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Journal of Pediatric Surgery (2007) 42, E27–E30

Clinical features and HLXB9 gene mutation of a sporadicChinese Currarino’s syndrome caseYing Liang, Jun Wang, Wei Cai*

Department of Pediatric Surgery, Xin Hua Hospital Affiliated to School of Medicine, Shanghai Jiaotong University,Shanghai 200092, China

⁎ Corresponding author. Tel.: +86 21E-mail address: caiw204@yahoo.com

0022-3468/$ – see front matter © 2007doi:10.1016/j.jpedsurg.2007.03.062

AbstractBackground/Purpose: The Currarino's syndrome (CS), which is characterized by sacral bonyanomalies, anorectal malformation, and a presacral mass, is associated with mutations of HLXB9gene. The aim of this study was to clearly define the clinical manifestations and molecular anomalies ofCS in China.Methods: We studied the medical history and clinical manifestations of a child with presacral mass.Genomic DNA was extracted from lymphocytes, and mutation analysis of the HLXB9 gene wasconducted by using polymerase chain reaction and direct sequencing in the child and her parents.Results: A previously unreported heterozygous missense mutation of HLXB9 gene was detected inthe child.Conclusions: The HLXB9 gene mutation could take place in sporadic cases of CS without a typicalhemisacrum.© 2007 Elsevier Inc. All rights reserved.

Index words

Currarino's syndrome;HLXB9 gene;Mutation

The Currarino's syndrome (CS) has been described as theassociation of presacral mass, anorectal malformation, andsacral bony defect by Currarino et al [1] in 1981. Patientsaffected by CS display phenotypic variability, and thespectrum of phenotypes ranges from multiple systemmalformations to asymptomatic features [2,3]. However,patients always exhibit the typical scimitar sacrum [3,4].Recently, mutations of HLXB9 gene have been reported to beextensively involved in the pathogenesis of CS [5-7].

In this study, we described the clinical characteristics of aChinese CS case and analyzed the HLXB9 gene mutation.

6501 1627; fax: +86 21 6501 1627..cn (W. Cai).

Elsevier Inc. All rights reserved.

1. Patients and methods

1.1. Case report

A 16-month-old girl had suffered from persistentconstipation since birth. A rectal examination showed noanal stenosis and ectopic anus. Barium enema found themildly dilated rectum because of the suspected narrow of theanal canal end and no obvious morphological changes inthe transverse colon and descending colon. Therefore, shewas admitted to the hospital for the first time with suspecteddiagnosis of Hirschsprung's disease. Magnetic resonanceimaging (MRI) demonstrated a presacral mass in associationwith sacrococcygeal hypoplasia (Fig. 1). The presacral masswas removed and detected to be a kind of dermoid cyst byhistochemical staining. The symptom of constipation wasrelieved after the surgical excision, and the child was

Fig. 1 Sagittal section (A) and axial section (B) of MRI showedthe presence of the presacral mass and sacrococcygeal hypoplasia.

ig. 2 Spinal image of MRI after excision of the presacral mass.

ig. 3 Pelvic x-ray film showing coccygeal defect with associatedcal loading.

E28 Y. Liang et al.

dismissed. Rectal dilatation was performed to size 24Fduring the follow-up period.

Seven months later, the child was admitted to the hospitalagain because of the recurrence of constipation. A similarobservation was reported according to barium enema andsuggested the possible existence of Hirschsprung's disease.Rectal pressure was measured and indicated no response tointernal sphincter relaxation reflux. Magnetic resonanceimaging demonstrated the postexcision changes and enlargedbladder (Fig. 2). Pelvic x-ray examination was performedafter the administration of glycerin enema. The film showedcoccygeal defect with associated fecal loading (Fig. 3). Thetypical hemisacrum was not observed in this case. Retentionof 10-mL urine was determined by ultrasonography. Rectalsuction biopsy was performed and showed the presence ofparasympathetic intrinsic ganglion cells in the submucosaland myenteric plexuses. Therefore, Hirschsprung's diseasewas excluded and Lynn's rectal myectomy was carried out.

The parents of the child had no complaint of intractableconstipation and neither other family members. The parentswere offered a pelvic x-ray, but no sacral bony anomalieswere indicated.

F

1.2. Mutation analysis of the HLXB9 gene

Blood was drawn from the child and her parents. GenomicDNA was isolated from lymphocytes by a QIAamp DNABlood Mini kit (QIAGEN, Hilden, Germany) after theprotocol of the manufacturer. Polymerase chain reaction(PCR) primers were synthesized according to what waspreviously described to screen the coding regions and basicpromoter of the HLXB9 gene [5,7]. Table 1 summarizes the

Ffe

Table 1 Primer sequences and PCR conditions for amplification and sequencing of the HLXB9 gene

Primer Sequence (5′→3′) Size (bp) Tm (°C) Comments

HB9-Ex1A F a: CCGCACACGG CCGCGTCGCC CGCCACCGGG 439 66 2 mmol/L MgCl2, 10% DMSORa: CGGCGGCGGC AGCGGCCGCT GCGCCCGGAT

HB9-Ex1B F: CGGCACGGGC GGCGGGCACG GGGGGCCCCA 500 66 2 mmol/L MgCl2, 10% DMSORa: GCAGCTCTTC CCCCGCTCGC TGGGAGCCAA

HB9-Ex2 F: GCCTCACTGC CCTTTGGTC 283 61 2 mol/L betaine (Sigma, St Louis, MO)R a: CAGCGCTAGA TGCCTCAGA

HB9-Ex3 F a: CCTCCCCATT CTGCTTCTAA 700 56 GC-rich PCR system(TaKaRa), 2 mol/L betaine (Sigma, St Louis, MO)R: CCCTTGCTTA AAAGGGAAGC

HB9-PR F a: GCTCAGGAGC GTGGAACC 724 61 2 mmol/L MgCl2, 10% DMSORa: GCGGGCTCGC GGAGTCAG

DMSO indicates dimethyl sulfoxide.a Primers used for sequencing.

