Ultrasound Obstet Gynecol 2014; 44: 719–721 Published online 27 October 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/uog.13381
First-trimester diagnosis of Meckel–Gruber syndrome by fetal ultrasound with molecular identification of CC2D2A mutations by next-generation sequencing D. JONES*, F. FIOZZO*, B. WATERS†, D. McKNIGHT‡ and S. BROWN* *Department of Obstetrics, Gynecology and Reproductive Sciences, University of Vermont, Burlington, VT, USA; †Department of Pathology, University of Vermont, Burlington, VT, USA; ‡GeneDx Inc., Gaithersburg, MD, USA
K E Y W O R D S: fetal malformation; gene mutation; Meckel–Gruber syndrome; next-generation sequencing
ABSTRACT We describe a first-trimester ultrasound examination in which the finding of fetal encephalocele and the cystic appearance of the kidneys raised suspicion of Meckel–Gruber syndrome (MKS). On the basis of sonographic findings, the patient elected termination of pregnancy, and post-termination studies using next-generation sequencing of a gene panel revealed two mutations (one previously described and the other novel) in the gene CC2D2A. Mutations in CC2D2A are known to cause MKS and Joubert syndrome, thus providing molecular confirmation of the clinical suspicion of MKS and opening the possibility for future prenatal diagnosis. This case highlights the ability to detect important anomalies in the first trimester using ultrasound, even in low-risk situations. It also demonstrates the growing role of new sequencing technologies in fetal testing. Copyright © 2014 ISUOG. Published by John Wiley & Sons Ltd.
CASE REPORT A 33-year-old woman, gravida 1, with unremarkable family history presented at 12 weeks’ gestation for first-trimester combined aneuploidy screening. Up to that point the pregnancy had been uneventful. A transvaginal scan at 8 weeks’ gestation was normal and confirmed menstrual dating. At the 12-week ultrasound evaluation, the fetus was noted to have protrusion of tissue from the occipital region of the skull consistent with a posterior encephalocele (Figure 1a). Both kidneys were noted to be cystic (Figures 1b and c). There was no evidence of polydactyly and the remainder of the findings were normal. The patient was informed that the findings might indicate a sporadic chromosome abnormality or could be the consequence of recessively inherited gene mutations
(e.g. Meckel–Gruber syndrome (MKS)). It was also noted that there might not be a recognizable genetic cause for the abnormalities. In light of the findings, the patient was informed that the prognosis was very poor regardless of the etiology. She elected immediate termination of pregnancy and desired comprehensive genetic evaluation. Termination was performed by suction dilatation and curettage so that gross inspection of the skull was impossible; however, at the time of the procedure fetal hands and feet were normal in appearance. Pathological examination of fetal kidneys confirmed them to be cystic (Figure 1d). Following termination, tissue from the placenta was submitted to GeneDx Inc. (Gaithersburg, MD, USA), a commercial laboratory, for chromosome microarray and comprehensive gene mutation testing using a multiple gene panel designed to include genes known to be involved in MKS, Joubert syndrome and other overlapping disorders. Findings of the chromosome microarray analysis were reported as normal. Next-generation sequencing (NGS) revealed two known mutations (pathogenic or expected to be pathogenic) in the CC2D2A gene. One of the mutations, c.3774dupT (E1259fsX1 or E1259X), which is predicted to replace glutamic acid at position 1259 with a stop codon, was previously reported in an individual with MKS1 . The other mutation, c4550 C > G, which changes threonine at amino acid position 1517 to serine (T1517S), was novel. Follow-up studies showed that each parent carried one of these mutations. The parents were informed that the combination of ultrasound, pathological and molecular genetic findings was consistent with a diagnosis of recessively inherited MKS and that the risk of recurrence was therefore expected to be 25%. Although the phenotype of a second affected pregnancy would be expected to be similar to that of the first pregnancy, the couple was also informed
Correspondence to: Dr S. Brown, University of Vermont, Given C258, 89 Beaumont, Burlington, VT 05405, USA (e-mail: [email protected]) Accepted: 26 March 2014
Copyright © 2014 ISUOG. Published by John Wiley & Sons Ltd.
