Skip to content

Advertisement

You're viewing the new version of our site. Please leave us feedback.

Learn more

BMC Medical Genetics

Open Access
Open Peer Review

This article has Open Peer Review reports available.

How does Open Peer Review work?

Breakpoints and deleted genes identification of ring chromosome 18 in a Chinese girl by whole-genome low-coverage sequencing: a case report study

  • Hui Yao1,
  • Chuanchun Yang3,
  • Xiaoli Huang1,
  • Luhong Yang1,
  • Wei Zhao2, 3,
  • Dan Yin2, 3,
  • Yuan Qin1,
  • Feng Mu2, 3,
  • Lin Liu2, 3,
  • Ping Tian1,
  • Zhisheng Liu1 and
  • Yun Yang2, 3, 4Email author
Contributed equally
BMC Medical GeneticsBMC series – open, inclusive and trusted201617:49

https://doi.org/10.1186/s12881-016-0307-1

Received: 4 July 2015

Accepted: 14 June 2016

Published: 22 July 2016

Abstract

Background

Ring chromosome 18 [r(18)] is formed by 18p- and 18q- partial deletion and generates a ring chromosome. Loss of critical genes on each arm of chromosome 18 may contribute to the specific phenotype, and the clinical spectrum varieties may heavily depend on the extent of the genomic deletion. The aim of this study is to identify the detailed breakpoints location and the deleted genes result from the r18.

Case presentation

Here we describe a detailed diagnosis of a seven-year-old Chinese girl with a ring chromosome 18 mutation by a high-throughput whole-genome low-coverage sequencing approach without karyotyping and other cytogenetic analysis. This method revealed two fragment heterozygous deletions of 18p and 18q, and further localized the detailed breakpoint sites and fusion, as well as the deleted genes.

Conclusions

To our knowledge, this is the first report of a ring chromosome 18 patient in China analyzed by whole-genome low-coverage sequencing approach. Detailed breakpoints location and deleted genes identification help to estimate the risk of the disease in the future. The data and analysis here demonstrated the feasibility of next-generation sequencing technologies for chromosome structure variation including ring chromosome in an efficient and cost effective way.

Keywords

Ring chromosomeWhole-genome low-coverage sequencingDetailed breakpointsDetailed diagnosis

Background

Ring chromosome 18 [r(18)] is formed from breakage of both ends of the chromosome and the break ends generate a ring chromosome [1]. Individuals with r(18) have 18p and 18q partial deletions and according phenotype, such as microcephaly, mental deficiency, hypotonia, and congenital heart defects [2, 3]. Short stature, microcephaly, mental deficiency, craniofacial dysmorphism and extremity abnormalities are the most commonly reported features in patients with r(18). The phenotype with r(18) syndrome is highly variable and depends on the combination of 18p- syndrome and 18q- syndrome. Loss of critical genes on each arm of chromosome 18 may contribute to the specific symptoms, and the clinical spectrum varieties may heavily depend on the extent of the genomic deletion [4].

Whole-genome low-coverage sequencing has been reported previously by our group to accurately detect chromosomal structural variation-associated breakpoints and affected region without cytogenetic analysis on patients [5].

In the current study, we applied whole-genome low-coverage sequencing to characterize the ring chromosome 18 mutation at a molecular level in a Chinese young girl for the first time. We described the full profile of clinical examination, genetic characterizations, and clinical treatment report. We localized the genomic breakpoints as well as identified the deleted genes. The deletion of the genes and detailed breakpoint identified help to understand the genotype- correlation and estimate the risk of the disease in the future.

Case presentation

The patient was born to non-consanguineous at the year of 2006. The patient was born at 40 weeks gestation with a birth weight of 3,050 g and length of 49 cm. At 2 years of age, she was found shorter than children of the same age. In April 2008, she was diagnosed hypothyroidism in the local clinic. Replacement of thyroid hormone (levothyroxine) was started for the treatment of autoimmune hypothyroidism. Unregular treatment lasted one year and discontinue by parents themselves.

