wu, Prenatal Diagnosis Center of Xiamen’s Maternal & Child Health Care Hospital, Xiamen, Fujian, China. E-mail: qichang [email protected]
Q. Wu et al.
A comprehensive multidisciplinary assessment is important for proper genetic counseling.
MATERIALS AND METHODS
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We diagnosed 10 cases of fetal OI between November 2010 and December 2012. The detected gestational ages of these cases ranged from 19 to 27 weeks, in women ranging from 20 to 35 years of age. Among these women, seven were primigravida and three were multigravida. These women were in non-consanguineous marriages and had a normal course of pregnancy; their family histories were devoid of other reports of skeletal malformations. Prenatal Ultrasound Evaluation The aim of prenatal ultrasound evaluations is to predict fetal prognosis. A poor prognosis, neonatal or infantile lethality, was determined using the following criteria. First, sonographic measurements that demonstrated a fetal femur and/or humerus length that was >4 standard deviations below the mean femoral length (FL) and/or humeral length (HL) for the gestational age, as predicted by Hadlock et al. [3]. Second, ultrasound findings such as a narrow thorax, protuberant abdomen, bent bones, bone mineralization, multiple bone fractures, polyhydramnios, and other associated malformations were considered. Because of the poor, even lethal, outcomes predicted by these findings, the families decided to terminate the pregnancies. In China, pregnancy can be terminated in the third trimester. Before termination of each pregnancy, cordocentesis was done for fetal karyotyping and molecular analysis. Postnatal Radiographic Diagnosis The radiological diagnostic criteria, based on the description by Sillence et al. [4] in 1979, are summarized in Table 1.
Table 1.
Radiological diagnostic criteria of Osteogenesis imperfecta types I–IV.
Type
I
Skull
Wormian bones
Ribs
No fractures
Extremities
Normal modeling at birth
Vertebrae
Normal at birth
Other
Usually no congenital fractures or osteopenia
II
III
IV
Severely diminished mineralization, wormian bones Short, broadened ribs with continuous beading/fractures Thick, short, crumpled long bone shafts Platyspondyly at birth Generalized osteopenia, multiple fractures with callus formation
Diminished mineralization, wormian bones
Diminished mineralization, sometimes with wormian bones No congenital fractures
Thin ribs with discontinuous beading/fractures
Short, deformed or Bowing of the long long, tubular bones bones Platyspondyly at Normal at birth birth Generalized Generalized osteopenia osteopenia, multiple fractures with callus formation
Fetal and Pediatric Pathology
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Multidisciplinary Assessment of Fetal Osteogenesis Imperfect
Molecular Genetic Analysis of COL1A1 and COL1A2 Qualified genomic DNA fragments were randomly interrupted (Covaris, Woburn, MA, USA) to produce 200–250 bp random pieces, and then adaptors were ligated to the fragmented DNA. The extracted DNA fragments involved amplification and purification using ligation-mediated polymerase chain reaction (LM-PCR), purified, and hybridized to the 2.1-M human microarray chips (Roche NimbleGen, Madison, WI, USA) for enrichment, and the non-hybridized fragments were washed out. The DNA libraries used high-fidelity DNA polymerase to amplify using highthroughput sequencing in an HiSeq2000 (Illumina) sequencing platform. The sequencing data and original images were analyzed using the CASAVA Software 1.7 (Illumina) for base calling adopting the default parameters, and the sequence of each individual was generated as 90-bp paired-end reads. Then, the bioinformatics analysis began from the sequencing data generated by the Illumina pipeline using Burrows–Wheeler Aligner (BWA), which generated the alignment output in sequence alignment/map (SAM) format. (http://samtools.sourceforge.net/SAM1.pdf). Finally, to test the mutation sites further, PolyPhen (http://genetics.bwh.harvard.edu/pph/) and SIFT (http://sift.jcvi.org/www/siftchrcoords submit. html) software were utilized to predict the pathogenic effect of the function of a protein.
RESULTS The results of ultrasound findings, radiographic examinations, and molecular genetic analyses of the 10 fetuses are shown in Table 2. The fetal karyotypes of the 10 fetuses were normal.
Table 2.
Details of the referral and evaluation findings of 10 cases.
