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NKX2.5 mutation identification on exome sequencing in a patient with heterotaxy
European Journal of Medical Genetics 57 (2014) 558e561
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Clinical report
NKX2.5 mutation identification on exome sequencing in a patient with heterotaxy Kosuke Izumi a, b, *, Sarah Noon a, Alisha Wilkens a, Ian D. Krantz a, c a
Division of Human Genetics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA Research Center for Epigenetic Disease, Institute for Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan c The Perelman School of Medicine at The University of Pennsylvania, Philadelphia, PA, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 March 2014 Accepted 3 August 2014 Available online 10 August 2014
Exome sequencing enables us to screen most of the protein coding genes in an unbiased way, this technique represents an ideal tool to identify previously under- or unappreciated phenotypes associated with known disease genes and genetic disorders. Here we present an illustrative case that required exome sequencing to identify a genetic alteration associated with the clinical features. The phenotype of the proband included heterotaxy, double outlet right ventricle, common atrioventricular canal, total anomalous pulmonary venous connection, asplenia, failure to thrive and short stature. Exome sequencing demonstrated a frameshift mutation c.397_400del (p.P133GfsTer 42) in NKX2.5. Although a single previous case of heterotaxy was reported in a large familial case of NKX2.5, heterotaxy is not clinically appreciated to be a part of the phenotypic spectrum associated with NKX2.5 mutations. This case report demonstrates the utility of exome sequencing in expanding a phenotypic spectrum of a known Mendelian disorder. We predict that this type of unexpected identification of mutations in known-disease associated genes in patients with atypical or expanded phenotypes will occur with increasing frequency as the use of exome and genome sequencing become more common tools in diagnosing patients with syndromic and non-syndromic foms of structural birth defects. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Atrioventricular canal Double outlet right ventricle Intestinal malrotation
1. Introduction Traditionally, the phenotypic spectrum of a genetic disorder had been defined by screening for mutations in an identified disease gene within a cohort of individuals with an overlapping clinical phenotype. The recent introduction of exome sequencing has revolutionized our ability to define the phenotypic spectrum associated with specific disease genes. Since exome sequencing enables us to screen most of the protein coding genes contained within the genome in an unbiased way, this technique represents an ideal tool to identify previously unappreciated phenotypes associated with a known disease gene and/or genetic disorder. This unbiased screening approach has already begun to unravel the wide phenotypic spectrum of Mendelian disorders [Gripp et al., 2013; Yu et al., 2013].
* Corresponding author. Research Center for Epigenetic Disease, Institute for Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan. Tel.: þ81 3 5841 0756; fax: þ81 3 5841 0757. E-mail address: [email protected] (K. Izumi). http://dx.doi.org/10.1016/j.ejmg.2014.08.003 1769-7212/Ó 2014 Elsevier Masson SAS. All rights reserved.
Here we present such an example by documenting the identification of a frameshift mutation in the NKX2.5 gene in a proband with heterotaxy. Heterotaxy is a disorder of lefteright axis determination, and is genetically heterogeneous condition. Molecular basis of heterotaxy has not yet been fully elucidated, although several genes whose mutations are associated with heterotaxy have been identified, and those include ZIC3, NODAL, CFC1, ACVR2B and LEFTY2 [Shiraishi and Ichikawa, 2012]. Heterotaxy was reported once before associated with an NKX2.5 in a case embedded within a larger familial report, however individual features, more commonly seen in constellation in heterotaxy, such as congenital heart defects (CHD) and asplenia, have been described in association with NKX2.5 mutations [Koss et al., 2012; Stallmeyer et al., 2010; Watanabe et al., 2002]. These previously reported cases along with the one reported here suggest a causal relationship between NKX2.5 mutations and heterotaxy.