At 6 and half years of age (March,2013), she came to our hospital for short stature. At the time of our first evaluation, she had a short stature problem (height: 90.7 cm [−6.0SD, equivalently 50 percentile of 2–2.5 years old], weight: 12.0 kg [<3 percentile, equivalently 50 percentile of 2–2.5 years old]). The general examination phenotypes of this patient include intellectual disability with IQ = 70, hypoactive, poor appetite, hypotonia, short neck without webbing, short fingers and toes, much shorter fifth finger, sparse hair and dry skin. She had dry stool once every 1 ~ 3 days. No goiter, lymphadenopathy or hepatosplenomegaly were noted.

The facial appearance of the patient was including flat midface, puffy eyelids, hypertelorism, epicanthic fold, flat nasal bridge, and micrognathia. Wide mouth, downturned corners of mouth, thick lips, large protruding ears ptosis and upslanting palpebral ptosis were also noted (Fig. 1). High narrow palate and several cavities in teeth were observed. In addition, she suffered from bronchitis and otitis media frequently, without serious infections. Auscultation revealed no heart murmur and normal respiratory sounds.
Fig. 1

Abnormalities of the craniofacial appearance. Facial appearance of the patient at age 7, showing flat midface, puffy eyelids, hypertelorism, epicanthic fold, flat nasal bridge, and micrognathia. a frontal view. b lateral view

Serological examination results showed normal liver and kidney functions but abnormal thyroid function, which prompted central autoimmune hypothyroidism and autoimmune thyroiditis. The thyroid auto antibodies were positive. Both TPO-Ab and TG-Ab were extremely high. The levels of IGF-1 and IGF-BP3 decreased drastically. IgA was slightly increased. E2、PROG、PRL and TESTO were all normal (data not show). Flow cytometry detection of T cell subgroup revealed that CD3 and CD8 + T were slightly higher (Additional file 1: Table S1). After euthyrox therapy, her total cholesterol and the triglyceride were back to normal levels, but the lipoprotein-α was still high (494.2 mg/l, reference range: 0-300 mg/l), the IGF-1 still low (29.8 ng/ml, reference range:64-345 ng/ml).

The abdominal color ultrasound results showed normal liver, uterus and ovaries. The sizes of both kidneys were smaller than normal. (left kidney: 6.2 cm × 2.8 cm, right kidney: 5.7 cm × 2.4 cm). The thyroid color ultrasound revealed that the thyroid was enlarged and its echo was not uniform. The thyroid isthmus was 0.5 cm thick (left lobe thyroid: 2.9 cm × 1.0 cm × 1.2 cm, right lobe thyroid: 3.3 cm × 1.0 cm × 1.3 cm) accompanied with uneven internal spots and echoes, like a network. The cardiac color ultrasound showed that the structure, shape and valves of the heart had no obvious abnormality.

The MRI image results revealed that the Pituitary height was 1.0 mm, much smaller than the normal size, and the neurohypophysis was not seen clearly, which indicated pituitary dysplasia (Fig. 2).
Fig. 2

Pituitary gland on MRI. The MRI image results revealed that the Pituitary height was 1.0 mm, much smaller than the normal size, and the neurohypophysis was not seen clearly, which indicated pituitary dysplasia. a Coronal MRI scan of Pituitary Gland. b Sagittal MRI scan of Pituitary Gland

Following informed consent, hromosomal analysis was performed on peripheral blood lymphocyte cultures. The result of conventional karyotyping was 46, XX, r (18) (Fig. 3). No chromosomal anomaly was detected in either of parent by karyotyping analysis (data not shown).
Fig. 3

Patient’s Karyotype analysis result by G-banding technique. The result of conventional karyotyping showed 46, XX, r(18) (arrow indicated)

Patient’s genomic DNA was extracted from peripheral blood using Qiagen DNA extraction kit and then was used to construct DNA libraries and to do sequencing assay including base calling. After removing reads containing sequencing adaptors and low quality reads, the high quality pair-end reads were aligned to the NCBI human reference genome (hg19, GRCh37.1) using SOAP2 [6]. Only uniquely mapped reads were remained in the following analysis.

The ring chromosome variation could be discovered using chimeric read pairs, which are paired-end reads that mapped to two different chromosomes.. The detail steps are listed in our previous published study [5].

