Pycnodysostosis: Novel Variants in CTSK and Occurrence of Giant Cell Tumor

Abstract

Pycnodysostosis is an autosomal recessive skeletal dysplasia caused by pathogenic variants in the cathepsin K ( CTSK ) gene. We report seven patients from four unrelated families with this condition in whom we have identified three novel pathogenic variants, c.120 + 1G > T in intron 2, c.399 + 1G > A in intron 4, and c.148T > G (p.W50G) in exon 2, and a known variant, c.568C > T (p.Q190*) in exon 5 of CTSK . We present the clinical, radiographic, and molecular findings of all individuals with molecularly proven pycnodysostosis from the present cohort. We also report the occurrence of giant cell tumor in the skull of a patient with this condition.

Introduction

Pycnodysostosis (OMIM 265800) was first described by Maroteaux and Lamy in 1962 as an autosomal recessive condition with short stature, relatively moderate bone brittleness, and craniofacial deformity with a delayed fusion of fontanels. ¹ Incidence of pycnodysostosis is 1 in 1.7 million individuals. ² Other features include fractures, osteolysis of the distal phalanges, dysplastic nails, clavicular dysplasia, congenital pseudarthrosis of clavicle, spondylolysis, frontal and parietal bossing, beaked nose, prominent eyes, bluish sclera, hypoplasia of maxilla and mandible, craniosynostosis, nonpneumatized paranasal sinuses, Arnold–Chiari malformation, delayed eruption of permanent teeth, persistence of deciduous teeth, dental crowding, dental malocclusion, obtuse mandibular angle, and grooved palate. ³ Conductive hearing loss due to otosclerosis is also reported. ⁴ Pycnodysostosis is caused by pathogenic variants in cathepsin K ( CTSK ), which is mapped to chromosome 1q21 and plays an important role in osteoclast function. ⁵ The CTSK has 8 exons, and the translated protein has 329 amino acids with a molecular mass of 36 kDa. CTSK is a lysosomal cysteine proteinase and is involved in the remodeling and resorption of bone tissue. It is predominantly expressed in the osteoclasts. It is also shown to be significantly expressed in human breast cancer cells where it apparently is conducive to tumor invasion. ⁶ To date, there have been approximately 55 different pathogenic variants reported in CTSK causing pycnodysostosis in the literature. ^{7 8 9 10 11 12 13 14 15 16 17 18 19} It includes 35 missense variants, 5 nonsense variants, 10 frameshift variants, 4 splice site variants, and 1 stop variant.

In this study, we performed mutation analysis in seven Indian patients from four unrelated families with clinical diagnoses of pycnodysostosis and found three novel CTSK variants and one known pathogenic variant in them. We also observed a giant cell tumor (GCT) in the occipital bone of one patient.

Materials and Methods

Appropriate informed consents were obtained from the patients and families for this study. Detailed clinical and radiological findings were recorded. Genomic DNA was isolated from peripheral blood using phenol–chloroform method. All the coding exons and flanking introns of CTSK (NM_000396.1) were amplified using polymerase chain reaction (PCR) with specifically designed primers. PCR amplicons were purified and sequenced by ABI PRISM 3130 genetic analyzer (Life Technologies, Foster City, California, United States). Multiple sequence alignment and in silico prediction of pathogenicity using MutationTaster, SIFT, and PolyPhen2 were used for bioinformatics analyses. This study was approved by the respective institutional ethics committees.

Results

We evaluated seven patients from four unrelated families. All of them were consanguineous ( Supplementary Material , available in the online version). The common clinical features include frontal prominence, micrognathia, short stature, and stubby digits ( Fig. 1 ). Skeletal survey revealed dense skull, persistently open anterior fontanel, delayed closure of the suture, obtuse angle of the mandible, brachydactyly, acro-osteolysis of hands, and osteosclerosis. Patient (P1) had a swelling over the occipital region and magnetic resonance imaging (MRI) of the brain revealed a focal, heterogeneously enhancing altered signal intensity extracalvarial lesion in the subgaleal and subcutaneous planes in the left occipital region ( Supplementary Material , available in the online version). The swelling over the occipital region was surgically removed. Histopathological evaluation established the diagnosis of GCT ( Supplementary Material , available in the online version). Following surgery, the tumor recurred. Clinical and radiological features of patients described here are summarized in Table 1 .

Fig. 1.

Facial profiles of patients with pycnodysostosis. (Patient [P] 1, A, B; P2, C, D; P4, E; P5, F, G; P6 H, I; P7, J). All of them share prominent forehead, frontal and parietal bossing, and micrognathia. P1 also shows scar of operated giant cell tumor.

Table 1. Clinical features of patients (P1-P5) with pycnodysostosis.