CASE REPORT
720
Jones et al.
Figure 1 (a) Mid-sagittal view of fetus, showing protrusion of tissue at the posterior skull and location of the encephalocele (arrow). (b) Parasagittal view of right fetal kidney (arrowheads) with apparent cysts. (c) Parasagittal view of left fetal kidney (arrowheads), also with cystic structures. (d) Hematoxylin and eosin-stained coronal section of entire fetal kidney, showing obvious cysts. A, anterior; C, cephalad; H, hilum of kidney; P, posterior.
about phenotypic variability. CC2D2A mutations have also been described in Joubert syndrome, for which ultrasound findings would be expected to be much more subtle. Therefore, prenatal diagnosis, including first-trimester molecular diagnosis, was offered for subsequent pregnancies and the possibility of preimplantation genetic diagnosis was discussed and offered.
DISCUSSION MKS is a recessive condition, most commonly characterized by cystic kidneys, posterior encephalocele, polydactyly and hepatic fibrosis2 . Recent advances in molecular genetic diagnosis have shown that MKS can be caused by mutations in at least five different genes1 . Importantly, it is now clear that all of the genes involved in MKS affect the structure or function of the cilium, a hair-like organelle present on the surface of most cell types. Recently, diseases due to disorders of the structure and/or function of the cilium have been termed ‘ciliopathies’. Therefore, MKS is best thought of as being part of the large spectrum of ‘ciliopathy’ disorders that includes MKS, Joubert syndrome, COACH (cerebellar
Copyright © 2014 ISUOG. Published by John Wiley & Sons Ltd.
vermis hypoplasia, oligophrenia, ataxia, coloboma, hepatic fibrosis) syndrome, nephronophthisis, Senior–Løken syndrome and others (see Hildebrandt et al. for a review)3 . Among the ciliopathies, MKS manifests with the most striking fetal malformations, making it amenable to diagnosis by prenatal imaging. Ultrasonographic prenatal diagnosis of MKS was first accomplished in 19824 . Over the ensuing years most reported prenatal diagnoses have been in the second trimester, although first-trimester diagnoses have been described as well5 – 7 . In general, confirmation of the diagnosis of MKS has been based on pathological findings and family history. Therefore, the case we present is unusual, not only because it was diagnosed as early as 12 weeks’ gestation, but also because it occurred in a low-risk patient with no prior history and because the diagnosis was confirmed by post-termination molecular genetic testing. This case highlights the growing importance of NGS in clinical diagnosis. Given the number and complexity of genes involved in MKS or in brain malformations in general, it would be impractical and not cost effective to analyze sequentially candidate genes using conventional sequencing methods. The clinical availability
Ultrasound Obstet Gynecol 2014; 44: 719–721.