2. Case report This proband was born to a 22-year-old G3P2 to 3 mother following 38 4/7 weeks of pregnancy. The mother had HIV infection
K. Izumi et al. / European Journal of Medical Genetics 57 (2014) 558e561
(discovered upon the conception) and received antiviral treatment with HAART (Kaletra and Combivir) during the pregnancy. The mother denied any teratogen exposures such as cocaine. Prenatal ultrasound identified a complex congenital heart defect, and further work up suggested the presence of heterotaxy spectrum disorder, although there was no evidence of a situs abnormality. An amniocentesis showed a normal 46,XY karytype and was negative for the 22q11 deletion by fluorescence in situ hybridization (FISH) analysis. The proband was delivered vaginally, and Apgar scores were 8 and 9 at 1 and 5 min, respectively. Birth weight was less than 10th centile, and 50th centile for 34.5 weeks gestation, length was 15th centile and head circumference was less than 10th centile and 50th centile for 34 weeks gestation. Upon birth, three preauricular tags, and a skin tag on the nose over right nares were identified. His postnatal cardiac evaluation demonstrated the presence of cyanotic heart disease due to double outlet right ventricle, common atrioventricular canal, total anomalous pulmonary venous connection and mild aortic arch hypoplasia (Fig. 1A). Treatment with prostaglandin was initiated upon birth for the management of CHD. Postnatal brain MRI at 1 week of age demonstrated dysmorphic midline structures, including dysgenesis of the splenium of the corpus callosum with a large anterior midline pericallosal lipoma (Fig. 1C). His abdominal organ position was also distorted, and liver was centrally located and spleen was not identified (Fig. 1B). He was also found to have intestinal
559
malformation, and he underwent a Ladd procedure and gastrostomy tube placement at 3 weeks old. In addition to his CHD, he had a history of arrhythmia. At 1 week of age, he had an episode of ectopic atrial tachycardia, and was started on digoxin treatment. However, ectopic atrial tachycardia persisted, and propranolol was added, with resolution of the arrhythmia. Postnatal radiograph revealed the presence of multiple segmentation defects of the vertebrae (Fig. 1D). This proband has demonstrated normal development. His other medical problems include failure to thrive (FTT) and short stature. Family history is unremarkable, and the mother does not have any known cardiovascular problems. At 20 months of age, his height was below 10th centile, and 50th centile for 11 months old, his weight was below 5th centile, and 50th centile for 7 months old, and head circumference was 3rd centile. His physical examination was unremarkable except for epicanthal folds (Fig. 2). Prior to the exome sequencing ZIC3 mutational analysis was ordered, however it did not reveal any pathological mutations in the coding region or in the exoneintron boundaries. Genome wide SNP array also did not reveal any pathological copy number alterations. Clinical exome sequencing was ordered through the Baylor College of Medicine (BCM) Whole Genome Laboratory (Houston, TX, USA) at the age of 20 months. Details of the clinical exome sequencing have been reported previously by Yang et al., and additional information can be found at the BCM Whole Genome
Fig. 1. Clinical manifestations of the proband. A) Congenital heart defects demonstrated by three-dimensional MRI. The ventricular arterial alignment was double outlet right ventricle. The atrioventricular connection was complete common AV canal. B) Abdominal MRI. Liver is centrally located with hepatic veins joining the inferior vena cava from the right and left sides of the spine. C) Brain MRI demonstrated a large anterior midline pericallosal lipoma. D) Vertebral segmentation defects at T7 and L3 spine.
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K. Izumi et al. / European Journal of Medical Genetics 57 (2014) 558e561
Fig. 2. Facial profile of the proband. Note epicanthal folds.
