Letter to Editor

Genetic Diagnosis Using Whole Exome Analysis in Two Cases with Malignant Osteopetrosis of Infancy


  • Korcan Demir
  • Özlem Nalbantoğlu
  • Kadri Karaer
  • Hüseyin Anıl Korkmaz
  • Melek Yıldız
  • Selma Tunç
  • Behzat Özkan

Received Date: 10.11.2015 Accepted Date: 12.11.2015 J Clin Res Pediatr Endocrinol 2015;7(4):356-357

Keywords: mutation,TCIRG1,CLCN7,osteopetrosis,whole exome analysis

To the Editor,

Malignant osteopetrosis of infancy (MOI) is a life-threatening form of osteopetrosis characterized by dense, sclerotic and fragile bones, impairment of bone marrow function, entrapment of cranial nerves, and growth retardation (1,2). The underlying mechanism leading to MOI is possibly an aberration in either the differentiation or function of osteoclasts. Until now, homozygous or compound heterozygous mutations in seven genes (TNFRSF11A, TNFSF11, TCIRG1, CLCN7, OSTM1, SNX10, and PLEKHM1) have been found in 80% of children with MOI (2). Detection of the exact cause and provision of genetic counselling via individual mutation analysis of all these genes would be expensive and time-consuming. Whole exome sequencing is being increasingly used given that its cost and the time needed for analysis are similar to that of single-gene sequencing (3,4). In addition, whole exome sequencing offers the probability to detect novel causative genes in the remaining 20% of patients with MOI.

Our first patient, a 9-day-old male infant, was referred to our institution with hypocalcemia (calcium, 5.4 mg/dL). His hemoglobin level was 8.6 g/dL and platelet count was 130 000/mm3. His parents were not relatives. There was no similar case in the family. His weight (3060 g, 25th-50th percentile), length (46 cm, 10th-25th percentile), and head circumference (36 cm, 10th percentile) measurements were normal, and no pathological examination finding was noted. Skeletal survey demonstrated sclerotic bones.

The second patient was a 7-day-old female infant with hepatosplenomegaly and hypocalcemia (calcium, 6.8 mg/dL). Her parents were relatives, and one of her elder brothers had died at age four months from complications of osteopetrosis without a genetic diagnosis. Physical examination revealed normal weight (3045 g, 25th-50th percentile), length (47.5 cm, 10th-25th percentile), and head circumference (37 cm, 25th percentile), and hepatosplenomegaly. She had thrombocytopenia (139.000/mm3) but no anemia or leukopenia. Radiographic findings revealed a dense skeleton, and the diagnosis of osteopetrosis was suggested.

In these two patients, we employed whole exome analysis with particular attention to the seven candidate genes. The DNA samples of the patients were extracted from peripheral blood. Exome sequencing data generated in Genotypic (India) using HiSeq 2,500 sequencer were analyzed in the Intergen Genetics Centre. In the first case, 31.382 variants were detected. Among the possible causative genes, a novel heterozygous mutation (c.718G>A, p.Gly240Arg), which was strongly predicted to be a disease-causing mutation with in silico analyses with MutationTaster (mutationtaster.org), SIFT (sift.jcvi.org), PolyPhen-2 (genetics.bwh.harvard.edu/pph2), was detected in CLCN7 gene (Figure 1a). Whole gene MiSeq next-generation sequencing of CLCN7 confirmed the above-mentioned mutation and detected another novel frameshift mutation as well (Figure 1b), resulting in a compound heterozygous state (c.398_401delTTGG, p.Ile133Argfs*49 and c.718G>A, p.Gly240Arg). In the second case, 32.529 variants were detected. A previously reported homozygous nonsense mutation p.Gln746* (c.2236C>T) in TCIRG1 was detected and confirmed using MiSeq next-generation sequencing (Figure 1c). Genetic counselling was provided, and pre-implantation genetic testing was recommended for both families.

Whole exome analysis is a useful method for diseases in which multiple genes play a role in the etiology. However, it should be kept in mind that Sanger sequencing/next-generation sequencing may be needed when a heterozygous mutation is detected by whole exome sequencing in autosomal recessively inherited candidate genes.

Authorship Contributions

Concept: Korcan Demir, Behzat Özkan, Design: Korcan Demir, Data Collection or Processing: Korcan Demir, Kadri Karaer, Hüseyin Anıl Korkmaz, Melek Yıldız, Selma Tunç, Analysis or Interpretation: Kadri Karaer, Korcan Demir, Literature Search: Korcan Demir, Özlem Nalbantoğlu, Writing: Korcan Demir, Özlem Nalbantoğlu, Conflict of Interest: No conflict of interest was declared by the authors, Peer-review: Internal peer-reviewed, Financial Disclosure: The authors declared that this study has received no financial support.

1. Del Fattore A, Cappariello A, Teti A. Genetics, pathogenesis and complications of osteopetrosis. Bone 2008;42:19-29.
2. Sobacchi C, Schulz A, Coxon FP, Villa A, Helfrich MH. Osteopetrosis: genetics, treatment and new insights into osteoclast function. Nat Rev Endocrinol 2013:9:522-536. Epub 2013 Jul 23
3. Sui W, Ou M, Liang J, Ding M, Chen J, Liu W, Xiao R, Meng X, Wang L, Pan X, Zhu P, Xue W, Zhang Y, Lin H,Li F, Zhang J, Dai Yl. Rapid gene identification in a Chinese osteopetrosis family by whole exome sequencing. Gene 2013:516:311-315. Epub 2013 Jan 4
4. Campeau PM, Lu JT, Sule G, Jiang MM, Bae Y, Madan S, Högler W, Shaw NJ, Mumm S, Gibbs RA, Whyte MP, Lee BH. Whole-exome sequencing identifies mutations in the nucleoside transporter gene SLC29A3 in dysosteosclerosis, a form of osteopetrosis. Hum Mol Genet 2012:21:4904-4909. Epub 2012 Aug 8