	Family 1	Family 2	Family 3		Family 4
	P1	P2	P3	P4	P5
Age at current evaluation (y)	26	5	13	7	3.5
Gender	Female	Male	Male	Female	Female
Height in cm (SD)	128 (–6)	NA	131 (–4)	NA	78 (–5)
Consanguinity	+	+	+	+	+
Clinical features
Short stature	+	NA	+	NA	+
Frontal prominence	+	+	+	+	+
Occipital prominence	–	+	NA	NA	–
Micrognathia	+	+	+	+	+
Retrognathia	+	+	–	–	–
Prominent nose	+	+	+	–	–
Wrinkled skin over dorsa of fingers	+	+	NA	+	–
Radiological features
Dense skull	+	+	NA	NA	+
Persistent open anterior fontanel	+	+	NA	NA	+
Wormian bones	+	+	NA	NA	–
Fracture	–	+	+	–	–
Delayed suture closure	+	+	NA	NA	+
Absent frontal sinus	+	+	NA	NA	–
Obtuse angle of mandible	+	+	NA	NA	+
Aplasia of clavicle	–	–	–	NA	–
Hypoplasia of clavicle	+	+	+	NA	–
Brachydactyly	+	+	+	+	–
Acro-osteolysis of hands	+	+	+	+	–
Scoliosis	+	+	+	NA	–
Osteosclerosis	+	+	+	+	+
Narrow ilia	NA	+	+	+	–
Genotype
Homozygous variants in CTSK gene	c.120 + 1G > T	c.399 + 1G > A	c.148T > G	c.148T > G	c.568C > T

Abbreviations: +, present; –,absent; CTSK , cathepsin K; NA: not available; P, patient; SD, standard deviation.

Mutation Analysis

Genomic deoxyribonucleic acid (DNA) from all seven patients was sequenced for the entire coding sequence and the splice sites of CTSK . Subsequent DNA analysis revealed three novel homozygous sequence variants: c.120 + 1G > T in intron 2 (P1), c.399 + 1G > A in intron 4 (P2), c.148T > G (p.W50G) in exon 2 (P3 and P4) ( Fig. 2A–I ). A known variant, c.568C > T (p.Q190*) in exon 5, was detected in P5, P6, and P7 ( Fig. 2J–N ). ²⁰ The biallelic segregation was confirmed in parents in all three families. All four variants were not present in the Exome Aggregation Consortium (ExAC) database. Multiple sequence alignment performed by Clustal Omega for the homologs of human CTSK gene product showed that tryptophan amino acid is highly conserved among the species, whereas it has been replaced by glycine in P3 and P4 from family 3. This variant was also found to be damaging using pathogenicity prediction tools such as MutationTaster, SIFT, and PolyPhen-2.

Fig. 2.

Electropherograms of families 1, 2, 3 and 4. Patient (P) 1 show homozygous splice site variant in intron 2 of cathepsin K ( CTSK ) gene ( A ), and the father is heterozygous for the same ( B ). P2 shows homozygous splice site variation in intron 4 ( C ), whereas parents are heterozygous for the same variant ( D,E ). P3 and P4 of family 3 show homozygous missense variation in exon 2 of CTSK gene ( F,G ), whereas parents are heterozygous for the same variant ( H,I ). P5 to P7 of family 4 show homozygous missense variation in exon 5 of CTSK gene ( J–L ), whereas parents are heterozygous for the same variant ( M, N ).

Discussion

In this study, the most common clinical features observed in all seven patients are frontal prominence and micrognathia. Radiological features include persistent anterior and posterior fontanels, osteosclerosis, acro-osteolysis of distal phalanges, and brachydactyly. Other features include a prominent nose, wrinkled skin over dorsa of the fingers, hypoplasia of clavicle, the presence of Wormian bones, absent frontal sinus, scoliosis, and narrow ilia. Additionally, we observed GCT in the left occipital region in P1 which could be, however, coincidental.

GCT in patients with pycnodysostosis has not been reported previously, and the mechanism of tumorigenesis is yet to be identified. GCT of bone is generally benign and histologically characterized by the presence of osteoclast-like cells that are multinucleated. Although it is a benign lesion and locally aggressive, it can reappear after surgical resection. ²¹ Lindeman et al established that CTSK is primarily responsible for osteolytic activity in GCT. ²²

Sequence analysis of CTSK in seven patients with clinical diagnosis of pycnodysostosis identified three novel variants and one previously known variant. Two patients had homozygous splice site pathogenic variants (c.120 + 1G > T in intron 2 and c.399 + 1G > A in intron 4), and another two patients belonging to the same family had a homozygous missense pathogenic variant c.148T > G in exon 2 of the CTSK . Splice site variants disrupt the canonical splice site in introns 2 and 4 in the CTSK . Canonical splice site variants are known to alter gene function by various mechanisms. The variant observed in the third family was a missense variant, c.148T > G, and that results in an amino acid substitution from tryptophan to glycine at the 50th codon in a highly conserved position. In silico analysis tools were consistent in predicting that the variant might damage CTSK protein function. In the fourth family, a known nonsense variant, c.568C > T, identified results in premature termination of translation at a residue 190. ²⁰

Very few mutation proven cases of pycnodysostosis have been reported from India. ^{8 9} To date, only four splice site variants in the CTSK gene are reported in the literature. ^{7 16 23 24} Therefore, our study widens the mutation spectrum of pycnodysostosis. Although CTSK has been associated with GCT in patients without pycnodysostosis, GCT in a patient with pycnodysostosis in our study has not been previously reported. This report expands the clinical spectrum of this condition as well.

Funding Statement

Funding This work is funded by the Department of Science and Technology through the project titled “Application of autozygosity mapping and exome sequencing to identify genetic basis of disorders of skeletal development” (SB/SO/HS/005/2014).