Finally, we identified two partial deletions which are a portion of 18p from 1 bp to nearly 3,881,000 bp (3.88 Mb), and a portion of 18q from nearly 73,239,191 bp to terminal (4.83 Mb) base on the bioinformatics results. Both ends of chromosome 18 showed heterozygous terminal deficiency (Fig. 4). The remaining sequence of chromosome 18 generated a ring from breakage and subsequent fusion of both chromosome arms. The two breakpoints located in 18p11.31 band and 18q23 band respectively. The detailed breakpoint sites were validated to be at 3,880,565 bp and at 73,239,237 bp of chr18 respectively by Sanger sequencing. Besides, we also found a 20 bp insertion between the fusion breakpoints (Fig. 5). There were 19 genes deleted at chromosome 18 (pter → p11.31) and 12 genes deleted at chromosome 18 (q23 → qter) (Tables 1 and 2).
Fig. 4

The Copy Number Ratio of Chromosome 18. The blue line indicates the normal diploid copy number ratio, and the window size is 5 kb. Both ends of chromosome 18 has a partial deletion, the copy number ratio is 0.5 showed heterozygous terminal deficiency. Centromere starts from 150Kb to 180Kb

Fig. 5

The Sanger sequence alignment around the breakpoints of chromosome 18. We found there are a 20 bp insertion between the fusion breakpoints