Laboratory website [Yang et al., 2013]. DNA extracted from peripheral blood was used for exome sequencing. Exome capture was performed using a BCM custom-designed NimblegenÒ capture reagent, and next generation sequencing was performed on an IlluminaÒ platform next-generation sequencer. This clinical exome sequencing analysis provides the mean exome coverage of 100120X and 95% of the exome is covered at >20X. Variant filtering was performed by using the custom-designed BCM Human Genome Sequencing Center (HGSC) annotation pipeline. Interpretation of data was performed according to the American College of Medical Genetics and Genomics (ACMG) guidelines and the patient’s phenotype. The BCM Whole Genome Laboratory reports variants under two main categories, “deleterious mutation related to the disease phenotype” and “variant of unknown significance related to the disease phenotype” [Yang et al., 2013]. The clinical exome sequencing demonstrated a frameshift mutation c.397_400del (p.P133GfsTer 42) in NKX2.5 along with three other variants of unknown clinical significance in disease genes related to the clinical phenotype (CCDC39, DNAH5 and DNAI2 genes (Table 1), although there none of the variants were categorized as “deleterious mutation related to the disease phenotype”. These four variants were confirmed by Sanger sequencing, and all were found to be inherited from one of biological parents. The exome coverage for heterotaxy genes was as follows. All of the coding regions of NODAL, CFC1, ACVR2B and CRELD1 are covered >20X, and 90.4% of LEFTY2 was covered >20X. 3. Discussion Here we present a case of heterotaxy due to a frameshift mutation in NKX2.5, identified by clinical exome sequencing. Sanger
sequencing and exome sequencing did not show the presence of any pathogenetic variants in previously reported heterotaxy genes such as ZIC3, NODAL, CFC1, ACVR2B, CRELD1 and LEFTY2. Although the exome coverage for LEFTY2 was not complete, the phenotype of the proband appears to be different from heterotaxy cases due to LEFTY2 mutations, since LEFTY2 mutations cause left isomerism [Kosaki et al., 1999]. Although NKX2.5 frameshift mutation was inherited from the mother who does no have a known cardiac phenotype (she has declined formal evaluation to date), we concluded that this variant is associated with this proband’s heterotaxy phenotype due to the following reasons. First, similar frameshift mutations of NKX2.5 have been documented in association with heterotaxy in a single prior patient from a large pedigree of various cardiac manifestations [Watanabe et al., 2002]. Second, mutations of NKX2.5 are associated with cardiac malformations commonly seen in patients with heterotaxy such as transposition of great arteries and double-outlet right ventricle [De Luca et al., 2010; McElhinney et al., 2003]. Third, mutations in the NKX2.5 gene were recently found in patients with asplenia, which is a part of heterotaxy phenotype, suggesting the possible association of NKX2.5 mutations in the pathogenesis of laterality disorders [Koss et al., 2012]. Although the same mutation of NKX2.5 was also found in the apparently unaffected mother, such intrafamilial phenotypic variation is well-known in individuals with NKX2.5 mutations [Elliott et al., 2003; Watanabe et al., 2002]. In addition to the NKX2.5 mutation, exome sequencing identified three additional heterozygous variants of unknown significance in three recessive genes (a missense mutation in the CCDC39 gene, a missense mutation in the DNAH5 gene, and a splice site mutation in the DNAI2 gene). These three variants are listed in dbSNP, although the population frequencies of these variants are
Table 1 List of the variants in disease genes related to clinical phenotype. Gene (RefSeq number)
Inheritance pattern of the disease associated with mutations
Location
Nucleotide alteration
Amino acid alteration
dbSNP ID (population allele frequency)
Zygosity
Inheritance
NKX2.5 (NM_001166176) CCDC39 (NM_181426) DNAH5 (NM_001369) DNAI2 (NM_023036)
AD AR AR AR
Exon2 Exon11 Exon52 Intronic
c.397_400del c.A1433G c.G8805C c.468-4G>T
p.P133GfsTer 42 p.Q478R p.M2935I N/A
Not listed rs115545935 (0.395%) rs77874614 (0.282%) rs146462823 (0.640%)
Het Het Het Het
Maternal Paternal Maternal Maternal
K. Izumi et al. / European Journal of Medical Genetics 57 (2014) 558e561
