Clin Genet 2016: 89: 109–114 Printed in Singapore. All rights reserved
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: 10.1111/cge.12572
Short Report
Phenotype analysis impacts testing strategy in patients with Currarino syndrome Cuturilo G., Hodge J.C., Runke C.K., Thorland E.C., Al-Owain M.A., Ellison J.W., Babovic-Vuksanovic D. Phenotype analysis impacts testing strategy in patients with Currarino syndrome. Clin Genet 2016: 89: 109–114. © John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2015 Currarino syndrome (OMIM 175450) presents with sacral, anorectal, and intraspinal anomalies and presacral meningocele or teratoma. Autosomal dominant loss-of-function mutations in the MNX1 gene cause nearly all familial and 30% of sporadic cases. Less frequently, a complex phenotype of Currarino syndrome can be caused by microdeletions of 7q containing MNX1. Here, we report one familial and three sporadic cases of Currarino syndrome. To determine the most efficient genetic testing approach for these patients, we have compared results from MNX1 sequencing, chromosomal microarray, and performed a literature search with analysis of genotype–phenotype correlation. Based on the relationship between the type of mutation (intragenic MNX1 mutations vs 7q microdeletion) and the presence of intellectual disability, growth retardation, facial dysmorphism, and associated malformations, we propose a testing algorithm. Patients with the classic Currarino triad of malformations but normal growth, intellect, and facial appearance should have MNX1 sequencing first, and only in the event of a normal result should the clinician proceed with chromosomal microarray testing. In contrast, if growth delay and/or facial dysmorphy and/or intellectual disability are present, chromosomal microarray should be the first method of choice for genetic testing. Conflict of interest
All authors declare no conflicts of interest
G. Cuturiloa,b , J.C. Hodgec,d , C.K. Runkec , E.C. Thorlandc , M.A. Al-Owaine,f , J.W. Ellisong and D. Babovic-Vuksanovicc,h a Faculty of Medicine, University of Belgrade, Belgrade, Serbia, b Department of Medical Genetics, University Children’s Hospital, Belgrade, Serbia, c Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA, d Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA, e Department of Medical Genetics, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia, f Department of Anatomy and Cell Biology, College of Medicine, Alfaisal University, Riyadh, Saudi Arabia, g Department of Genetics, Kaiser Permanente Medical Center, San Francisco, CA, USA, and h Department of Medical Genetics, Mayo Clinic, Rochester, MN, USA
Key words: chromosomal microarray – Currarino syndrome – distal 7q deletions – MNX1 gene – sequencing – testing algorithm Corresponding author: Dr Dusica Babovic-Vuksanovic, MD, Professor of Medical Genetics and Pediatrics, Chair, Department of Medical Genetics, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. Tel.: +507 284 3215; fax: +507 284 1067; e-mail: [email protected] Received 11 November 2014, revised and accepted for publication 12 February 2015
Currarino syndrome is the triad consisting of partial sacral agenesis with intact first sacral vertebra (‘sickle-shaped sacrum’), a presacral mass, and anorectal malformation. Other associated congenital malformations and/or developmental delay have been described (1–5). The syndrome is caused by haploinsufficiency
of the motor neuron and pancreas homeobox 1 (MNX1) gene on chromosome 7q36.3 that encodes a nuclear protein, homeodomain-containing transcription factor (6). Various loss-of-function intragenic mutations, as well as deletions of the 7q36 region, have been reported in association with Currarino syndrome (2, 7). The current
109
Cuturilo et al. diagnostic gold standard for these (micro)deletions is chromosomal microarray analysis, while most intragenic point mutations or small insertions/deletions can be detected by sequencing (2, 3, 7, 8). We propose a diagnostic algorithm for patients with suspected Currarino syndrome based on phenotype–genotype assessment and the extensive literature review. Materials and methods Sanger sequencing and quantitative real-time PCR of MNX1
For patient 1, PCR amplification and direct sequencing of all coding exons and flanking intronic sequences of MNX1 were carried out, followed by gene dosage analysis by quantitative real-time PCR (qPCR) with five intragenic amplicons in exons 1 (three amplicons), 2, and 3 (9). Whole-genome array
Chromosomal microarray testing on peripheral blood specimens from patients 2, 3, and 4 occurred in the Mayo Clinic Cytogenetics Laboratory using the 4 × 44 K oligonucleotide-based whole-genome cytogenomic platform (Agilent Technologies, Santa Clara, CA). Fluorescence in situ hybridization (FISH) and/or karyotype analysis using standard methods were used for confirmation of microarray findings.