Maternal age (y)
Gestational age (weeks)
Ultrasound finding
Radiographic diagnosis
Molecular analysis results
F1
26
23
Type II, Figure 1(a)
F2
26
25
FL, 2.1 cm; bent bones, narrow thorax FL, 1.6 cm; narrow thorax
F3
27
23
Type II, Figure 1(c)
F4
20
19
FL, 1.5 cm; diminished mineralization FL, 1.3 cm; bent bones, narrow thorax
F5
25
25
F6
27
F7
No gene mutation found COL1A1, p.Gly689Asp, c.2066G>A, Gly>Asp COL1A1, p.Gly695Ser, c.2083G>A, Gly>Ser COL1A1, p.Gly419Glu, c.1256G>A, Gly>Glu COL1A1, p.Val1289Leu, c.3865G>A, Val>Leu COL1A2, p.Arg906His c.2717G>A, Arg>His No gene mutation found No gene mutation found COL1A2, p.Gly1042Ser, c.3124G>A, Gly>Ser COL1A1, p.Gly1187Arg, c.3559G>C, Gly>Arg
Case
Type II, Figure 1(b)
Type II, Figure 1(d)
Type II, Figure 1(e)
27
FL, 1.9 cm; narrow thorax, diminished mineralization FL, 2.7 cm; bent bones
23
25
FL, 1.9 cm; bent bones
Type III, Figure 2(b)
F8
25
26
FL, 2.0 cm; bent bones
Type III, Figure 2(c)
F9
25
26
FL, 1.7 cm; bent bones
Type III, Figure 2(d)
F10
28
22
FL, 1.4 cm; bent bones
Type III, Figure 2(e)
FL, femoral length. C Informa Healthcare USA, Inc. Copyright
Type III, Figure 2(a)
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Q. Wu et al.
Figure 1. Perinatal OI type II. Radiography shows the notable under mineralization of the calvarium. The ribs show multiple fractures in a discontinuous pattern with normal rib parts in between callus formation (discontinuous beading). The long bones of the lower extremities are broadened, deformed and shortened as a result of multiple fractures.
DISCUSSION Prenatal Ultrasound Evaluation The skeletal dysplasias are a heterogenous group of over 350 distinct disorders of skeletogenesis. Many are manifested in the prenatal period, making them amenable to ultrasound diagnosis. The fetal skeleton is readily visualized by two-dimensional ultrasound at 14 weeks of gestation, and measurement of the fetal femora and humeri are considered part of any basic midtrimester ultrasound evaluation [5]. Any fetus showing femora or humeri length measurements less than the 5th centile or −2 SD from the mean in the second trimester should be evaluated at a center with expertise in evaluating the entire fetal skeleton. A series of fetal ultrasound parameters must be visualized and plotted against normative values. These measurements should include the fetal cranium, abdominal circumference, mandible, clavicle, scapula, chest circumference, and all fetal long bones. In addition, close attention should be paid to the fetal facial profile, the presence and shape of the vertebral bodies, and the shape and Fetal and Pediatric Pathology
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Multidisciplinary Assessment of Fetal Osteogenesis Imperfect
Figure 2. Perinatal OI type III. Radiography shows diminished but visible mineralization of the calvarium. The ribs are slender without fractures. Multiple fractures of femora can be observed.
mineralization pattern of the fetal calvarium and skeleton, as these can be helpful in differentiating the potential disorders. Krakow et al. reported [6] the utilization of ultrasound to analyze a constellation of findings, enabling a differential diagnosis. In this study, we reported 10 cases of fetal OI, all with long bone measurements more than 4 SD below the mean, strongly indicative of severe short-limb dwarfism. One of the characteristics of lethal OI is severe micromelia, typically with irregular bending of the long bones and ribs due to multiple intrauterine fractures. The affected bones are very short and irregular, and the thorax is narrow. The spine is also sometimes irregular in appearance, with platyspondyly due to fractures of the vertebrae. Most authors have indicated that these ultrasound findings are not necessarily pathognomic for distinguishing or diagnosing definite OI [7]. Our experience with prenatal ultrasound evaluation indicates that the FL is the basic and most important parameter for detecting skeletal dysplasia and for predicting fetal prognosis. The most important determination made by ultrasound in our study was that all cases had a poor prognosis, with either neonatal or infantile lethality probable. C Informa Healthcare USA, Inc. Copyright
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Q. Wu et al.