E29Clinical features and HLXB9 gene mutation

primer sequence and PCR condition for each reaction.Briefly, PCR reactions were carried out in a total volume of50 μL containing approximately 200 ng genomic DNA,1× PCRbuffer, 0.5 μmol/L of each primer, 200 μmol/L ofeach deoxynucleotide triphosphates (dNTP), and 1 U TaqDNA polymerase (TaKaRa, Japan). Amplification conditionwas as follows: 94°C for 5 minutes (1 cycle), 94°C for1 minute, Tm for 1 minute, 72°C for 1 minute (35 cycles), and72°C for 10 minutes (1 cycle). The PCR products werepurified and sequenced by using an ABI PRISM Big Dyeassay (Applied Biosystems, Foster City, CA).

2. Results

The heterozygous missense 754 C N A (Q252 K, Q =Gln = glutamine, K = Lys = lysine) mutation was discoveredin the child (Fig. 4). This mutation is located in the DNAbinding domain where several missense mutations have beenreported [6,8]. No HLXB9 gene mutation was detected in herparents. Therefore, the child presenting with presacral massand HLXB9 gene mutation is a sporadic CS case.

3. Discussion

The symptoms of CS are highly variable. The usualpresenting symptom in child is constipation, which is caused

Fig. 4 A, Electrophoregram from the child indicated a hetero-zygous C N A mutation at cDNA base pair position 754 (markedwith an asterisk). B, A normal electrophoregram from her parents.

by anorectal malformations [9]. In this case, physicalexamination and biopsy analysis provided no evidence ofthe existence of anal stenosis or Hirschsprung's disease. Thepossible explanation might be owing to the failure of analsphincter relaxation.

Patients affected by CS always exhibit the typical scimitarsacrum, but not in our case. Only the hypoplastic sacrum andcoccyx were detected by MRI. Belloni et al [3] have shownthat of the 5 specific categories of sacrococcygeal anomalies,only hemisacrum is caused by mutations in the HLXB9 gene.However, we report here that a mutation of HLXB9 gene wasdetected in the child without hemisacrum. Therefore,although the presence of a scimitar sacrum is essential,molecular analysis of the HLXB9 gene is necessary forproper diagnosis of CS.

The HLXB9 gene has 3 exons encoding a 403–amino acidprotein HB9. The HB9 protein contains a homeodomainpreceded by a highly conserved 82–amino acid domain.Most of the reported missense mutations of HLXB9 geneaffect the homeobox domain [7,8]. Our report providesfurther evidence of the relevance of HLXB9 missensemutation in homeobox domain. It is known that proteinsencoded by homeobox-containing genes are sequence-specific DNA binding proteins and function as a transcrip-tion factor regulating gene expression in both developing andadult tissues. Ross et al [5] have demonstrated that HLXB9gene was expressed in the sacral region during embryogen-esis. Thus, it is likely that an amino acid change in thehomeobox domain of HB9 protein is critical to the sacralagenesis in CS.

References

[1] Currarino G, Coln D, Votteler T. Triad of anorectal, sacral, and presacralanomalies. Am J Roentgen 1981;137:395-8.

[2] Emans PJ, Kootstra G, Marcelis CL, et al. The Currarino triad: thevariable expression. J Pediatr Surg 2005;40:1238-42.

[3] Belloni E, Martucciello G, Verderio D, et al. Involvement of the HLXB9homeobox gene in Currarino syndrome. Am J Hum Genet 2000;66:312-9.

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[4] Lynch SA, Wang Y, Strachan T, et al. Autosomal dominant sacralagenesis: Currarino syndrome. J Med Genet 2000;37:561-6.

[5] Ross AJ, Ruiz-Perez V,Wang Y, et al. A homeobox gene, HLXB9, is themajor locus for dominantly inherited sacral agenesis. Nat Genet1998;20:358-61.

[6] Hagan DM, Ross AJ, Strachan T, et al. Mutation analysis and embryonicexpression of the HLXB9 Currarino syndrome gene. Am J Hum Genet2000;66:1504-15.

[7] Garcia-Barcelo M, So MT, Lau DK, et al. Population differences in thepolyalanine domain and 6 new mutations in HLXB9 in patients withCurrarino syndrome. Clin Chem 2006;52:46-52.

[8] Wang RY, Jones JR, Chen S, et al. A previously unreported mutation ina Currarino syndrome kindred. Am J Med Genet 2006;140A:1923-30.

[9] Kochling J, Karbasiyan M, Reis A. Spectrum of mutations andgenotype-phenotype analysis in Currarino syndrome. Europ J HumGenet 2001;9:599-605.