exon 1 of the MNX1 gene was detected using sequencing. Parental testing was suggested, but not completed. Patient 2
A boy (Fig. 1b) was born at term to a healthy, unrelated couple. He had anal stenosis, sacral agenesis, tethered spinal cord, hypoplastic urethra, hypospadias, grades II–III/grade V right vesicoureteral reflux, and a neurogenic bladder. Infancy was characterized by pronounced feeding difficulties, poor weight gain, and global developmental delay. A left retinal coloboma, mild conductive hearing loss, and mild pulmonary valve stenosis were disclosed during follow-up. Karyotyping and subtelomeric FISH studies revealed a 46,XY,add(7)(q32).ish der(7)t(7;13)(q34; q347qter–,13qter+) result. Chromosomal microarray disclosed a 20-Mb deletion containing 222 genes including BRAF, EZH2, KCNH2, PRKAG2, SHH, LMBR1, MNX1, and DNAJB6. In addition, a terminal duplication of approximately 960 kilobases of the region 13q34-qter was present. The mother’s karyotype was normal, while father was not available for testing. The child has been monitored for heart rhythm disturbances since infancy because of haploinsufficiency of the KCNH2 gene, which has been associated with a chronic QT syndrome; no episodes of LQT2-triggered ventricular arrhythmias have been observed so far. Patient 3
Analysis of the literature
A focused literature analysis was performed in PubMed to identify all articles reporting an association of the Currarino phenotype with causative mutations of any type. ‘Currarino syndrome’ was the only key phrase used. The time period analyzed was between 1998 (when the molecular basis of Currarino syndrome was discovered) and 2014. Only the articles reporting unclassified genomic variants or asymptomatic carriers were excluded. Results Clinical, chromosome, whole-genome array, and molecular results Patient 1
The full-term male was diagnosed at birth with an imperforate anus, posterior urethral valve, ureterohydronephrosis, undescended left testis, and sacrococcygeal teratoma. He had no facial dysmorphism and his growth and development were normal (Fig. 1a). Both mother and maternal grandmother were born with an imperforate anus and C-shaped sacrum, without any other congenital abnormalities or developmental difficulties. Using qPCR, a 43-bp heterozygous frameshift deletion of the 5′ end and middle of exon 1 of the MNX1 was identified. In addition, an unclassified, heterozygous single-nucleotide substitution c.415C > G (p.L139V) in
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A 3-year-old boy was evaluated for anal stenosis, myelomeningocele, terminal cord lipoma, tethered cord, syringomyelia, and partial sacral agenesis with a normally formed lumbar vertebra. He had perineal hypospadias, left undescended testis, encopresis, and prolonged QT interval on EKG. His cognitive and motor developmental were severely delayed; he was small for age. Dysmorphic features included microcephaly, prominent ears, down-slanted palpebral fissures, broad nasal bridge and deep filtrum. Chromosomal microarray analysis revealed a terminal deletion of 153 oligonucleotide probes of the 7q36.1-qter region spanning approximately 9.2 Mb and a terminal duplication of 64 oligonucleotide probes within 14q32.32-qter region spanning approximately 3.5 Mb as a result of an unbalanced 7;14 translocation. Parental karyotypes and FISH testing showed normal results. The deleted interval at 7q contains approximately 72 genes, including dosage-sensitive KCNH2, PRKAG2, SHH, MNX1, and DNAJB6. According to OMIM, the deleted interval at 14q does not contain genes haploinsufficiency of which produces congenital malformations or intellectual disability. Patient 4
The male patient was born at term after an uncomplicated pregnancy to a young couple (first cousins) from the Middle East. He had low birth weight and had sacral agenesis, tethered spinal cord, and a sacral mass (intraspinal lipoma; Fig. 1d,e). At the age of 3 years, he
Diagnostic algorithm for Currarino syndrome (a)
(b)
(d)
(c)
(e)
Fig. 1. Facial features. (a) Patient 1 with normal cranial and facial appearance. (b) Patient 2 with microcephaly, low frontal hair line, ptosis of the left eyelid, depressed nasal bridge, prominent ears, prominent premaxillary area, full cheeks, smooth philtrum, hypoplastic mandible, and groove between lower lip and chin. (c) Patient 4 with low frontal hair line, large and cupped ears, and short and smooth philtrum. Lumbar spine magnetic resonance imaging (MRI) of patient 4. (d) The sagittal T1 and (e) sagittal T2 views of lumbar spine MRI shows a 0.8 × 0.8 × 2.0 cm intraspinal lipoma (arrows) at the L5-S1 level. Only the body of S1 is visualized, while the rest of the sacrum and coccygeal vertebrae are absent. Note the tethered spinal cord above the mass.