Postnatal Radiographic Diagnosis OI is a heritable skeletal disorder characterized by bone fragility and, often, short stature. The spectrum of clinical severity is broad, ranging from nearly asymptomatic individuals with a mild predisposition toward fractures, normal stature, and a normal life-span, to neonates with severe bone deformities, mobility impairments, and very short stature to prenatal lethality [8]. Because a specific diagnosis of fetal skeletal dysplasia, by prenatal sonographic examination, is a difficult and subjective task, postnatal radiographic examination is the major method for distinguishing the different disorders. Radiographs provide more information regarding the appearance of the spine, skull, pelvis, the length and shape of the long bones, and the presence of overall calcification or other abnormalities. These observations provide the best proof for making a specific diagnosis. Classically, four types of OI are distinguishable, as described by Sillence et al. in 1979 [4]. This classification is based on clinical features, primarily radiological features. The radiological diagnostic criteria are summarized in Table 1. We diagnosed five cases of type II and five cases of type III OI in the present study, according to the criteria indicated in the table. Molecular Genetic Testing of COL1A1 and COL1A2 OI comprises a group of heterogenous disorders, with an estimated 90% of cases due to a causative variant in the COL1A1 or COL1A2 genes, and with the remaining 10% due to causative recessive variants in eight known genes, or in other, currently unknown genes [9]. COL1A1/2-related OI is inherited in an autosomal dominant manner. The proportion of cases caused by a de novo COL1A1 or COL1A2 mutation varies by disease severity; virtually 100% of perinatal lethal OI, and close to 100% of progressively deforming OI are the result of de novo mutations [10]. Gonadal mosaicism may be present in 3–5% of cases [11]. The COL1A1 and COL1A2 genes code for alpha-1 and alpha-2 type I collagen. Type I collagen is a heterotrimer consisting of two alpha-1 chains and one alpha-2 chain. It is initially synthesized as a proalpha chain with a propeptide at each end. The propeptides are necessary for proalpha chain association and triple helix formation. The triple helical domains are composed of uninterrupted repeats of the Gly-X-Y tripeptide [12]. The formation of the triple helix requires a glycine residue at every third position in the chains because glycine is the only amino acid small enough to fit into the restricted space at the inside of the helix. OI types II–IV are usually caused by sequence variants in either COL1A1 or COL1A2 that result in substitutions for glycine residues in the uninterrupted Gly-X-Y triplet repeat of the 1014-residue triple-helical domains encoded by each gene [13]. Two exclusively lethal regions in the alpha-1 chain (helix positions 691–823 and 910–964) and eight clusters of lethality in the alpha-2 chain have been identified [14]. These lethal areas align with the major ligand-binding regions for extracellular matrix proteins, which might explain the lethal outcome of mutations occurring at these sites. Even though the identification of some “lethal regions” provides a useful rule of thumb, there are many exceptions to the rule. So far, more than 1000 distinct variants in the COL1A1 and COL1A2 gene have been identified to cause OI [15]. In our study, 7 of the 10 cases were identified to have distinct heterozygous type I collagen mutations. Of the seven samples in which collagen mutations were identified, five had causative mutations in COL1A1 and two in COL1A2. Among the five distinct COL1A1 causative mutations, four had not been previously seen, and these four cases were radiographically diagnosed as type II OI. The two distinct COL1A2 causative mutations that were identified in our study have not been previously reported, and both were radiographically diagnosed as type III OI. We identified the mutations in samples from four subjects with type II and three subjects with type III OI. Very little is known Fetal and Pediatric Pathology
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Multidisciplinary Assessment of Fetal Osteogenesis Imperfect
about how a specific collagen type I mutation leads to a particular phenotype. Some studies indicate that glycine substitutions in the alpha-1 chains of type I collagen are more frequent than mutations in the alpha-2 chain and are more likely to have a lethal outcome [16]. Our results not only support this view, but also contribute to the understanding of the etiology of OI by providing data to evaluate and refine current models relating genotype to phenotype and by providing an unbiased indication of the relative frequency of mutations in the OI-associated genes. The use of prenatal ultrasound examination to differentiate and diagnose fetal OI can be challenging. At present, a definitive diagnosis of fetal OI is initially based on morphological, but mostly radiological, features. Advances in molecular genetics have helped elucidate the biological basis and the genotype–phenotype correlations of OI, making this technique amenable to invasive prenatal diagnoses, in addition to the classical morphological diagnostic approach. Although the occurrence of individual skeletal dysplasias such as OI may be rare, as a group, they account for a significant number of newborns with congenital anomalies. The definitive diagnosis of the type of skeletal dysplasia should be accomplished by multidisciplinary assessment, and it is paramount for proper genetic counseling. ACKNOWLEDGEMENT This study was supported by the Science and Technology Committee of Fujian Province (2011D008). Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