very low (Table 1). The disorder associated with genetic mutations in these three genes is ciliary dyskinesia [Loges et al., 2008; Merveille et al., 2011; Olbrich et al., 2002]. The exome coverage of these genes was very high; hence, it was unlikely that second mutations in these genes were missed. Although the phenotype of this proband did not fit well with typical symptoms of ciliary dyskinesia, it is intriguing that heterotaxy is occasionally seen in individuals with mutations in ciliary dyskinesia genes including DNAH5 and CCDC39 [Kennedy et al., 2007; Merveille et al., 2011]. In a case described by Watanabe et al., there was significant intrafamilial phenotypic variation, suggesting the presence of modifier genetic or environmental factors [Watanabe et al., 2002]. For example the missense variant in CCDC39, which was inherited from the father, may have served as a modifier for this proband’s phenotype, given that the mother does not have heterotaxy. Aside from heterotaxy-related birth defects, the proband presented with several additional manifestations such as vertebral defects, midline brain malformations, short stature and FTT. Among these, only vertebral defects and scoliosis has been described as an occasional associated anomaly of heterotaxy [Ticho et al., 2000]. Therefore, it remains to be determined whether midline brain malformation, short stature and FTT are associated with the NKX2.5 mutation or caused by other etiologies. Because the proband’s exome sequencing was performed as a clinical test, only genetic variants in diseaseassociated genes were reported. Hence, it is possible that this probands have additional genetic mutation(s) resulting in brain malformation, short stature and FTT. This case report demonstrates the utility of exome sequencing in expanding a phenotypic spectrum of a known Mendelian disorder. Because heterotaxy is not the phenotype which is known to be associated with NKX2.5 mutations, the single gene mutational analysis of NKX2.5 was not considered clinically. The unexpected discovery of mutations in known-disease associated genes, and the resultant expansion of the associated phenotypes, will continue to rise as the use of exome or genome sequencing becomes more commonplace. Such discoveries will lead to a better understanding of the phenotypic spectrum of genetic disorders.
Web resource dbSNP: http://www.ncbi.nlm.nih.gov/SNP/ BCM Whole Genome Laboratory: https://www.bcm.edu/ research/medical-genetics-labs/wholegenomelab
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Acknowledgements The authors thank BCM Whole Genome Laborary for providing the detailed information about the clinical exome analysis of the proband.
References De Luca A, Sarkozy A, Consoli F, Ferese R, Guida V, Dentici ML, et al. Familial transposition of the great arteries caused by multiple mutations in laterality genes. Heart 2010;96:673e7. http://dx.doi.org/10.1136/hrt.2009.181685. Elliott DA, Kirk EP, Yeoh T, Chandar S, McKenzie F, Taylor P, et al. Cardiac homeobox gene NKX2-5 mutations and congenital heart disease: associations with atrial septal defect and hypoplastic left heart syndrome. J Am Coll Cardiol 2003;41: 2072e6. Gripp KW, Ennis S, Napoli J. Exome analysis in clinical practice: expanding the phenotype of Bartsocas-Papas syndrome. Am J Med Genet A 2013;161A:1058e 63. http://dx.doi.org/10.1002/ajmg.a.35913. Kennedy MP, Omran H, Leigh MW, Dell S, Morgan L, Molina PL, et al. Congenital heart disease and other heterotaxic defects in a large cohort of patients with primary ciliary dyskinesia. Circulation 2007;115:2814e21. http://dx.doi.org/ 10.1161/CIRCULATIONAHA.106.649038. Kosaki K, Bassi MT, Kosaki R, Lewin M, Belmont J, Schauer G, et al. Characterization and mutation analysis of human LEFTY A and LEFTY B, homologues of murine genes implicated in left-right axis development. Am J Hum Genet 1999;64: 712e21. http://dx.doi.org/10.1086/302289. Koss M, Bolze A, Brendolan A, Saggese M, Capellini TD, Bojilova E, et al. Congenital asplenia in mice and humans with mutations in a Pbx/Nkx2-5/p15 module. Dev Cell 2012;22:913e26. http://dx.doi.org/10.1016/j.devcel.2012.02.009. Loges NT, Olbrich H, Fenske L, Mussaffi H, Horvath J, Fliegauf M, et al. DNAI2 mutations cause primary ciliary dyskinesia with defects in the outer dynein arm. Am J Hum Genet 2008;83:547e58. http://dx.doi.org/10.1016/j.ajhg.2008.10.001. McElhinney DB, Geiger E, Blinder J, Benson DW, Goldmuntz E. NKX2.5 mutations in patients with congenital heart disease. J Am Coll Cardiol 2003;42:1650e5. Merveille A-C, Davis EE, Becker-Heck A, Legendre M, Amirav I, Bataille