was short statured, had generalized hypotonia, microcephaly, severe developmental delay, and facial dysmorphism (Fig. 1c). Conventional chromosome and metaphase FISH studies disclosed a complex chromosome 7 rearrangement denoted as 46,XY,dup(7)(q32q36.1).ish der(7) (pter+,qter–). Chromosomal microarray analysis confirmed a 17.2-Mb interstitial duplication of the 7q33-q36.2 region, followed immediately by a 4.4-Mb terminal deletion of the 7q36.2 containing 20 genes (including SHH, LMBR1, and MNX1). Parental studies were normal. Results of literature analysis
The Table 1 gives an overview of patients with Currarino syndrome reported in the literature (1998–2014) with their phenotypic characteristics and causative mutations. Discussion
Currarino syndrome (OMIM 175450) is characterized by a variable phenotype as a consequence of a wide
range of MNX1 mutations and their variable expressivity (2, 3, 5, 8). The syndrome is caused by haploinsufficiency of the MNX1 gene arising from either intragenic mutations or distal 7q deletions of different sizes (3, 6). Mutations in MNX1 result in complex phenotypes including (i) hemisacrum or bifid sacrum with neurogenic bladder, recurrent urinary infections, or incontinence; (ii) presacral masses such as anterior meningocele, enteric cyst, or presacral teratoma; and (iii) anorectal malformations including atresia, stenosis, and fistula frequently complicated with chronic constipation or incontinence (2, 10, 11). Once this classical triad of Currarino syndrome is recognized, a thorough search for associated malformations and developmental difficulties as well as a genetic evaluation should be initiated. In this study, we describe four new patients with Currarino syndrome (one familial and three sporadic cases) and present an extensive review of literature (Table 1). Including the literature review, 205 patients with Currarino syndrome were analyzed for the presence of intellectual disability, growth retardation, facial dysmorphy, and associated malformations. We investigated whether there was a difference in the frequency distribution
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Cuturilo et al. Table 1. Phenotypic characteristics and causative mutations of patients with Currarino syndrome reported in the literature between 1998 and 2014 and from this study. Articles were excluded that reported unclassified genomic variants or asymptomatic carriers only Intellectual Growth Facial disability retardation dysmorphy Pathogenic cytogenetic deletions 20-Mb deletion of 7q32-qter and 960-Kb duplication of 13q34-qter 9.2-Mb deletion of 7q36.1-qter and 3.5-Mb duplication of 14q32.32-qter 4.4-Mb deletion of 7q36.2-qter and 17.2-Mb duplication of 7q33-q36.2 8.8-Mb deletion of 7q36 and 10.3-Mb duplication of 7q34-q35 46,XY inv7(p15.1;q34); 46,XY der(7)t(7;12) (q36.12;q24.21).pat; 46,XX; ins(4;7)(q25;q31.3q21.3) 46,XX/46,XX,add(7q36).ish der(7)(q36),t(2,7)(p22;q36) 46, XY,del(7)(q36.1).ish del(7)(q36.3q36.3) 2.7-Mb deletion Microdeletion including MNX1 genea
Associated anomalies
Number of patients
Reference Number
+
+
+
+
1
This study
+
+
+
+
1
This study
+
+
+
+
1
This study
+
+
+
+
1
(4)
2 of 3 pts
1 of 3 pts
2 of 3 pts
2 of 3 pts
3
(2)
n/a + n/a +
n/a + n/a 1 of 3 pts
+ + + 1 of 3 pts
n/a + + (urogenital) –
1 1 1 3
+ n/a
n/a n/a
n/a n/a
n/a n/a
3 1
(16) (3) (17) (5) and references therein (18) (11)
–
–
–
3
(19)
n/a – – – –
n/a – – – –
n/a – – – –
1 of 3 pts (holoprosencephaly) n/a – – – –
1 3 3 1 2
(20) This study (8) (21) (22)
– –
– –
2 12
Frameshift point mutations
–
–
–
(23) (24), (14), (25), and (26) (27)
Point mutations not further defined
–
–
–
n/a –
n/a –
n/a –
– – –
– – –
– – –
n/a
n/a
n/a
Microdeletion of 7qa Deletion 7qa Pathogenic MNX1 intragenic mutations Partial deletionsa
Insertions Missense mutation and intragenic deletion-insertion Missense point mutations Nonsense point mutations
Other intragenic mutations not further defined
– – 1 of 12 pts c – Hirschsprung disease –
n/a 1 of 6 pts (Hirschsprung disease) 5b – 3 of 50 pts (urogenital) n/a
1 20
36 6
(19), (28), (23), (29), (30), (31), and (32) (11), (18) (7)
23 (2) 24 (10/24)d (28) and (10) 50 (5/50)d (5) and (6) 1
(33)
n/a, not available; pts, patients. a Precise characterization of the mutation not provided in some of the articles. b Müllerian duplication in all, scoliosis in one, aortic stenosis in one. c Subtile facial dysmorphy (fetal pathology report). d Asymptomatic patients with radiologic findings only.
of these phenotypic characteristics between Currarino patients with MNX1 intragenic mutations (n = 188) and those with distal 7q deletions (n = 17). Intellectual disability
Data indicating intellectual status were available in 150/188 patients with intragenic mutations and 14/17 patients with 7q deletions. Intellectual disability was
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present in none of the patients with intragenic MNX1 mutations, and 13/14 (92.9%) of patients with larger distal 7q deletions 5 (12, 13) (Table 1). This implies that mutations limited to the MNX1 gene do not represent a risk of developmental delay; however, while for most of reported patients the exact age is not known, many were below age 1 year at time of evaluation. The lack of information about their prospective development represents a research bias of our study.
Diagnostic algorithm for Currarino syndrome
Fig. 2. Testing algorithm for patients with Currarino phenotype.
