Genetic diagnosis of skeletal dysplasias causing short stature in children

When should a pediatric endocrinologist consider the possibility of skeletal dysplasia when evaluating a child with short stature?

The main clues to the diagnosis of skeletal dysplasia in a child brought with short stature include the presence of disproportion, multiple fractures detected clinically or on X-rays, unresponsiveness to calcium and vitamin D therapy in a child with rickets, and X-rays showing significant metaphyseal or epiphyseal abnormalities or the presence of dislocations. The presence of additional cardiac, digital, or ear abnormalities may point to an underlying syndrome.

What are the common forms of skeletal dysplasia presenting with short stature?

The most common skeletal dysplasias usually seen in a pediatric clinic include ACH, mucopolysaccharidoses, and spondyloepiphyseal dysplasias (SEDs). Other causes of short stature such as celiac disease, hypothyroidism, and chronic renal disease should be ruled out by performing appropriate tests.

What are the steps in making a diagnosis and genetic testing of skeletal dysplasias?

Although some skeletal and joint involvements have variable phenotypes, most of the commoner ones present with short stature.^[6,7] The initial clinical evaluation includes finding any disproportion which involves measuring limb lengths and the upper segment to the lower segment ratio. The radiological survey for skeletal dysplasia involves doing X-rays of the upper limbs, lower limbs, pelvis, spine, and hands (including wrist) to look for the type of involvement – rhizomelic, mesomelic or acromelic; or if there are additional defects in the spine.

The confirmation of the diagnosis would require biochemical and molecular evaluation in most cases apart from radiological characterization. The biochemical tests include serum calcium, phosphorus, and alkaline phosphatase levels. Urinary biomarkers such as pyridinolines or collagen breakdown products are tested in some specific cases. Bone-specific alkaline phosphatase can also be done in some selected cases. Enzyme testing on heparinized blood samples confirms the type of MPS in most cases. Deoxyribonucleic acid testing may be needed in some cases to confirm the diagnosis if enzyme testing is not available or shows borderline values. [Table 3] lists the common disorders encountered in pediatric practice. Figure 1 shows bowing seen in osteogenesis imperfecta (OI) and metaphyseal dysplasia, and Figure 2 depicts the phenotypic presentation in hereditary multiple exostosis. Dysostosis seen in MPS patients is shown in Figure 3.

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Figure 1:

Radiograph showing osteopenia, fractures, and bowing in osteogenesis imperfecta (a) and bowing of femur and metaphyseal flaring and irregularities in Schmid metaphyseal dysplasia (b).

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Figure 2:

The swellings over the lower limb (a) and X-ray showing bony projections or osteochondromas at the upper end of the tibia and lower ends of the femur (b) in hereditary multiple exostosis.

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Figure 3:

Dysostosis seen on X-rays in mucopolysaccharidosis (MPS). There is kyphosis, anterior beaking and platyspondyly in MPS IV type A (a) and shortening, irregularity, and proximal pointing of metacarpals in hand X-ray in the same patient (b).

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A simple and practical approach to the genetic diagnosis is illustrated in the flowchart [Figure 4].

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Figure 4:

Flowchart depicting the clinical approach to genetic testing in skeletal dysplasias.

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Is there a phenotype-genotype correlation in the clinical presentations?

There is significant phenotypic variability in the clinical presentations, though the gene involved may be the same. This is known as phenotypic heterogeneity of that gene-related skeletal disorder. Two examples include the fibroblast growth factor receptor 3 gene (FGFR3) and collagen type 2 alfa 1 (COL2A1) gene.

The phenotypes related to the FGFR3 gene include ACH, a common skeletal dysplasia causing short stature, thanatophoric dysplasia, which is a lethal skeletal dysplasia and CATSHL syndrome in which there is tall stature along with camptodactyly, scoliosis, and hearing loss.^[8] In the SADDAN phenotype, there is severe ACH, along with developmental delay and acanthosis nigricans, and early respiratory failure leads to mortality. Thus, thanatophoric dysplasia, ACH, and hypochondroplasia (HCH) are all the result of pathogenic variants in the FGFR3. ACH can be easily diagnosed by Sanger sequencing. The two common mutations FGFR3:c.1138G >A and c.1138G >C in this gene account for 98% of patients with ACH leading to p.Gly380Arg change in the protein. In SADDAN, there is a different mutation, c.1949A >T.