Meckel–Gruber mutations of NGS panels for potentially relevant genes made it possible to provide a firm diagnosis of MKS after termination of pregnancy which, in turn, allows for specific testing in future pregnancies. We anticipate that NGS will have an increasing role in the diagnosis of genetic disorders associated with serious or fatal fetal abnormalities. The gene identified in this family, CC2D2A, was first described in Joubert syndrome and MKS in 20088,9 . Since then, further reports have confirmed the role of CC2D2A in both MKS and Joubert syndrome, while other studies have elucidated the role of CC2D2A in ciliary function10 . The present case also illustrates a common question that arises in the context of genetic testing, particularly with multigene panels: how does one determine whether a sequence variant is benign or is a disease-causing mutation? In this case, the pathogenicity of c.377dupT was established based on its nature; frameshift mutations are expected to result in a loss of normal protein function either through protein truncation or nonsense-mediated mRNA decay. For the novel missense variant T1517S, several pieces of evidence strongly implicate this sequence variant as being a bona fide mutation. First, threonine at this position is conserved across multiple vertebrate species, including zebrafish and, among invertebrates, is conserved in fruit flies and other insects. Second, SIFT (SIFT.jcvi.org) and Polyphen (genetics.bwh.harvard.edu/pph2/), two algorithms used for determining whether an amino acid change will be deleterious to protein function, both indicate a high probability that T1517S is deleterious. Additionally, T1517S is not reported in the 1000 Genomes database (1000.genomes.org) or the NHLBI Exome Sequencing Project (http://evs.gs.washington.edu/EVS/), indicating that it is not a common benign variant in these populations. Based on these criteria, T1517S was reported as ‘likely to be a disease-causing mutation’ by the testing laboratory. In conclusion, this case illustrates that fetal ultrasound findings can be used in combination with post-termination NGS to provide specific molecular diagnoses. In this case,
Copyright © 2014 ISUOG. Published by John Wiley & Sons Ltd.
721
the patient will benefit from the opportunity to have molecular diagnosis in future pregnancies.
REFERENCES 1. Otto EA, Ramaswami G, Janssen S, Chaki M, Allen SJ, Zhou W, Airik R, Hurd TW, Ghosh AK, Wolf MT, Hoppe B, Neuhaus TJ, Bockenhauer D, Milford DV, Soliman NA, Antignac C, Saunier S, Johnson CA, Hildebrandt F; GPN Study Group. Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy. J Med Genet 2011; 48: 105–116. 2. Fraser FC, Lytwyn A. Spectrum of anomalies in the Meckel syndrome, or: ‘Maybe there is a malformation syndrome with at least one constant anomaly’. Am J Med Gen 1981; 9: 67–73. 3. Hildebrandt F, Benzing T, Katsanis N. Ciliopathies. N Engl J Med 2011; 364: 1533–1543. 4. Schmidt W, Kubli F. Early diagnosis of severe congenital malformations by ultrasonography. J Perinat Med 1982; 10: 233–241. 5. Eckmann-Scholz C, Jonat W, Zerres K, Ortiz-Bruchle N. Earliest ultrasound findings and description of splicing mutations in Meckel–Gruber syndrome. Arch Gynecol Obstet 2012; 286: 917–921. 6. Ickowicz V, Eurin D, Maugey-Laulom B, Didier F, Garel C, Gubler MC, Laquerri`ere A, Avni EF. Meckel–Gruber syndrome: sonography and pathology. Ultrasound Obstet Gynecol 2006; 27: 296–300. 7. Liu SS, Cheong ML, She BQ, Tsai MS. First-trimester ultrasound diagnosis of Meckel-Gruber syndrome. Acta Obstet Gynecol Scand 2006; 85: 757–759. 8. Noor A, Windpassinger C, Patel M, Stachowiak B, Mikhailov A, Azam M, Irfan M, Siddiqui ZK, Naeem F, Paterson AD, Lutfullah M, Vincent JB, Ayub M. CC2D2A, encoding a coiled-coil and C2 domain protein, causes autosomal-recessive mental retardation with retinitis pigmentosa. Am J Hum Genet 2008; 82: 1011–1018. 9. Tallila J, Jakkula E, Peltonen L, Salonen R, Kestila M. Identification of CC2D2A as a Meckel syndrome gene adds an important piece to the ciliopathy puzzle. Am J Hum Genet 2008; 82: 1361–1367. 10. Williams CL, Li C, Kida K, Inglis PN, Mohan S, Semenec L, Bialas NJ, Stupay RM, Chen N, Blacque OE, Yoder BK, Leroux MR. MKS and NPHP modules cooperate to establish basal body/transition zone membrane associations and ciliary gate function during ciliogenesis. J Cell Biol 2011; 192: 1023–1041.
Ultrasound Obstet Gynecol 2014; 44: 719–721.