Growth delay
We obtained information about growth characteristics for 150/188 patients with intragenic mutations and 11/17 patients with larger 7q deletions and found growth deficiency in 0/150 and 7/11 (63.6%), respectively (Table 1). Thus, the presence of growth delay in a patient with Currarino syndrome raises a possibility for a larger chromosome 7q deletion, while normal growth parameters have less predictive value in terms of potential causative mutation. Facial dysmorphy
Facial morphology was evaluated in 150/188 patients with intragenic mutations and 13/17 patients with larger 7q deletions. Dysmorphic facial features were noted in 1/150 (0.6%) and 10/13 (76.9%) patients, respectively (Table 1). Only one patient with an intragenic MNX1 mutation, a fetus from a pregnancy terminated at 20 weeks of gestation, had subtle facial dysmorphism consisting of hypertelorism and retrognathism (14). These results suggest that facial dysmorphology is a strong indicator of the presence of a larger 7q deletion in Currarino patients, while intragenic mutations of the MNX1 gene rarely, if ever, produce dysmorphic facial features. Associated malformation
Besides the classical Currarino triad, the most common malformations reported in patients with Currarino syndrome are terminal spinal cord malformations (e.g. low laying conus, short and thickened filum, tethered cord, and spinal lipoma) and urogenital anomalies (2, 11). The available data from 150/188 patients with intragenic mutations and 12/17 patients with larger 7q deletions were analyzed. Associated anomalies were found in 11/150 (7.3%) and 8/12 (66.6%), respectively (Table 1). These data suggest that disclosure of congenital malformations, besides the classical Currarino
triad, positively predicts the presence of a larger 7q deletion. Chromosomal microarray (array-CGH or SNPmicroarray) has become the test of first choice in patients with multiple congenital malformations, especially in those with intellectual disability or in young infants in whom mental development is unpredictable (15). Even in situations when a specific deletion/duplication syndrome is suspected, this technique is often employed based on a phenotypic overlap between syndromes. Thus, a clinician could easily order chromosomal microarray testing in patients with the classical Currarino triad; however, the result will be negative in the majority of cases with an intragenic MNX1 mutation. Considering the results shown in this study, we believe that sequencing rather than chromosomal microarray analysis should be carried out in cases with normal growth, development, and facial appearance. However, in the case of a normal sequencing result, the clinician should undoubtedly proceed with chromosomal microarray, especially because other clinical features, including long QT syndrome, can be associated with 7q deletions. In contrast, if growth delay and/or facial dysmorphy and/or developmental delay are verified on examination, especially in the presence of associated malformations, chromosomal microarray is a method of first choice for genetic testing (Fig. 2). In conclusion, we suggest that in the era of more advanced techniques for genetic testing, careful clinical examination remains important in the everyday practice of genetics. This is manifested in the case of Currarino syndrome, as appreciation of facial morphology and early recognition of disturbances of growth or development could lead to a less expensive and more efficient, individualized approach for diagnostic testing. References 1. Currarino G, Coln D, Votteler T. Triad of anorectal, sacral, and presacral anomalies. AJR Am J Roentgenol 1981: 137: 395–398. 2. Cretolle C, Pelet A, Sanlaville D et al. Spectrum of HLXB9 gene mutations in Currarino syndrome and genotype-phenotype correlation. Hum Mutat 2008: 29: 903–910.
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Cuturilo et al. 3. Horn D, Tonnies H, Neitzel H et al. Minimal clinical expression of the holoprosencephaly spectrum and of Currarino syndrome due to different cytogenetic rearrangements deleting the Sonic Hedgehog gene and the HLXB9 gene at 7q36.3. Am J Med Genet A 2004: 128A: 85–92. 4. Pavone P, Ruggieri M, Lombardo I et al. Microcephaly, sensorineural deafness and Currarino triad with duplication-deletion of distal 7q. Eur J Pediatr 2010: 169: 475–481. 5. Hagan DM, Ross AJ, Strachan T et al. Mutation analysis and embryonic expression of the HLXB9 Currarino syndrome gene. Am J Hum Genet 2000: 66: 1504–1515. 6. Ross AJ, Ruiz-Perez V, Wang Y et al. A homeobox gene, HLXB9, is the major locus for dominantly inherited sacral agenesis. Nat Genet 1998: 20: 358–361. 7. Garcia-Barcelo MM, Lui VC, So MT et al. MNX1 (HLXB9) mutations in Currarino patients. J Pediatr Surg 2009: 44: 1892–1898. 8. Holm I, Monclair T, Lundar T, Stadheim B, Prescott TE, Eiklid KL. A 5.8 kb deletion removing the entire MNX1 gene in a Norwegian family with Currarino syndrome. Gene 2013: 518: 457–460. 9. Borozdin W, Boehm D, Leipoldt M et al. SALL4 deletions are a common cause of Okihiro and acro-renal-ocular syndromes and confirm haploinsufficiency as the pathogenic mechanism. J Med Genet 2004: 41: e113. 10. Kochling J, Karbasiyan M, Reis A. Spectrum of mutations and genotype-phenotype analysis in Currarino syndrome. Eur J Human Genet 2001: 9: 599–605. 11. Monclair T, Lundar T, Smevik B, Holm I, Orstavik KH. Currarino syndrome at Rikshospitalet 1961–2012. Tidsskr Nor Laegeforen 2013: 133: 2364–2368. 12. Li S, Mattar P, Dixit R et al. RAS/ERK signaling controls proneural genetic programs in cortical development and gliomagenesis. J Neurosci 2014: 34: 2169–2190. 13. Qi C, Liu S, Qin R et al. Coordinated regulation of dendrite arborization by epigenetic factors CDYL and EZH2. J Neurosci 2014: 34: 4494–4508. 14. Cretolle C, Sarnacki S, Amiel J et al. Currarino syndrome shown by prenatal onset ventriculomegaly and spinal dysraphism. Am J Med Genet A 2007: 143A: 871–874. 15. South ST, Lee C, Lamb AN, Higgins AW, Kearney HM. ACMG Standards and Guidelines for constitutional cytogenomic microarray analysis, including postnatal and prenatal applications: revision 2013. Genet Med 2013: 15: 901–909. 16. Le Caignec C, Winer N, Boceno M et al. Prenatal diagnosis of sacrococcygeal teratoma with constitutional partial monosomy 7q/trisomy 2p. Prenat Diagn 2003: 23: 981–984. 17. Coutton C, Poreau B, Devillard F et al. Currarino syndrome and HPE microform associated with a 2.7-Mb deletion in 7q36.3 excluding SHH gene. Mol Syndromol 2014: 5: 25–31.