REFERENCES [1] Schramm T, Gloning KP, Minderer S, et al. Prenatal sonographic diagnosis of skeletal dysplasis. Ultrasound Obstet Gynecol 2009;l34:160–170. [2] Byers PH, Tsipouras P, Bonadio JF, et al. Perinatal lethal Osteogenesis imperfecta (OI typeII): a biochemically heterogeneous disorder usually due to new mutations in the genes for type 1 collagen. Am J Hum Genet 1988;42:237–248. [3] Hadlock FP, Deter RL, Harrist RB, Park SK. Estimating fetal age: computer-assisted analysis of multiple fetal growth parameters. Radiology 1984;152:497–501. [4] Sillence DO, Senn A, Danks DM. Genetic heterogeneity in Osteogenesis imperfecta. J Med Genet 1979; 16:101–116. [5] Bahado-Singh RO, Raymond M. The 20-week scan: beyond biometry and anatomy. Clin Obstet Gynecol 2007;50:478–486. [6] Krakow D, Lachman RS, Rimoin DL. Guidelines for the prenatal diagnosis of fetal skeletal dysplasias. Genet Med 2009; 11:127–133. [7] Superti-Furga A, Bonafe L, Rimoin DL. Molecular-pathogenetic classification of genetic disorders of the skeleton. Am J Med Genet 2001;106:282–293. [8] Rauch F, Glorieux FH. Osteogenesis imperfecta. The Lancet 2004; 363:1377–1385. [9] Van Dijk FS, Cobben JM, Kariminejad A, et al. Osteogenesis imperfecta: a review with clinical examples. Mol Syndromol 2011;2:1–20. [10] Pepin M, Atkinson M, Starman BJ, Byers PH. Strategies and outcomes of prenatal diagnosis for Osteogenesis imperfecta: a review of biochemical and molecular studies completed in 129 pregnancies. Prenat Diagn 1997;17:559–570. [11] Steiner RD, Adsit J, Basel D. COL1A1/2-related Osteogenesis imperfecta. Gene ReviewsTM [Internet]. Seattle, WA: University of Washington; 1993–2013. [12] Ben Amor IM, Glorieux FH, Fauch F. Genotype-phenotype correlations in autosomal dominant Osteogenesis imperfecta. J Osteoporosis 2011;540178. C Informa Healthcare USA, Inc. Copyright
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[13] Bodian DL, Chan TF, Poon A, et al. Mutation and polymorphism spectrum in Osteogenesis imperfecta type II: implications for genotype-phenotype relationships. Hum Mol Genet 2009; 18:463–471. [14] Marini JC, Forlino A, Cabral WA, et al. Consortium for Osteogenesis imperfecta mutations in the helical domain of type I collagen: regions rich in lethal mutations align with collagen binding sites for integrins and proteoglycans. Hum Mutat 2007; 28:209–221. [15] Van Dijk FS, Byers PH, Dalgleish R, et al. EMQN best practice guidelines for the laboratory diagnosis of Osteogenesis imperfecta. Eur J Hum Genet 2012;20:11–19. [16] Rauch F, Lalic L, Roughley P, Glorieux FH. Genotype-phenotype correlations in nonlethal Osteogenesis imperfecta caused by mutations in the helical domain of collagen type I. Eur J Hum Genet 2010; 18:642–647.
Fetal and Pediatric Pathology
ORIGINAL ARTICLE
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Diagnosis of Fetal Osteogenesis imperfecta by Multidisciplinary Assessment: A Retrospective Study of 10 Cases Qichang Wu,1 Wenbo Wang,1 Lin Cao,2 Li Sun,1 Yasong Xu,1 and Xiaohong Zhong1 1
Prenatal Diagnosis Center of Xiamen’s Maternal & Child Health Care Hospital, Xiamen, Fujian, China; 2 Clinical laboratory Center of Beijing Genomics Institute at Shenzhen, Shenzhen, China
Objective: To describe our 2 year experience in diagnosing prenatal-onset osteogenesis imperfecta (OI) by multidisciplinary assessment. Methods: We retrospectively analyzed 10 cases of fetal OI by using prenatal ultrasound evaluation, postnatal radiographic diagnosis, and molecular genetic testing of COL1A1/2. Results: By postnatal radiographic examination, five patients were diagnosed with type II OI and five were diagnosed with type III OI. A causative variant in the COL1A1 gene was found in four cases of type II and one case of type III OI; a causative variant in the COL1A2 gene was found in two cases of type III OI. Conclusion: The definitive diagnosis of fetal OI should be accomplished using a multidisciplinary assessment, which is paramount for proper genetic counseling. With the discovery of COL1A1/2 gene variants as a cause of OI, sequence analysis of these genes will add to the diagnostic process. Keywords: osteogenesis imperfecta, ultrasound, radiograph, collagen type I, gene, COL1A1/2