First-trimester diagnosis of Meckel–Gruber syndrome by fetal ultrasound with molecular identification of CC2D2A mutations by next-generation sequencing D. JONES*, F. FIOZZO*, B. WATERS†, D. McKNIGHT‡ and S. BROWN* *Department of Obstetrics, Gynecology and Reproductive Sciences, University of Vermont, Burlington, VT, USA; †Department of Pathology, University of Vermont, Burlington, VT, USA; ‡GeneDx Inc., Gaithersburg, MD, USA
K E Y W O R D S: fetal malformation; gene mutation; Meckel–Gruber syndrome; next-generation sequencing
ABSTRACT We describe a first-trimester ultrasound examination in which the finding of fetal encephalocele and the cystic appearance of the kidneys raised suspicion of Meckel–Gruber syndrome (MKS). On the basis of sonographic findings, the patient elected termination of pregnancy, and post-termination studies using next-generation sequencing of a gene panel revealed two mutations (one previously described and the other novel) in the gene CC2D2A. Mutations in CC2D2A are known to cause MKS and Joubert syndrome, thus providing molecular confirmation of the clinical suspicion of MKS and opening the possibility for future prenatal diagnosis. This case highlights the ability to detect important anomalies in the first trimester using ultrasound, even in low-risk situations. It also demonstrates the growing role of new sequencing technologies in fetal testing. Copyright © 2014 ISUOG. Published by John Wiley & Sons Ltd.
CASE REPORT A 33-year-old woman, gravida 1, with unremarkable family history presented at 12 weeks’ gestation for first-trimester combined aneuploidy screening. Up to that point the pregnancy had been uneventful. A transvaginal scan at 8 weeks’ gestation was normal and confirmed menstrual dating. At the 12-week ultrasound evaluation, the fetus was noted to have protrusion of tissue from the occipital region of the skull consistent with a posterior encephalocele (Figure 1a). Both kidneys were noted to be cystic (Figures 1b and c). There was no evidence of polydactyly and the remainder of the findings were normal. The patient was informed that the findings might indicate a sporadic chromosome abnormality or could be the consequence of recessively inherited gene mutations
(e.g. Meckel–Gruber syndrome (MKS)). It was also noted that there might not be a recognizable genetic cause for the abnormalities. In light of the findings, the patient was informed that the prognosis was very poor regardless of the etiology. She elected immediate termination of pregnancy and desired comprehensive genetic evaluation. Termination was performed by suction dilatation and curettage so that gross inspection of the skull was impossible; however, at the time of the procedure fetal hands and feet were normal in appearance. Pathological examination of fetal kidneys confirmed them to be cystic (Figure 1d). Following termination, tissue from the placenta was submitted to GeneDx Inc. (Gaithersburg, MD, USA), a commercial laboratory, for chromosome microarray and comprehensive gene mutation testing using a multiple gene panel designed to include genes known to be involved in MKS, Joubert syndrome and other overlapping disorders. Findings of the chromosome microarray analysis were reported as normal. Next-generation sequencing (NGS) revealed two known mutations (pathogenic or expected to be pathogenic) in the CC2D2A gene. One of the mutations, c.3774dupT (E1259fsX1 or E1259X), which is predicted to replace glutamic acid at position 1259 with a stop codon, was previously reported in an individual with MKS1 . The other mutation, c4550 C > G, which changes threonine at amino acid position 1517 to serine (T1517S), was novel. Follow-up studies showed that each parent carried one of these mutations. The parents were informed that the combination of ultrasound, pathological and molecular genetic findings was consistent with a diagnosis of recessively inherited MKS and that the risk of recurrence was therefore expected to be 25%. Although the phenotype of a second affected pregnancy would be expected to be similar to that of the first pregnancy, the couple was also informed
Correspondence to: Dr S. Brown, University of Vermont, Given C258, 89 Beaumont, Burlington, VT 05405, USA (e-mail: [email protected]) Accepted: 26 March 2014
Copyright © 2014 ISUOG. Published by John Wiley & Sons Ltd.