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18. Lynch SA, Wang Y, Strachan T, Burn J, Lindsay S. Autosomal dominant sacral agenesis: Currarino syndrome. J Med Genet 2000: 37: 561–566. 19. Belloni E, Martucciello G, Verderio D et al. Involvement of the HLXB9 homeobox gene in Currarino syndrome. Am J Hum Genet 2000: 66: 312–319. 20. Iyer RS, Khanna PC. Currarino syndrome. Pediatr Radiol 2010: 40 (Suppl 1): S102. 21. Lin YH, Huang RL, Lai HC. Presacral teratoma in a Curarrino syndrome woman with an unreported insertion in MNX1 gene. Taiwan J Obstet Gynecol 2011: 50: 512–514. 22. Kim IS, Oh SY, Choi SJ et al. Clinical and genetic analysis of HLXB9 gene in Korean patients with Currarino syndrome. J Hum Genet 2007: 52: 698–701. 23. Ciotti P, Mandich P, Bellone E et al. Currarino syndrome with pelvic neuroendocrine tumor diagnosed by post-mortem genetic analysis of tissue specimens. Am J Med Genet A 2011: 155A: 2750–2753. 24. Fleury J, Picherot G, Cretolle C et al. Currarino syndrome as an etiology of a neonatal Escherichia coli meningitis. J Perinatol 2007: 27: 589–591. 25. Wang Y, Wu Y. A novel HLXB9 mutation in a Chinese family with Currarino syndrome. Eur J Pediatr Surg 2012: 22: 243–245. 26. Wang RY, Jones JR, Chen S et al. A previously unreported mutation in a Currarino syndrome kindred. Am J Med Genet A 2006: 140: 1923–1930. 27. Volk A, Karbasiyan M, Semmler A et al. Adult index patient with Currarino syndrome due to a novel HLXB9 mutation, c.336dupG (p.P113fsX224), presenting with Hirschsprung’s disease, cephalgia, and lumbodynia. Birth Defects Res A Clin Mol Teratol 2007: 79: 249–251. 28. Markljung E, Adamovic T, Cao J et al. Novel mutations in the MNX1 gene in two families with Currarino syndrome and variable phenotype. Gene 2012: 507: 50–53. 29. Zu S, Winberg J, Arnberg F et al. Mutation analysis of the motor neuron and pancreas homeobox 1 (MNX1, former HLXB9) gene in Swedish patients with Currarino syndrome. J Pediatr Surg 2011: 46: 1390–1395. 30. Kiefer AS, Gupta P, Kirmani S, Schwartz K, Henry N, Fischer PR. Treatment-resistant meningitis leading to the diagnosis of Currarino syndrome: a case report. Pediatr Infect Dis J 2009: 28: 547–549. 31. Garcia-Barcelo M, So MT, Lau DK et al. Population differences in the polyalanine domain and 6 new mutations in HLXB9 in patients with Currarino syndrome. Clin Chem 2006: 52: 46–52. 32. Martucciello G, Torre M, Belloni E et al. Currarino syndrome: proposal of a diagnostic and therapeutic protocol. J Pediatr Surg 2004: 39: 1305–1311. 33. Sekaran P, Brindley N. A case of Currarino’s syndrome presenting as neonatal bowel obstruction. J Pediatr Surg 2012: 47: 1600–1603.
© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: 10.1111/cge.12572
Short Report
Phenotype analysis impacts testing strategy in patients with Currarino syndrome Cuturilo G., Hodge J.C., Runke C.K., Thorland E.C., Al-Owain M.A., Ellison J.W., Babovic-Vuksanovic D. Phenotype analysis impacts testing strategy in patients with Currarino syndrome. Clin Genet 2016: 89: 109–114. © John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2015 Currarino syndrome (OMIM 175450) presents with sacral, anorectal, and intraspinal anomalies and presacral meningocele or teratoma. Autosomal dominant loss-of-function mutations in the MNX1 gene cause nearly all familial and 30% of sporadic cases. Less frequently, a complex phenotype of Currarino syndrome can be caused by microdeletions of 7q containing MNX1. Here, we report one familial and three sporadic cases of Currarino syndrome. To determine the most efficient genetic testing approach for these patients, we have compared results from MNX1 sequencing, chromosomal microarray, and performed a literature search with analysis of genotype–phenotype correlation. Based on the relationship between the type of mutation (intragenic MNX1 mutations vs 7q microdeletion) and the presence of intellectual disability, growth retardation, facial dysmorphism, and associated malformations, we propose a testing algorithm. Patients with the classic Currarino triad of malformations but normal growth, intellect, and facial appearance should have MNX1 sequencing first, and only in the event of a normal result should the clinician proceed with chromosomal microarray testing. In contrast, if growth delay and/or facial dysmorphy and/or intellectual disability are present, chromosomal microarray should be the first method of choice for genetic testing. Conflict of interest
All authors declare no conflicts of interest
G. Cuturiloa,b , J.C. Hodgec,d , C.K. Runkec , E.C. Thorlandc , M.A. Al-Owaine,f , J.W. Ellisong and D. Babovic-Vuksanovicc,h a Faculty of Medicine, University of Belgrade, Belgrade, Serbia, b Department of Medical Genetics, University Children’s Hospital, Belgrade, Serbia, c Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA, d Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, USA, e Department of Medical Genetics, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia, f Department of Anatomy and Cell Biology, College of Medicine, Alfaisal University, Riyadh, Saudi Arabia, g Department of Genetics, Kaiser Permanente Medical Center, San Francisco, CA, USA, and h Department of Medical Genetics, Mayo Clinic, Rochester, MN, USA
Key words: chromosomal microarray – Currarino syndrome – distal 7q deletions – MNX1 gene – sequencing – testing algorithm Corresponding author: Dr Dusica Babovic-Vuksanovic, MD, Professor of Medical Genetics and Pediatrics, Chair, Department of Medical Genetics, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. Tel.: +507 284 3215; fax: +507 284 1067; e-mail: [email protected] Received 11 November 2014, revised and accepted for publication 12 February 2015