G, et al. CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs. Nat Genet 2011;43:72e8. http://dx.doi.org/10.1038/ng.726. Olbrich H, Häffner K, Kispert A, Völkel A, Volz A, Sasmaz G, et al. Mutations in DNAH5 cause primary ciliary dyskinesia and randomization of left-right asymmetry. Nat Genet 2002;30:143e4. http://dx.doi.org/10.1038/ng817. Shiraishi I, Ichikawa H. Human heterotaxy syndrome e from molecular genetics to clinical features, management, and prognosis e. Circ J 2012;76:2066e75. Stallmeyer B, Fenge H, Nowak-Göttl U, Schulze-Bahr E. Mutational spectrum in the cardiac transcription factor gene NKX2.5 (CSX) associated with congenital heart disease. Clin Genet 2010;78:533e40. http://dx.doi.org/10.1111/j.13990004.2010.01422.x. Ticho BS, Goldstein AM, Van Praagh R. Extracardiac anomalies in the heterotaxy syndromes with focus on anomalies of midline-associated structures. Am J Cardiol 2000;85:729e34. Watanabe Y, Benson DW, Yano S, Akagi T, Yoshino M, Murray JC. Two novel frameshift mutations in NKX2.5 result in novel features including visceral inversus and sinus venosus type ASD. J Med Genet 2002;39:807e11. Yang Y, Muzny DM, Reid JG, Bainbridge MN, Willis A, Ward PA, et al. Clinical wholeexome sequencing for the diagnosis of mendelian disorders. N Engl J Med 2013;369:1502e11. http://dx.doi.org/10.1056/NEJMoa1306555. Yu TW, Chahrour MH, Coulter ME, Jiralerspong S, Okamura-Ikeda K, Ataman B, et al. Using whole-exome sequencing to identify inherited causes of autism. Neuron 2013;77:259e73. http://dx.doi.org/10.1016/j.neuron.2012.11.002.
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Clinical report
NKX2.5 mutation identification on exome sequencing in a patient with heterotaxy Kosuke Izumi a, b, *, Sarah Noon a, Alisha Wilkens a, Ian D. Krantz a, c a
Division of Human Genetics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA Research Center for Epigenetic Disease, Institute for Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan c The Perelman School of Medicine at The University of Pennsylvania, Philadelphia, PA, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 March 2014 Accepted 3 August 2014 Available online 10 August 2014
Exome sequencing enables us to screen most of the protein coding genes in an unbiased way, this technique represents an ideal tool to identify previously under- or unappreciated phenotypes associated with known disease genes and genetic disorders. Here we present an illustrative case that required exome sequencing to identify a genetic alteration associated with the clinical features. The phenotype of the proband included heterotaxy, double outlet right ventricle, common atrioventricular canal, total anomalous pulmonary venous connection, asplenia, failure to thrive and short stature. Exome sequencing demonstrated a frameshift mutation c.397_400del (p.P133GfsTer 42) in NKX2.5. Although a single previous case of heterotaxy was reported in a large familial case of NKX2.5, heterotaxy is not clinically appreciated to be a part of the phenotypic spectrum associated with NKX2.5 mutations. This case report demonstrates the utility of exome sequencing in expanding a phenotypic spectrum of a known Mendelian disorder. We predict that this type of unexpected identification of mutations in known-disease associated genes in patients with atypical or expanded phenotypes will occur with increasing frequency as the use of exome and genome sequencing become more common tools in diagnosing patients with syndromic and non-syndromic foms of structural birth defects. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Atrioventricular canal Double outlet right ventricle Intestinal malrotation
1. Introduction Traditionally, the phenotypic spectrum of a genetic disorder had been defined by screening for mutations in an identified disease gene within a cohort of individuals with an overlapping clinical phenotype. The recent introduction of exome sequencing has revolutionized our ability to define the phenotypic spectrum associated with specific disease genes. Since exome sequencing enables us to screen most of the protein coding genes contained within the genome in an unbiased way, this technique represents an ideal tool to identify previously unappreciated phenotypes associated with a known disease gene and/or genetic disorder. This unbiased screening approach has already begun to unravel the wide phenotypic spectrum of Mendelian disorders [Gripp et al., 2013; Yu et al., 2013].