CASE REPORT
720
Jones et al.
Figure 1 (a) Mid-sagittal view of fetus, showing protrusion of tissue at the posterior skull and location of the encephalocele (arrow). (b) Parasagittal view of right fetal kidney (arrowheads) with apparent cysts. (c) Parasagittal view of left fetal kidney (arrowheads), also with cystic structures. (d) Hematoxylin and eosin-stained coronal section of entire fetal kidney, showing obvious cysts. A, anterior; C, cephalad; H, hilum of kidney; P, posterior.
about phenotypic variability. CC2D2A mutations have also been described in Joubert syndrome, for which ultrasound findings would be expected to be much more subtle. Therefore, prenatal diagnosis, including first-trimester molecular diagnosis, was offered for subsequent pregnancies and the possibility of preimplantation genetic diagnosis was discussed and offered.
DISCUSSION MKS is a recessive condition, most commonly characterized by cystic kidneys, posterior encephalocele, polydactyly and hepatic fibrosis2 . Recent advances in molecular genetic diagnosis have shown that MKS can be caused by mutations in at least five different genes1 . Importantly, it is now clear that all of the genes involved in MKS affect the structure or function of the cilium, a hair-like organelle present on the surface of most cell types. Recently, diseases due to disorders of the structure and/or function of the cilium have been termed ‘ciliopathies’. Therefore, MKS is best thought of as being part of the large spectrum of ‘ciliopathy’ disorders that includes MKS, Joubert syndrome, COACH (cerebellar
Copyright © 2014 ISUOG. Published by John Wiley & Sons Ltd.
vermis hypoplasia, oligophrenia, ataxia, coloboma, hepatic fibrosis) syndrome, nephronophthisis, Senior–Løken syndrome and others (see Hildebrandt et al. for a review)3 . Among the ciliopathies, MKS manifests with the most striking fetal malformations, making it amenable to diagnosis by prenatal imaging. Ultrasonographic prenatal diagnosis of MKS was first accomplished in 19824 . Over the ensuing years most reported prenatal diagnoses have been in the second trimester, although first-trimester diagnoses have been described as well5 – 7 . In general, confirmation of the diagnosis of MKS has been based on pathological findings and family history. Therefore, the case we present is unusual, not only because it was diagnosed as early as 12 weeks’ gestation, but also because it occurred in a low-risk patient with no prior history and because the diagnosis was confirmed by post-termination molecular genetic testing. This case highlights the growing importance of NGS in clinical diagnosis. Given the number and complexity of genes involved in MKS or in brain malformations in general, it would be impractical and not cost effective to analyze sequentially candidate genes using conventional sequencing methods. The clinical availability
Ultrasound Obstet Gynecol 2014; 44: 719–721.
Meckel–Gruber mutations of NGS panels for potentially relevant genes made it possible to provide a firm diagnosis of MKS after termination of pregnancy which, in turn, allows for specific testing in future pregnancies. We anticipate that NGS will have an increasing role in the diagnosis of genetic disorders associated with serious or fatal fetal abnormalities. The gene identified in this family, CC2D2A, was first described in Joubert syndrome and MKS in 20088,9 . Since then, further reports have confirmed the role of CC2D2A in both MKS and Joubert syndrome, while other studies have elucidated the role of CC2D2A in ciliary function10 . The present case also illustrates a common question that arises in the context of genetic testing, particularly with multigene panels: how does one determine whether a sequence variant is benign or is a disease-causing mutation? In this case, the pathogenicity of c.377dupT was established based on its nature; frameshift mutations are expected to result in a loss of normal protein function either through protein truncation or nonsense-mediated mRNA decay. For the novel missense variant T1517S, several pieces of evidence strongly implicate this sequence variant as being a bona fide mutation. First, threonine at this position is conserved across multiple vertebrate species, including zebrafish and, among invertebrates, is conserved in fruit flies and other insects. Second, SIFT (SIFT.jcvi.org) and Polyphen (genetics.bwh.harvard.edu/pph2/), two algorithms used for determining whether an amino acid change will be deleterious to protein function, both indicate a high probability that T1517S is deleterious. Additionally, T1517S is not reported in the 1000 Genomes database (1000.genomes.org) or the NHLBI Exome Sequencing Project (http://evs.gs.washington.edu/EVS/), indicating that it is not a common benign variant in these populations. Based on these criteria, T1517S was reported as ‘likely to be a disease-causing mutation’ by the testing laboratory. In conclusion, this case illustrates that fetal ultrasound findings can be used in combination with post-termination NGS to provide specific molecular diagnoses. In this case,
Copyright © 2014 ISUOG. Published by John Wiley & Sons Ltd.