Currarino syndrome is the triad consisting of partial sacral agenesis with intact first sacral vertebra (‘sickle-shaped sacrum’), a presacral mass, and anorectal malformation. Other associated congenital malformations and/or developmental delay have been described (1–5). The syndrome is caused by haploinsufficiency
of the motor neuron and pancreas homeobox 1 (MNX1) gene on chromosome 7q36.3 that encodes a nuclear protein, homeodomain-containing transcription factor (6). Various loss-of-function intragenic mutations, as well as deletions of the 7q36 region, have been reported in association with Currarino syndrome (2, 7). The current
109
Cuturilo et al. diagnostic gold standard for these (micro)deletions is chromosomal microarray analysis, while most intragenic point mutations or small insertions/deletions can be detected by sequencing (2, 3, 7, 8). We propose a diagnostic algorithm for patients with suspected Currarino syndrome based on phenotype–genotype assessment and the extensive literature review. Materials and methods Sanger sequencing and quantitative real-time PCR of MNX1
For patient 1, PCR amplification and direct sequencing of all coding exons and flanking intronic sequences of MNX1 were carried out, followed by gene dosage analysis by quantitative real-time PCR (qPCR) with five intragenic amplicons in exons 1 (three amplicons), 2, and 3 (9). Whole-genome array
Chromosomal microarray testing on peripheral blood specimens from patients 2, 3, and 4 occurred in the Mayo Clinic Cytogenetics Laboratory using the 4 × 44 K oligonucleotide-based whole-genome cytogenomic platform (Agilent Technologies, Santa Clara, CA). Fluorescence in situ hybridization (FISH) and/or karyotype analysis using standard methods were used for confirmation of microarray findings.
exon 1 of the MNX1 gene was detected using sequencing. Parental testing was suggested, but not completed. Patient 2
A boy (Fig. 1b) was born at term to a healthy, unrelated couple. He had anal stenosis, sacral agenesis, tethered spinal cord, hypoplastic urethra, hypospadias, grades II–III/grade V right vesicoureteral reflux, and a neurogenic bladder. Infancy was characterized by pronounced feeding difficulties, poor weight gain, and global developmental delay. A left retinal coloboma, mild conductive hearing loss, and mild pulmonary valve stenosis were disclosed during follow-up. Karyotyping and subtelomeric FISH studies revealed a 46,XY,add(7)(q32).ish der(7)t(7;13)(q34; q347qter–,13qter+) result. Chromosomal microarray disclosed a 20-Mb deletion containing 222 genes including BRAF, EZH2, KCNH2, PRKAG2, SHH, LMBR1, MNX1, and DNAJB6. In addition, a terminal duplication of approximately 960 kilobases of the region 13q34-qter was present. The mother’s karyotype was normal, while father was not available for testing. The child has been monitored for heart rhythm disturbances since infancy because of haploinsufficiency of the KCNH2 gene, which has been associated with a chronic QT syndrome; no episodes of LQT2-triggered ventricular arrhythmias have been observed so far. Patient 3
Analysis of the literature
A focused literature analysis was performed in PubMed to identify all articles reporting an association of the Currarino phenotype with causative mutations of any type. ‘Currarino syndrome’ was the only key phrase used. The time period analyzed was between 1998 (when the molecular basis of Currarino syndrome was discovered) and 2014. Only the articles reporting unclassified genomic variants or asymptomatic carriers were excluded. Results Clinical, chromosome, whole-genome array, and molecular results Patient 1
The full-term male was diagnosed at birth with an imperforate anus, posterior urethral valve, ureterohydronephrosis, undescended left testis, and sacrococcygeal teratoma. He had no facial dysmorphism and his growth and development were normal (Fig. 1a). Both mother and maternal grandmother were born with an imperforate anus and C-shaped sacrum, without any other congenital abnormalities or developmental difficulties. Using qPCR, a 43-bp heterozygous frameshift deletion of the 5′ end and middle of exon 1 of the MNX1 was identified. In addition, an unclassified, heterozygous single-nucleotide substitution c.415C > G (p.L139V) in
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A 3-year-old boy was evaluated for anal stenosis, myelomeningocele, terminal cord lipoma, tethered cord, syringomyelia, and partial sacral agenesis with a normally formed lumbar vertebra. He had perineal hypospadias, left undescended testis, encopresis, and prolonged QT interval on EKG. His cognitive and motor developmental were severely delayed; he was small for age. Dysmorphic features included microcephaly, prominent ears, down-slanted palpebral fissures, broad nasal bridge and deep filtrum. Chromosomal microarray analysis revealed a terminal deletion of 153 oligonucleotide probes of the 7q36.1-qter region spanning approximately 9.2 Mb and a terminal duplication of 64 oligonucleotide probes within 14q32.32-qter region spanning approximately 3.5 Mb as a result of an unbalanced 7;14 translocation. Parental karyotypes and FISH testing showed normal results. The deleted interval at 7q contains approximately 72 genes, including dosage-sensitive KCNH2, PRKAG2, SHH, MNX1, and DNAJB6. According to OMIM, the deleted interval at 14q does not contain genes haploinsufficiency of which produces congenital malformations or intellectual disability. Patient 4