* Corresponding author. Research Center for Epigenetic Disease, Institute for Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan. Tel.: þ81 3 5841 0756; fax: þ81 3 5841 0757. E-mail address: [email protected] (K. Izumi). http://dx.doi.org/10.1016/j.ejmg.2014.08.003 1769-7212/Ó 2014 Elsevier Masson SAS. All rights reserved.
Here we present such an example by documenting the identification of a frameshift mutation in the NKX2.5 gene in a proband with heterotaxy. Heterotaxy is a disorder of lefteright axis determination, and is genetically heterogeneous condition. Molecular basis of heterotaxy has not yet been fully elucidated, although several genes whose mutations are associated with heterotaxy have been identified, and those include ZIC3, NODAL, CFC1, ACVR2B and LEFTY2 [Shiraishi and Ichikawa, 2012]. Heterotaxy was reported once before associated with an NKX2.5 in a case embedded within a larger familial report, however individual features, more commonly seen in constellation in heterotaxy, such as congenital heart defects (CHD) and asplenia, have been described in association with NKX2.5 mutations [Koss et al., 2012; Stallmeyer et al., 2010; Watanabe et al., 2002]. These previously reported cases along with the one reported here suggest a causal relationship between NKX2.5 mutations and heterotaxy.
2. Case report This proband was born to a 22-year-old G3P2 to 3 mother following 38 4/7 weeks of pregnancy. The mother had HIV infection
K. Izumi et al. / European Journal of Medical Genetics 57 (2014) 558e561
(discovered upon the conception) and received antiviral treatment with HAART (Kaletra and Combivir) during the pregnancy. The mother denied any teratogen exposures such as cocaine. Prenatal ultrasound identified a complex congenital heart defect, and further work up suggested the presence of heterotaxy spectrum disorder, although there was no evidence of a situs abnormality. An amniocentesis showed a normal 46,XY karytype and was negative for the 22q11 deletion by fluorescence in situ hybridization (FISH) analysis. The proband was delivered vaginally, and Apgar scores were 8 and 9 at 1 and 5 min, respectively. Birth weight was less than 10th centile, and 50th centile for 34.5 weeks gestation, length was 15th centile and head circumference was less than 10th centile and 50th centile for 34 weeks gestation. Upon birth, three preauricular tags, and a skin tag on the nose over right nares were identified. His postnatal cardiac evaluation demonstrated the presence of cyanotic heart disease due to double outlet right ventricle, common atrioventricular canal, total anomalous pulmonary venous connection and mild aortic arch hypoplasia (Fig. 1A). Treatment with prostaglandin was initiated upon birth for the management of CHD. Postnatal brain MRI at 1 week of age demonstrated dysmorphic midline structures, including dysgenesis of the splenium of the corpus callosum with a large anterior midline pericallosal lipoma (Fig. 1C). His abdominal organ position was also distorted, and liver was centrally located and spleen was not identified (Fig. 1B). He was also found to have intestinal
559
malformation, and he underwent a Ladd procedure and gastrostomy tube placement at 3 weeks old. In addition to his CHD, he had a history of arrhythmia. At 1 week of age, he had an episode of ectopic atrial tachycardia, and was started on digoxin treatment. However, ectopic atrial tachycardia persisted, and propranolol was added, with resolution of the arrhythmia. Postnatal radiograph revealed the presence of multiple segmentation defects of the vertebrae (Fig. 1D). This proband has demonstrated normal development. His other medical problems include failure to thrive (FTT) and short stature. Family history is unremarkable, and the mother does not have any known cardiovascular problems. At 20 months of age, his height was below 10th centile, and 50th centile for 11 months old, his weight was below 5th centile, and 50th centile for 7 months old, and head circumference was 3rd centile. His physical examination was unremarkable except for epicanthal folds (Fig. 2). Prior to the exome sequencing ZIC3 mutational analysis was ordered, however it did not reveal any pathological mutations in the coding region or in the exoneintron boundaries. Genome wide SNP array also did not reveal any pathological copy number alterations. Clinical exome sequencing was ordered through the Baylor College of Medicine (BCM) Whole Genome Laboratory (Houston, TX, USA) at the age of 20 months. Details of the clinical exome sequencing have been reported previously by Yang et al., and additional information can be found at the BCM Whole Genome