Table 1

Genes and their genomic location within the deleted segment at 18p

Gene Symbol

Gene ID

Chromosome

Start Position

End Position

Strand

USP14

NM_001037334

chr18

158482

213739

+

NM_005151

THOC1

NM_005131

chr18

214519

268059

-

COLEC12

NM_130386

chr18

319354

500729

-

CETN1

NM_004066

chr18

580368

581524

+

NM_014410

NM_199167

C18orf56

NM_001012716

chr18

649619

658340

-

TYMS

NM_001071

chr18

657603

673499

+

ENOSF1

NM_202758,

chr18

670323

712517

-

NM_001126123

NM_017512

YES1

NM_005433

chr18

721591

812327

-

ADCYAP1

NM_001099733

chr18

904943

912173

+

NM_001117

METTL4

NM_022840

chr18

2537523

2571489

-

NDC80

NM_006101

chr18

2571509

2616634

+

SMCHD1

NM_015295

chr18

2655885

2805015

+

EMILIN2

NM_032048

chr18

2847027

2914090

+

LPIN2

NM_014646

chr18

2916991

3011945

-

MYOM1

NM_003803

chr18

3066804

3220106

-

NM_019856

MYL12A

NM_006471

chr18

3247527

3256234

+

MYL12B

NM_033546

chr18

3262110

3278282

+

NM_001144944

NM_001144945

TGIF1

NM_174886

chr18

3412071

3458406

+

NM_173207

NM_173209

NM_173208

NM_003244,

NM_170695

NM_173210

NM_173211

DLGAP1

NM_001003809

chr18

3498836

3845296

-

NM_004746

Table 2

Genes and their genomic location within the deleted segment at 18q

Gene Symbol

Gene ID

Chromosome

Start Position

End Position

Strand

ZNF516

NM_014643

chr18

74069636

74207146

-

NM_007345

MBP

NM_001025081

chr18

74690788

74729055

-

NM_001025090

NM_002385

NM_001025101

NM_001025100

GALR1

NM_001480

chr18

74962007

74982096

+

ATP9B

NM_198531

chr18

76829396

77138282

+

NFATC1

NM_172390

chr18

77155771

77228177

+

NM_006162

NM_172388

NM_172387

NM_172389

CTDP1

NM_004715

chr18

77439800

77514510

+

NM_048368

NM_001202504

PQLC1

NM_001146343

chr18

77662419

77711653

-

NM_001146345

NM_025078

HSBP1L1

NM_001136180

chr18

77724581

77730822

+

TXNL4A

NM_006701

chr18

77732866

77748532

-

RBFA

NM_001171967

chr18

77794345

77810652

+

NM_024805

ADNP2

NM_014913

chr18

77866914

77898228

+

PARD6G

NM_032510

chr18

77915116

78005397

-

Discussion

Ring chromosome 18 syndrome is a rare human cytogenetic abnormality. The syndrome is formed from breakage of both ends of the chromosome and the break ends generate a ring chromosome. The phenotype with r(18) syndrome is highly variable and depends on the combination of 18p- syndrome and 18q- syndrome [7]. The deletion of the short arm of chromosome 18 became a well-known chromosomal aberration after first discovery by de Grouchy in 1963 [2]. In 2009, Patricia et al. analyzed 18q in a high resolution level using aCGH, although they clarified the detailed breakpoint location, the deleted genes result from breakage of 18q were not able to be identified [8]. Normally, people use conventional karyotyping, FISH or aCGH to analysis chromosome aberrations, however, these methods have their limitations of revealing responsible critical genes and clarifying the genotype-phenotype correlations. Whole-genome low-coverage sequencing analysis could solve these problems at a base-level resolution.

Immunoglobulin A deficiency is frequently associated with ring chromosome 18 syndrome [9]. However, IgA deficiency was not noted in our patient, and further our patient appears features of central autoimmune hypothyroidism and small pituitary glands. The pituitary glands of our patient appeared morphologically small on head magnetic resonance imaging, while the thyroid showed morphologically normal on ultrasound. After receiving 10 months hormone therapy (levothyroxine), the IGF-1, T3 and T4 levels were still low, indicating that small pituitary invoked some functional defects, which resulted in the negative feedback failure of Hypothalamus-hypophysis-thyroid axis (HHTA).

There were totally 31 genes deleted at the del(18p) and del(18q) region. Some of them are very important for the physiological activity of the cells. Such as the USP14 gene that encodes a member of the ubiquitin-specific processing (UBP) family of proteases that is a deubiquitinating enzyme (DUB). Mice with a mutation that results in reduced expression of the ortholog of this protein are retarded for growth [10]. Gripp et al. [11] concluded that TGIF1 links the NODAL signaling pathway to the bifurcation of the human forebrain and the establishment of ventral midline structures. The GALR1 gene is widely expressed in the brain and spinal cord, as well as in peripheral sites such as the small intestine and heart [12]. Mutations in CTDP1 gene are associated with congenital cataracts, facial dysmorphism and neuropathy syndrome (CCFDN) [13]. So that, the inactivity of these genes may results to neurodevelopment, craniofacial appearance, oral manifestations and brain development anomalies.

In this report, We have presented a ring chromosome 18 patient with two heterozygous deletions of 3.88 Mb and 4.83 Mb indentified by whole-genome low-coverage sequencing method. The deletion of the genes and ring closure of chromosome 18 contribute to the clinical picture of dysmorphogenesis and mental retardation. Detailed breakpoints location and deleted genes identification help to estimate the risk of the disease in the future. At the same time, further studies are needed to delineate the function of responsible critical genes and clarify the genotype-phenotype correlations. The report here demonstrated the feasibility of next-generation sequencing technologies for chromosomal structural variation including ring chromosome in an efficient and cost effective way, which would improve the detection and prediction of genotype and phenotypic outcomes to direct postnatal medical care.

Conclusions

In conclusion, we analyzed a ring chromosome 18 patient in China by whole-genome low-coverage sequencing method for the first time. We described the full profile of clinical examination, genetic characterizations, and clinical treatment report. We localized the genomic breakpoints as well as identified the deleted genes. Detailed breakpoints location and deleted genes identification help to estimate the risk of the disease in the future. The report here demonstrated the feasibility of next-generation sequencing technologies for chromosomal structural variation including ring chromosome in an efficient and cost effective way, which would improve the detection and prediction of genotype and phenotypic outcomes to direct postnatal medical care.

Consent

Written informed consent was obtained in accordance with the Institutional Review Board of Wuhan Maternal and Child Health Hospital and the Declaration of Helsinki. The parents permitted the publication of the case, their clinical details and images.

Declarations

Acknowledgments

We thank all participants involved in this study.

Authors’ contributions

HY, CY and YY managed the project. XH, LY, YQ and PT. collected and prepared the samples. FM and DY performed the sequencing. CY and WZ performed the bioinformatics analysis. HY and CY wrote the paper. LL, DY and YY revised the paper.