In the case of COL2A1 also, there are variable phenotypes, as shown in Figure 5. Over 700 variants causative of the phenotype have been reported in the COL2A1 gene, which is a large gene with 54 exons. These clinical phenotypes of type II collagenopathies include MED, Stanescu type SED, spondyloepimetaphyseal dysplasia, spondyloperipheral dysplasia, and Kniest dysplasia.^[9] The characteristic features include myopia, hearing loss, and cleft palate in addition to short stature. Glycine substitutions in the amino acid sequence of the protein account for over 60% of cases.^[9] In phenotypes involving large genes or multiple genes, like OI, high throughput sequencing technologies can be used.^[10] With the aid of massively parallel sequencing technologies, the entire exome (whole-exome sequencing) or specific exome regions (clinical exome sequencing and panel analysis) can be quickly sequenced in a single assay on the blood sample of a singular patient.^[11,12]

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Figure 5:

The phenotypes reported with pathogenic and likely pathogenic variants in the COL2A1 gene. Appropriate diagnosis requires detailed clinical and radiological evaluation.

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What are the therapeutic strategies for skeletal dysplasias?

The cornerstone of the treatment in OI includes the optimal management of fractures and the administration of bisphosphonates. These can be given by intravenous (IV) administration as for zoledronate and pamidronate or orally as for risedronate and neridronate therapy.^[10] Newer treatments on the horizon include denosumab and recombinant parathyroid hormone (PTH) but these are unlikely to be of significant benefit unless given in combination with bisphosphonates.

Similarly, there is another antibody being studied in OI – fresolimumab, which acts through transforming growth factor-beta inhibition. Anti-sclerostin therapy is also under consideration in OI and it leads to inhibition of the WNT-beta-catenin signaling pathway.^[14] It has been found to be promising in mouse models of OI. Another form of therapy under trial is an infusion of allogeneic fetal-tissue-derived mesenchymal stem cells prenatally and/or postnatally to improve bone strength (European multicentric trial).

There are conflicting results on growth hormone therapy in ACH and HCH. A newer drug that has been approved for the treatment of ACH is vosoritide, a recombinant C-type natriuretic peptide analog. It has been approved by the Food and Drug Administration of the USA for children above 5 years of age and also by the European Medicines Agency. Administration of this drug can increase the growth of the child with ACH by 1–1.5 cm/year.^[14]

Other agents being tried include soluble forms of FGFR3, meclizine, and PTH for improving longitudinal skeletal growth.^[15] Infigratinib inhibits phosphorylation of FGFR3 by selective tyrosine kinase inhibition and is undergoing trials in patients of 3–11 years of age.^[16]

Enzyme replacement therapy (ERT) is the mainstay of therapy in many forms of mucopolysaccharidoses such as MPS I, MPS II, and MPS VI. The predominant skeletal form of MPS is MPS type IV and it is of two subtypes – type A and type B. In MPS IV type A, ERT is approved and available from Biomarin Pharmaceuticals, though quite expensive.^[17] If financial support is available from the Government through the rare disease treatment policy or other channels for reimbursement of financial support, then this can be tried for some selected patients so that subsequent cardiopulmonary complications can be prevented.

Additional drugs used recently in different disorders with promising results include palovarotene in fibrodysplasia ossificans progressiva, burosumab in X-linked hypophosphatemic rickets, and asfotase alfa in hypophosphatasia.^[15,18] Palovarotene binds to retinoic acid receptor gamma as an agonist and blocks BMP and SMAD signaling pathways. Asfotase-alfa carries the active site of the tissue’s non-specific alkaline phosphatase; it acts as a replacement therapy with a selective role in bone mineralization. Furthermore, resveratrol is being tried in pseudo-ACH. It decreases the acetylation of SOD2 and ROS and also activates the AMPK pathway. N-acetyl-cysteine has been recently used in dystrophic dysplasia and found to be effective in mouse models.

Is prenatal diagnosis possible? How should we go about genetic counseling?

In severe phenotypes of skeletal disorders such as recurrent fractures and multiple dislocations, prenatal diagnosis can be performed once the diagnosis is confirmed and pathogenic mutations or variants are identified in previously affected children.^[19,20] Genetic counseling is important before genetic testing and also if prenatal diagnosis is being contemplated in the family. Genetic counseling can be done by a certified genetic counselor, nurse counselor, or clinical geneticist. In autosomal recessive disorders, the risk of recurrence would be 25% and in X-linked disorders, the risk of recurrence would be 50% for males if the mutation is inherited in the family.

The sample for prenatal testing is taken at 10–14 weeks by chorionic villus sampling and at 16–22 weeks by amniocentesis. Sanger sequencing is done for the previously identified variant in affected child or carrier parents. The report is usually available in 1–2 weeks, and if the fetus is affected medical termination of pregnancy can be advised.