The male patient was born at term after an uncomplicated pregnancy to a young couple (first cousins) from the Middle East. He had low birth weight and had sacral agenesis, tethered spinal cord, and a sacral mass (intraspinal lipoma; Fig. 1d,e). At the age of 3 years, he
Diagnostic algorithm for Currarino syndrome (a)
(b)
(d)
(c)
(e)
Fig. 1. Facial features. (a) Patient 1 with normal cranial and facial appearance. (b) Patient 2 with microcephaly, low frontal hair line, ptosis of the left eyelid, depressed nasal bridge, prominent ears, prominent premaxillary area, full cheeks, smooth philtrum, hypoplastic mandible, and groove between lower lip and chin. (c) Patient 4 with low frontal hair line, large and cupped ears, and short and smooth philtrum. Lumbar spine magnetic resonance imaging (MRI) of patient 4. (d) The sagittal T1 and (e) sagittal T2 views of lumbar spine MRI shows a 0.8 × 0.8 × 2.0 cm intraspinal lipoma (arrows) at the L5-S1 level. Only the body of S1 is visualized, while the rest of the sacrum and coccygeal vertebrae are absent. Note the tethered spinal cord above the mass.
was short statured, had generalized hypotonia, microcephaly, severe developmental delay, and facial dysmorphism (Fig. 1c). Conventional chromosome and metaphase FISH studies disclosed a complex chromosome 7 rearrangement denoted as 46,XY,dup(7)(q32q36.1).ish der(7) (pter+,qter–). Chromosomal microarray analysis confirmed a 17.2-Mb interstitial duplication of the 7q33-q36.2 region, followed immediately by a 4.4-Mb terminal deletion of the 7q36.2 containing 20 genes (including SHH, LMBR1, and MNX1). Parental studies were normal. Results of literature analysis
The Table 1 gives an overview of patients with Currarino syndrome reported in the literature (1998–2014) with their phenotypic characteristics and causative mutations. Discussion
Currarino syndrome (OMIM 175450) is characterized by a variable phenotype as a consequence of a wide
range of MNX1 mutations and their variable expressivity (2, 3, 5, 8). The syndrome is caused by haploinsufficiency of the MNX1 gene arising from either intragenic mutations or distal 7q deletions of different sizes (3, 6). Mutations in MNX1 result in complex phenotypes including (i) hemisacrum or bifid sacrum with neurogenic bladder, recurrent urinary infections, or incontinence; (ii) presacral masses such as anterior meningocele, enteric cyst, or presacral teratoma; and (iii) anorectal malformations including atresia, stenosis, and fistula frequently complicated with chronic constipation or incontinence (2, 10, 11). Once this classical triad of Currarino syndrome is recognized, a thorough search for associated malformations and developmental difficulties as well as a genetic evaluation should be initiated. In this study, we describe four new patients with Currarino syndrome (one familial and three sporadic cases) and present an extensive review of literature (Table 1). Including the literature review, 205 patients with Currarino syndrome were analyzed for the presence of intellectual disability, growth retardation, facial dysmorphy, and associated malformations. We investigated whether there was a difference in the frequency distribution
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Cuturilo et al. Table 1. Phenotypic characteristics and causative mutations of patients with Currarino syndrome reported in the literature between 1998 and 2014 and from this study. Articles were excluded that reported unclassified genomic variants or asymptomatic carriers only Intellectual Growth Facial disability retardation dysmorphy Pathogenic cytogenetic deletions 20-Mb deletion of 7q32-qter and 960-Kb duplication of 13q34-qter 9.2-Mb deletion of 7q36.1-qter and 3.5-Mb duplication of 14q32.32-qter 4.4-Mb deletion of 7q36.2-qter and 17.2-Mb duplication of 7q33-q36.2 8.8-Mb deletion of 7q36 and 10.3-Mb duplication of 7q34-q35 46,XY inv7(p15.1;q34); 46,XY der(7)t(7;12) (q36.12;q24.21).pat; 46,XX; ins(4;7)(q25;q31.3q21.3) 46,XX/46,XX,add(7q36).ish der(7)(q36),t(2,7)(p22;q36) 46, XY,del(7)(q36.1).ish del(7)(q36.3q36.3) 2.7-Mb deletion Microdeletion including MNX1 genea
Associated anomalies
Number of patients
Reference Number
+
+
+
+
1
This study
+
+
+
+
1
This study
+
+
+
+
1
This study
+
+
+
+
1
(4)
2 of 3 pts
1 of 3 pts
2 of 3 pts
2 of 3 pts
3
(2)
n/a + n/a +
n/a + n/a 1 of 3 pts
+ + + 1 of 3 pts
n/a + + (urogenital) –
1 1 1 3
+ n/a
n/a n/a
n/a n/a
n/a n/a
3 1
(16) (3) (17) (5) and references therein (18) (11)
–
–
–
3
(19)
n/a – – – –
n/a – – – –
n/a – – – –
1 of 3 pts (holoprosencephaly) n/a – – – –
1 3 3 1 2
(20) This study (8) (21) (22)
– –
– –
2 12
Frameshift point mutations
–
–
–
(23) (24), (14), (25), and (26) (27)
Point mutations not further defined
–
–
–
n/a –
n/a –
n/a –
– – –
– – –
– – –
n/a
n/a
n/a
Microdeletion of 7qa Deletion 7qa Pathogenic MNX1 intragenic mutations Partial deletionsa
Insertions Missense mutation and intragenic deletion-insertion Missense point mutations Nonsense point mutations
Other intragenic mutations not further defined
– – 1 of 12 pts c – Hirschsprung disease –
n/a 1 of 6 pts (Hirschsprung disease) 5b – 3 of 50 pts (urogenital) n/a
1 20
36 6
(19), (28), (23), (29), (30), (31), and (32) (11), (18) (7)
23 (2) 24 (10/24)d (28) and (10) 50 (5/50)d (5) and (6) 1
(33)
n/a, not available; pts, patients. a Precise characterization of the mutation not provided in some of the articles. b Müllerian duplication in all, scoliosis in one, aortic stenosis in one. c Subtile facial dysmorphy (fetal pathology report). d Asymptomatic patients with radiologic findings only.