INTRODUCTION Recent developments in prenatal ultrasonography have made it easy to detect fetal skeletal dysplasia, especially severe short-limb dwarfism. The differential diagnosis for fetal, severe, short-limbs should include achondrogenesis, thanatophoric dysplasia, osteogenesis imperfecta (OI), and other disorders. According to a recent study on prenatal sonographic diagnosis of fetal skeletal dysplasia, OI was the second most common disorder [1]. OI is an inherited bone fragility disorder with a wide range of clinical severities that are normally caused by mutations in COL1A1 or COL1A2, the genes that encode two type I collagen alpha chains [2]. With the discovery of COL1A1/2 gene variants as a cause of OI, we recognized that the sequence analysis of these genes should add to the diagnostic process. In order to specify a diagnosis, ultrasound, radiography, and molecular genetic examinations are often required. In this study, we describe our experience in diagnosing prenatal-onset OI by using a multidisciplinary assessment that included prenatal ultrasound evaluation, postnatal radiographic diagnosis, and molecular genetic testing of COL1A1/2. Received 28 January 2014; Revised 27 August 2014; accepted 2 September 2014. Address correspondence to Dr. Qichang Wu, Prenatal Diagnosis Center of Xiamen’s Maternal & Child Health Care Hospital, Xiamen, Fujian, China. E-mail: qichang [email protected]
Q. Wu et al.
A comprehensive multidisciplinary assessment is important for proper genetic counseling.
MATERIALS AND METHODS
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We diagnosed 10 cases of fetal OI between November 2010 and December 2012. The detected gestational ages of these cases ranged from 19 to 27 weeks, in women ranging from 20 to 35 years of age. Among these women, seven were primigravida and three were multigravida. These women were in non-consanguineous marriages and had a normal course of pregnancy; their family histories were devoid of other reports of skeletal malformations. Prenatal Ultrasound Evaluation The aim of prenatal ultrasound evaluations is to predict fetal prognosis. A poor prognosis, neonatal or infantile lethality, was determined using the following criteria. First, sonographic measurements that demonstrated a fetal femur and/or humerus length that was >4 standard deviations below the mean femoral length (FL) and/or humeral length (HL) for the gestational age, as predicted by Hadlock et al. [3]. Second, ultrasound findings such as a narrow thorax, protuberant abdomen, bent bones, bone mineralization, multiple bone fractures, polyhydramnios, and other associated malformations were considered. Because of the poor, even lethal, outcomes predicted by these findings, the families decided to terminate the pregnancies. In China, pregnancy can be terminated in the third trimester. Before termination of each pregnancy, cordocentesis was done for fetal karyotyping and molecular analysis. Postnatal Radiographic Diagnosis The radiological diagnostic criteria, based on the description by Sillence et al. [4] in 1979, are summarized in Table 1.
Table 1.
Radiological diagnostic criteria of Osteogenesis imperfecta types I–IV.
Type
I
Skull
Wormian bones
Ribs
No fractures
Extremities
Normal modeling at birth
Vertebrae
Normal at birth
Other
Usually no congenital fractures or osteopenia
II
III
IV
Severely diminished mineralization, wormian bones Short, broadened ribs with continuous beading/fractures Thick, short, crumpled long bone shafts Platyspondyly at birth Generalized osteopenia, multiple fractures with callus formation
Diminished mineralization, wormian bones
Diminished mineralization, sometimes with wormian bones No congenital fractures
Thin ribs with discontinuous beading/fractures
Short, deformed or Bowing of the long long, tubular bones bones Platyspondyly at Normal at birth birth Generalized Generalized osteopenia osteopenia, multiple fractures with callus formation
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Multidisciplinary Assessment of Fetal Osteogenesis Imperfect
Molecular Genetic Analysis of COL1A1 and COL1A2 Qualified genomic DNA fragments were randomly interrupted (Covaris, Woburn, MA, USA) to produce 200–250 bp random pieces, and then adaptors were ligated to the fragmented DNA. The extracted DNA fragments involved amplification and purification using ligation-mediated polymerase chain reaction (LM-PCR), purified, and hybridized to the 2.1-M human microarray chips (Roche NimbleGen, Madison, WI, USA) for enrichment, and the non-hybridized fragments were washed out. The DNA libraries used high-fidelity DNA polymerase to amplify using highthroughput sequencing in an HiSeq2000 (Illumina) sequencing platform. The sequencing data and original images were analyzed using the CASAVA Software 1.7 (Illumina) for base calling adopting the default parameters, and the sequence of each individual was generated as 90-bp paired-end reads. Then, the bioinformatics analysis began from the sequencing data generated by the Illumina pipeline using Burrows–Wheeler Aligner (BWA), which generated the alignment output in sequence alignment/map (SAM) format. (http://samtools.sourceforge.net/SAM1.pdf). Finally, to test the mutation sites further, PolyPhen (http://genetics.bwh.harvard.edu/pph/) and SIFT (http://sift.jcvi.org/www/siftchrcoords submit. html) software were utilized to predict the pathogenic effect of the function of a protein.