Competing interests

The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Wuhan Medical Care Center for Women and Children
(2)
BGI-Wuhan
(3)
BGI-Shenzhen
(4)
Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Zhengzhou University

References

  1. Wertelecki W, Gerald PS. Clinical and chromosomal studies of the 18q- syndrome. J Pediatr. 1971;78(1):44–52.View ArticlePubMedGoogle Scholar
  2. De Grouchy J, Leveque B, Debauchez C, Salmon C, Lamy M, Marie J. [17–18 Ring-Chromosomes and Congenital Malformations in a Young Girl]. Ann Genet. 1964;7:17–23.Google Scholar
  3. Cody JD, Ghidoni PD, DuPont BR, Hale DE, Hilsenbeck SG, Stratton RF, Hoffman DS, Muller S, Schaub RL, Leach RJ. Congenital anomalies and anthropometry of 42 individuals with deletions of chromosome 18q. Am J Med Genet. 1999;85(5):455–62.View ArticlePubMedGoogle Scholar
  4. Subrt I, Pokorny J. Familial occurrence of 18q. Humangenetik. 1970;10(2):181–7.PubMedGoogle Scholar
  5. Dong Z, Jiang L, Yang C, Hu H, Wang X, Chen H, Choy KW, Hu H, Dong Y, Hu B. A robust approach for blind detection of balanced chromosomal rearrangements with whole-genome low-coverage sequencing. Hum Mutat. 2014;35(5):625–36.View ArticlePubMedGoogle Scholar
  6. Li R, Yu C, Li Y, Lam TW, Yiu SM, Kristiansen K, Wang J. SOAP2: an improved ultrafast tool for short read alignment. Bioinformatics. 2009;25(15):1966–7.View ArticlePubMedGoogle Scholar
  7. Brkanac Z, Cody JD, Leach RJ, DuPont BR. Identification of cryptic rearrangements in patients with 18q- deletion syndrome. Am J Hum Genet. 1998;62(6):1500–6.View ArticlePubMedPubMed CentralGoogle Scholar
  8. Heard PL, Carter EM, Crandall AC, Sebold C, Hale DE, Cody JD. High resolution genomic analysis of 18q- using oligo-microarray comparative genomic hybridization (aCGH). Am J Med Genet A. 2009;149A(7):1431–7.View ArticlePubMedPubMed CentralGoogle Scholar
  9. Litzman J, Brysova V, Gaillyova R, Thon V, Pijackova A, Michalova K, Zemanova Z, Lokaj J. Agammaglobulinaemia in a girl with a mosaic of ring 18 chromosome. J Paediatr Child Health. 1998;34(1):92–4.View ArticlePubMedGoogle Scholar
  10. Wilson SM, Bhattacharyya B, Rachel RA, Coppola V, Tessarollo L, Householder DB, Fletcher CF, Miller RJ, Copeland NG, Jenkins NA. Synaptic defects in ataxia mice result from a mutation in Usp14, encoding a ubiquitin-specific protease. Nat Genet. 2002;32(3):420–5.View ArticlePubMedGoogle Scholar
  11. Gripp KW, Wotton D, Edwards MC, Roessler E, Ades L, Meinecke P, Richieri-Costa A, Zackai EH, Massague J, Muenke M. Mutations in TGIF cause holoprosencephaly and link NODAL signalling to human neural axis determination. Nat Genet. 2000;25(2):205–8.View ArticlePubMedGoogle Scholar
  12. Walli R, Schafer H, Morys-Wortmann C, Paetzold G, Nustede R, Schmidt WE. Identification and biochemical characterization of the human brain galanin receptor. J Mol Endocrinol. 1994;13(3):347–56.View ArticlePubMedGoogle Scholar
  13. Varon R, Gooding R, Steglich C, Marns L, Tang H, Angelicheva D, Yong KK, Ambrugger P, Reinhold A, Morar B. Partial deficiency of the C-terminal-domain phosphatase of RNA polymerase II is associated with congenital cataracts facial dysmorphism neuropathy syndrome. Nat Genet. 2003;35(2):185–9.View ArticlePubMedGoogle Scholar

Copyright

© The Author(s). 2016

Advertisement