Fig. 1. Clinical manifestations of the proband. A) Congenital heart defects demonstrated by three-dimensional MRI. The ventricular arterial alignment was double outlet right ventricle. The atrioventricular connection was complete common AV canal. B) Abdominal MRI. Liver is centrally located with hepatic veins joining the inferior vena cava from the right and left sides of the spine. C) Brain MRI demonstrated a large anterior midline pericallosal lipoma. D) Vertebral segmentation defects at T7 and L3 spine.
560
K. Izumi et al. / European Journal of Medical Genetics 57 (2014) 558e561
Fig. 2. Facial profile of the proband. Note epicanthal folds.
Laboratory website [Yang et al., 2013]. DNA extracted from peripheral blood was used for exome sequencing. Exome capture was performed using a BCM custom-designed NimblegenÒ capture reagent, and next generation sequencing was performed on an IlluminaÒ platform next-generation sequencer. This clinical exome sequencing analysis provides the mean exome coverage of 100120X and 95% of the exome is covered at >20X. Variant filtering was performed by using the custom-designed BCM Human Genome Sequencing Center (HGSC) annotation pipeline. Interpretation of data was performed according to the American College of Medical Genetics and Genomics (ACMG) guidelines and the patient’s phenotype. The BCM Whole Genome Laboratory reports variants under two main categories, “deleterious mutation related to the disease phenotype” and “variant of unknown significance related to the disease phenotype” [Yang et al., 2013]. The clinical exome sequencing demonstrated a frameshift mutation c.397_400del (p.P133GfsTer 42) in NKX2.5 along with three other variants of unknown clinical significance in disease genes related to the clinical phenotype (CCDC39, DNAH5 and DNAI2 genes (Table 1), although there none of the variants were categorized as “deleterious mutation related to the disease phenotype”. These four variants were confirmed by Sanger sequencing, and all were found to be inherited from one of biological parents. The exome coverage for heterotaxy genes was as follows. All of the coding regions of NODAL, CFC1, ACVR2B and CRELD1 are covered >20X, and 90.4% of LEFTY2 was covered >20X. 3. Discussion Here we present a case of heterotaxy due to a frameshift mutation in NKX2.5, identified by clinical exome sequencing. Sanger
sequencing and exome sequencing did not show the presence of any pathogenetic variants in previously reported heterotaxy genes such as ZIC3, NODAL, CFC1, ACVR2B, CRELD1 and LEFTY2. Although the exome coverage for LEFTY2 was not complete, the phenotype of the proband appears to be different from heterotaxy cases due to LEFTY2 mutations, since LEFTY2 mutations cause left isomerism [Kosaki et al., 1999]. Although NKX2.5 frameshift mutation was inherited from the mother who does no have a known cardiac phenotype (she has declined formal evaluation to date), we concluded that this variant is associated with this proband’s heterotaxy phenotype due to the following reasons. First, similar frameshift mutations of NKX2.5 have been documented in association with heterotaxy in a single prior patient from a large pedigree of various cardiac manifestations [Watanabe et al., 2002]. Second, mutations of NKX2.5 are associated with cardiac malformations commonly seen in patients with heterotaxy such as transposition of great arteries and double-outlet right ventricle [De Luca et al., 2010; McElhinney et al., 2003]. Third, mutations in the NKX2.5 gene were recently found in patients with asplenia, which is a part of heterotaxy phenotype, suggesting