RESULTS The results of ultrasound findings, radiographic examinations, and molecular genetic analyses of the 10 fetuses are shown in Table 2. The fetal karyotypes of the 10 fetuses were normal.
Table 2.
Details of the referral and evaluation findings of 10 cases.
Maternal age (y)
Gestational age (weeks)
Ultrasound finding
Radiographic diagnosis
Molecular analysis results
F1
26
23
Type II, Figure 1(a)
F2
26
25
FL, 2.1 cm; bent bones, narrow thorax FL, 1.6 cm; narrow thorax
F3
27
23
Type II, Figure 1(c)
F4
20
19
FL, 1.5 cm; diminished mineralization FL, 1.3 cm; bent bones, narrow thorax
F5
25
25
F6
27
F7
No gene mutation found COL1A1, p.Gly689Asp, c.2066G>A, Gly>Asp COL1A1, p.Gly695Ser, c.2083G>A, Gly>Ser COL1A1, p.Gly419Glu, c.1256G>A, Gly>Glu COL1A1, p.Val1289Leu, c.3865G>A, Val>Leu COL1A2, p.Arg906His c.2717G>A, Arg>His No gene mutation found No gene mutation found COL1A2, p.Gly1042Ser, c.3124G>A, Gly>Ser COL1A1, p.Gly1187Arg, c.3559G>C, Gly>Arg
Case
Type II, Figure 1(b)
Type II, Figure 1(d)
Type II, Figure 1(e)
27
FL, 1.9 cm; narrow thorax, diminished mineralization FL, 2.7 cm; bent bones
23
25
FL, 1.9 cm; bent bones
Type III, Figure 2(b)
F8
25
26
FL, 2.0 cm; bent bones
Type III, Figure 2(c)
F9
25
26
FL, 1.7 cm; bent bones
Type III, Figure 2(d)
F10
28
22
FL, 1.4 cm; bent bones
Type III, Figure 2(e)
FL, femoral length. C Informa Healthcare USA, Inc. Copyright
Type III, Figure 2(a)
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Figure 1. Perinatal OI type II. Radiography shows the notable under mineralization of the calvarium. The ribs show multiple fractures in a discontinuous pattern with normal rib parts in between callus formation (discontinuous beading). The long bones of the lower extremities are broadened, deformed and shortened as a result of multiple fractures.
DISCUSSION Prenatal Ultrasound Evaluation The skeletal dysplasias are a heterogenous group of over 350 distinct disorders of skeletogenesis. Many are manifested in the prenatal period, making them amenable to ultrasound diagnosis. The fetal skeleton is readily visualized by two-dimensional ultrasound at 14 weeks of gestation, and measurement of the fetal femora and humeri are considered part of any basic midtrimester ultrasound evaluation [5]. Any fetus showing femora or humeri length measurements less than the 5th centile or −2 SD from the mean in the second trimester should be evaluated at a center with expertise in evaluating the entire fetal skeleton. A series of fetal ultrasound parameters must be visualized and plotted against normative values. These measurements should include the fetal cranium, abdominal circumference, mandible, clavicle, scapula, chest circumference, and all fetal long bones. In addition, close attention should be paid to the fetal facial profile, the presence and shape of the vertebral bodies, and the shape and Fetal and Pediatric Pathology
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Multidisciplinary Assessment of Fetal Osteogenesis Imperfect
Figure 2. Perinatal OI type III. Radiography shows diminished but visible mineralization of the calvarium. The ribs are slender without fractures. Multiple fractures of femora can be observed.