721
the patient will benefit from the opportunity to have molecular diagnosis in future pregnancies.
REFERENCES 1. Otto EA, Ramaswami G, Janssen S, Chaki M, Allen SJ, Zhou W, Airik R, Hurd TW, Ghosh AK, Wolf MT, Hoppe B, Neuhaus TJ, Bockenhauer D, Milford DV, Soliman NA, Antignac C, Saunier S, Johnson CA, Hildebrandt F; GPN Study Group. Mutation analysis of 18 nephronophthisis associated ciliopathy disease genes using a DNA pooling and next generation sequencing strategy. J Med Genet 2011; 48: 105–116. 2. Fraser FC, Lytwyn A. Spectrum of anomalies in the Meckel syndrome, or: ‘Maybe there is a malformation syndrome with at least one constant anomaly’. Am J Med Gen 1981; 9: 67–73. 3. Hildebrandt F, Benzing T, Katsanis N. Ciliopathies. N Engl J Med 2011; 364: 1533–1543. 4. Schmidt W, Kubli F. Early diagnosis of severe congenital malformations by ultrasonography. J Perinat Med 1982; 10: 233–241. 5. Eckmann-Scholz C, Jonat W, Zerres K, Ortiz-Bruchle N. Earliest ultrasound findings and description of splicing mutations in Meckel–Gruber syndrome. Arch Gynecol Obstet 2012; 286: 917–921. 6. Ickowicz V, Eurin D, Maugey-Laulom B, Didier F, Garel C, Gubler MC, Laquerri`ere A, Avni EF. Meckel–Gruber syndrome: sonography and pathology. Ultrasound Obstet Gynecol 2006; 27: 296–300. 7. Liu SS, Cheong ML, She BQ, Tsai MS. First-trimester ultrasound diagnosis of Meckel-Gruber syndrome. Acta Obstet Gynecol Scand 2006; 85: 757–759. 8. Noor A, Windpassinger C, Patel M, Stachowiak B, Mikhailov A, Azam M, Irfan M, Siddiqui ZK, Naeem F, Paterson AD, Lutfullah M, Vincent JB, Ayub M. CC2D2A, encoding a coiled-coil and C2 domain protein, causes autosomal-recessive mental retardation with retinitis pigmentosa. Am J Hum Genet 2008; 82: 1011–1018. 9. Tallila J, Jakkula E, Peltonen L, Salonen R, Kestila M. Identification of CC2D2A as a Meckel syndrome gene adds an important piece to the ciliopathy puzzle. Am J Hum Genet 2008; 82: 1361–1367. 10. Williams CL, Li C, Kida K, Inglis PN, Mohan S, Semenec L, Bialas NJ, Stupay RM, Chen N, Blacque OE, Yoder BK, Leroux MR. MKS and NPHP modules cooperate to establish basal body/transition zone membrane associations and ciliary gate function during ciliogenesis. J Cell Biol 2011; 192: 1023–1041.
Ultrasound Obstet Gynecol 2014; 44: 719–721.