the possible association of NKX2.5 mutations in the pathogenesis of laterality disorders [Koss et al., 2012]. Although the same mutation of NKX2.5 was also found in the apparently unaffected mother, such intrafamilial phenotypic variation is well-known in individuals with NKX2.5 mutations [Elliott et al., 2003; Watanabe et al., 2002]. In addition to the NKX2.5 mutation, exome sequencing identified three additional heterozygous variants of unknown significance in three recessive genes (a missense mutation in the CCDC39 gene, a missense mutation in the DNAH5 gene, and a splice site mutation in the DNAI2 gene). These three variants are listed in dbSNP, although the population frequencies of these variants are
Table 1 List of the variants in disease genes related to clinical phenotype. Gene (RefSeq number)
Inheritance pattern of the disease associated with mutations
Location
Nucleotide alteration
Amino acid alteration
dbSNP ID (population allele frequency)
Zygosity
Inheritance
NKX2.5 (NM_001166176) CCDC39 (NM_181426) DNAH5 (NM_001369) DNAI2 (NM_023036)
AD AR AR AR
Exon2 Exon11 Exon52 Intronic
c.397_400del c.A1433G c.G8805C c.468-4G>T
p.P133GfsTer 42 p.Q478R p.M2935I N/A
Not listed rs115545935 (0.395%) rs77874614 (0.282%) rs146462823 (0.640%)
Het Het Het Het
Maternal Paternal Maternal Maternal
K. Izumi et al. / European Journal of Medical Genetics 57 (2014) 558e561
very low (Table 1). The disorder associated with genetic mutations in these three genes is ciliary dyskinesia [Loges et al., 2008; Merveille et al., 2011; Olbrich et al., 2002]. The exome coverage of these genes was very high; hence, it was unlikely that second mutations in these genes were missed. Although the phenotype of this proband did not fit well with typical symptoms of ciliary dyskinesia, it is intriguing that heterotaxy is occasionally seen in individuals with mutations in ciliary dyskinesia genes including DNAH5 and CCDC39 [Kennedy et al., 2007; Merveille et al., 2011]. In a case described by Watanabe et al., there was significant intrafamilial phenotypic variation, suggesting the presence of modifier genetic or environmental factors [Watanabe et al., 2002]. For example the missense variant in CCDC39, which was inherited from the father, may have served as a modifier for this proband’s phenotype, given that the mother does not have heterotaxy. Aside from heterotaxy-related birth defects, the proband presented with several additional manifestations such as vertebral defects, midline brain malformations, short stature and FTT. Among these, only vertebral defects and scoliosis has been described as an occasional associated anomaly of heterotaxy [Ticho et al., 2000]. Therefore, it remains to be determined whether midline brain malformation, short stature and FTT are associated with the NKX2.5 mutation or caused by other etiologies. Because the proband’s exome sequencing was performed as a clinical test, only genetic variants in diseaseassociated genes were reported. Hence, it is possible that this probands have additional genetic mutation(s) resulting in brain malformation, short stature and FTT. This case report demonstrates the utility of exome sequencing in expanding a phenotypic spectrum of a known Mendelian disorder. Because heterotaxy is not the phenotype which is known to be associated with NKX2.5 mutations, the single gene mutational analysis of NKX2.5 was not considered clinically. The unexpected discovery of mutations in known-disease associated genes, and the resultant expansion of the associated phenotypes, will continue to rise as the use of exome or genome sequencing becomes more commonplace. Such discoveries will lead to a better understanding of the phenotypic spectrum of genetic disorders.
Web resource dbSNP: http://www.ncbi.nlm.nih.gov/SNP/ BCM Whole Genome Laboratory: https://www.bcm.edu/ research/medical-genetics-labs/wholegenomelab
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Acknowledgements The authors thank BCM Whole Genome Laborary for providing the detailed information about the clinical exome analysis of the proband.
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