mineralization pattern of the fetal calvarium and skeleton, as these can be helpful in differentiating the potential disorders. Krakow et al. reported [6] the utilization of ultrasound to analyze a constellation of findings, enabling a differential diagnosis. In this study, we reported 10 cases of fetal OI, all with long bone measurements more than 4 SD below the mean, strongly indicative of severe short-limb dwarfism. One of the characteristics of lethal OI is severe micromelia, typically with irregular bending of the long bones and ribs due to multiple intrauterine fractures. The affected bones are very short and irregular, and the thorax is narrow. The spine is also sometimes irregular in appearance, with platyspondyly due to fractures of the vertebrae. Most authors have indicated that these ultrasound findings are not necessarily pathognomic for distinguishing or diagnosing definite OI [7]. Our experience with prenatal ultrasound evaluation indicates that the FL is the basic and most important parameter for detecting skeletal dysplasia and for predicting fetal prognosis. The most important determination made by ultrasound in our study was that all cases had a poor prognosis, with either neonatal or infantile lethality probable. C Informa Healthcare USA, Inc. Copyright
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Q. Wu et al.
Postnatal Radiographic Diagnosis OI is a heritable skeletal disorder characterized by bone fragility and, often, short stature. The spectrum of clinical severity is broad, ranging from nearly asymptomatic individuals with a mild predisposition toward fractures, normal stature, and a normal life-span, to neonates with severe bone deformities, mobility impairments, and very short stature to prenatal lethality [8]. Because a specific diagnosis of fetal skeletal dysplasia, by prenatal sonographic examination, is a difficult and subjective task, postnatal radiographic examination is the major method for distinguishing the different disorders. Radiographs provide more information regarding the appearance of the spine, skull, pelvis, the length and shape of the long bones, and the presence of overall calcification or other abnormalities. These observations provide the best proof for making a specific diagnosis. Classically, four types of OI are distinguishable, as described by Sillence et al. in 1979 [4]. This classification is based on clinical features, primarily radiological features. The radiological diagnostic criteria are summarized in Table 1. We diagnosed five cases of type II and five cases of type III OI in the present study, according to the criteria indicated in the table. Molecular Genetic Testing of COL1A1 and COL1A2 OI comprises a group of heterogenous disorders, with an estimated 90% of cases due to a causative variant in the COL1A1 or COL1A2 genes, and with the remaining 10% due to causative recessive variants in eight known genes, or in other, currently unknown genes [9]. COL1A1/2-related OI is inherited in an autosomal dominant manner. The proportion of cases caused by a de novo COL1A1 or COL1A2 mutation varies by disease severity; virtually 100% of perinatal lethal OI, and close to 100% of progressively deforming OI are the result of de novo mutations [10]. Gonadal mosaicism may be present in 3–5% of cases [11]. The COL1A1 and COL1A2 genes code for alpha-1 and alpha-2 type I collagen. Type I collagen is a heterotrimer consisting of two alpha-1 chains and one alpha-2 chain. It is initially synthesized as a proalpha chain with a propeptide at each end. The propeptides are necessary for proalpha chain association and triple helix formation. The triple helical domains are composed of uninterrupted repeats of the Gly-X-Y tripeptide [12]. The formation of the triple helix requires a glycine residue at every third position in the chains because glycine is the only amino acid small enough to fit into the restricted space at the inside of the helix. OI types II–IV are usually caused by sequence variants in either COL1A1 or COL1A2 that result in substitutions for glycine residues in the uninterrupted Gly-X-Y triplet repeat of the 1014-residue triple-helical domains encoded by each gene [13]. Two exclusively lethal regions in the alpha-1 chain (helix positions 691–823 and 910–964) and eight clusters of lethality in the alpha-2 chain have been identified [14]. These lethal areas align with the major ligand-binding regions for extracellular matrix proteins, which might explain the lethal outcome of mutations occurring at these sites. Even though the identification of some “lethal regions” provides a useful rule of thumb, there are many exceptions to the rule. So far, more than 1000 distinct variants in the COL1A1 and COL1A2 gene have been identified to cause OI [15]. In our study, 7 of the 10 cases were identified to have distinct heterozygous type I collagen mutations. Of the seven samples in which collagen mutations were identified, five had causative mutations in COL1A1 and two in COL1A2. Among the five distinct COL1A1 causative mutations, four had not been previously seen, and these four cases were radiographically diagnosed as type II OI. The two distinct COL1A2 causative mutations that were identified in our study have not been previously reported, and both were radiographically diagnosed as type III OI. We identified the mutations in samples from four subjects with type II and three subjects with type III OI. Very little is known Fetal and Pediatric Pathology