of these phenotypic characteristics between Currarino patients with MNX1 intragenic mutations (n = 188) and those with distal 7q deletions (n = 17). Intellectual disability
Data indicating intellectual status were available in 150/188 patients with intragenic mutations and 14/17 patients with 7q deletions. Intellectual disability was
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present in none of the patients with intragenic MNX1 mutations, and 13/14 (92.9%) of patients with larger distal 7q deletions 5 (12, 13) (Table 1). This implies that mutations limited to the MNX1 gene do not represent a risk of developmental delay; however, while for most of reported patients the exact age is not known, many were below age 1 year at time of evaluation. The lack of information about their prospective development represents a research bias of our study.
Diagnostic algorithm for Currarino syndrome
Fig. 2. Testing algorithm for patients with Currarino phenotype.
Growth delay
We obtained information about growth characteristics for 150/188 patients with intragenic mutations and 11/17 patients with larger 7q deletions and found growth deficiency in 0/150 and 7/11 (63.6%), respectively (Table 1). Thus, the presence of growth delay in a patient with Currarino syndrome raises a possibility for a larger chromosome 7q deletion, while normal growth parameters have less predictive value in terms of potential causative mutation. Facial dysmorphy
Facial morphology was evaluated in 150/188 patients with intragenic mutations and 13/17 patients with larger 7q deletions. Dysmorphic facial features were noted in 1/150 (0.6%) and 10/13 (76.9%) patients, respectively (Table 1). Only one patient with an intragenic MNX1 mutation, a fetus from a pregnancy terminated at 20 weeks of gestation, had subtle facial dysmorphism consisting of hypertelorism and retrognathism (14). These results suggest that facial dysmorphology is a strong indicator of the presence of a larger 7q deletion in Currarino patients, while intragenic mutations of the MNX1 gene rarely, if ever, produce dysmorphic facial features. Associated malformation
Besides the classical Currarino triad, the most common malformations reported in patients with Currarino syndrome are terminal spinal cord malformations (e.g. low laying conus, short and thickened filum, tethered cord, and spinal lipoma) and urogenital anomalies (2, 11). The available data from 150/188 patients with intragenic mutations and 12/17 patients with larger 7q deletions were analyzed. Associated anomalies were found in 11/150 (7.3%) and 8/12 (66.6%), respectively (Table 1). These data suggest that disclosure of congenital malformations, besides the classical Currarino
triad, positively predicts the presence of a larger 7q deletion. Chromosomal microarray (array-CGH or SNPmicroarray) has become the test of first choice in patients with multiple congenital malformations, especially in those with intellectual disability or in young infants in whom mental development is unpredictable (15). Even in situations when a specific deletion/duplication syndrome is suspected, this technique is often employed based on a phenotypic overlap between syndromes. Thus, a clinician could easily order chromosomal microarray testing in patients with the classical Currarino triad; however, the result will be negative in the majority of cases with an intragenic MNX1 mutation. Considering the results shown in this study, we believe that sequencing rather than chromosomal microarray analysis should be carried out in cases with normal growth, development, and facial appearance. However, in the case of a normal sequencing result, the clinician should undoubtedly proceed with chromosomal microarray, especially because other clinical features, including long QT syndrome, can be associated with 7q deletions. In contrast, if growth delay and/or facial dysmorphy and/or developmental delay are verified on examination, especially in the presence of associated malformations, chromosomal microarray is a method of first choice for genetic testing (Fig. 2). In conclusion, we suggest that in the era of more advanced techniques for genetic testing, careful clinical examination remains important in the everyday practice of genetics. This is manifested in the case of Currarino syndrome, as appreciation of facial morphology and early recognition of disturbances of growth or development could lead to a less expensive and more efficient, individualized approach for diagnostic testing. References 1. Currarino G, Coln D, Votteler T. Triad of anorectal, sacral, and presacral anomalies. AJR Am J Roentgenol 1981: 137: 395–398. 2. Cretolle C, Pelet A, Sanlaville D et al. Spectrum of HLXB9 gene mutations in Currarino syndrome and genotype-phenotype correlation. Hum Mutat 2008: 29: 903–910.
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