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Multidisciplinary Assessment of Fetal Osteogenesis Imperfect
about how a specific collagen type I mutation leads to a particular phenotype. Some studies indicate that glycine substitutions in the alpha-1 chains of type I collagen are more frequent than mutations in the alpha-2 chain and are more likely to have a lethal outcome [16]. Our results not only support this view, but also contribute to the understanding of the etiology of OI by providing data to evaluate and refine current models relating genotype to phenotype and by providing an unbiased indication of the relative frequency of mutations in the OI-associated genes. The use of prenatal ultrasound examination to differentiate and diagnose fetal OI can be challenging. At present, a definitive diagnosis of fetal OI is initially based on morphological, but mostly radiological, features. Advances in molecular genetics have helped elucidate the biological basis and the genotype–phenotype correlations of OI, making this technique amenable to invasive prenatal diagnoses, in addition to the classical morphological diagnostic approach. Although the occurrence of individual skeletal dysplasias such as OI may be rare, as a group, they account for a significant number of newborns with congenital anomalies. The definitive diagnosis of the type of skeletal dysplasia should be accomplished by multidisciplinary assessment, and it is paramount for proper genetic counseling. ACKNOWLEDGEMENT This study was supported by the Science and Technology Committee of Fujian Province (2011D008). Declaration of Interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
REFERENCES [1] Schramm T, Gloning KP, Minderer S, et al. Prenatal sonographic diagnosis of skeletal dysplasis. Ultrasound Obstet Gynecol 2009;l34:160–170. [2] Byers PH, Tsipouras P, Bonadio JF, et al. Perinatal lethal Osteogenesis imperfecta (OI typeII): a biochemically heterogeneous disorder usually due to new mutations in the genes for type 1 collagen. Am J Hum Genet 1988;42:237–248. [3] Hadlock FP, Deter RL, Harrist RB, Park SK. Estimating fetal age: computer-assisted analysis of multiple fetal growth parameters. Radiology 1984;152:497–501. [4] Sillence DO, Senn A, Danks DM. Genetic heterogeneity in Osteogenesis imperfecta. J Med Genet 1979; 16:101–116. [5] Bahado-Singh RO, Raymond M. The 20-week scan: beyond biometry and anatomy. Clin Obstet Gynecol 2007;50:478–486. [6] Krakow D, Lachman RS, Rimoin DL. Guidelines for the prenatal diagnosis of fetal skeletal dysplasias. Genet Med 2009; 11:127–133. [7] Superti-Furga A, Bonafe L, Rimoin DL. Molecular-pathogenetic classification of genetic disorders of the skeleton. Am J Med Genet 2001;106:282–293. [8] Rauch F, Glorieux FH. Osteogenesis imperfecta. The Lancet 2004; 363:1377–1385. [9] Van Dijk FS, Cobben JM, Kariminejad A, et al. Osteogenesis imperfecta: a review with clinical examples. Mol Syndromol 2011;2:1–20. [10] Pepin M, Atkinson M, Starman BJ, Byers PH. Strategies and outcomes of prenatal diagnosis for Osteogenesis imperfecta: a review of biochemical and molecular studies completed in 129 pregnancies. Prenat Diagn 1997;17:559–570. [11] Steiner RD, Adsit J, Basel D. COL1A1/2-related Osteogenesis imperfecta. Gene ReviewsTM [Internet]. Seattle, WA: University of Washington; 1993–2013. [12] Ben Amor IM, Glorieux FH, Fauch F. Genotype-phenotype correlations in autosomal dominant Osteogenesis imperfecta. J Osteoporosis 2011;540178. C Informa Healthcare USA, Inc. Copyright
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[13] Bodian DL, Chan TF, Poon A, et al. Mutation and polymorphism spectrum in Osteogenesis imperfecta type II: implications for genotype-phenotype relationships. Hum Mol Genet 2009; 18:463–471. [14] Marini JC, Forlino A, Cabral WA, et al. Consortium for Osteogenesis imperfecta mutations in the helical domain of type I collagen: regions rich in lethal mutations align with collagen binding sites for integrins and proteoglycans. Hum Mutat 2007; 28:209–221. [15] Van Dijk FS, Byers PH, Dalgleish R, et al. EMQN best practice guidelines for the laboratory diagnosis of Osteogenesis imperfecta. Eur J Hum Genet 2012;20:11–19. [16] Rauch F, Lalic L, Roughley P, Glorieux FH. Genotype-phenotype correlations in nonlethal Osteogenesis imperfecta caused by mutations in the helical domain of collagen type I. Eur J Hum Genet 2010; 18:642–647.
Fetal and Pediatric Pathology
