From the children tested, only five of them had utterly normal WES results and thus were correctly labelled as ISS. Eight of the patients had mutations that could potentially be the underlying cause of their short stature. One patient had a mutation unrelated to his short height, thus correctly diagnosed with ISS. The summary of the mutations is shown in table 1.
Mutations of unknown significance
The first patient, an eight-year-old girl with a height of 112 cm and an SD of -2.8 for her age with normal height parents. She was heterozygous for GHSR gene (NM_198407 exon2: c.847>T) that led to amino acid changes p.R283. This gene encodes a member of the G-protein receptors family16. The mutation was of uncertain significance, and its inheritance was either autosomal recessive (AR) or autosomal dominant (AD) pattern16. However, pathologic mutations of this gene could have developed isolated partial growth hormone deficiency (GHDP) in the individual17. The mutation in GHSR gene results in growth delay, short stature, and in some reported cases, episodes of abdominal pain, vomiting, ketosis, and hyperglycemia18. In our case report, with respect to the mutation of uncertain significance, the child’s phenotype, and her low levels of GH, we concluded that the mutation is likely pathologic in nature and the cause of her short stature. Given her low GH levels, hormonal therapy was initiated, and she showed a great response to the intervention.
The second patient was a 10-year-old girl with a height of 112 cm and an SD of -4.2 for her age, with a relatively short mother who had a height of 144 cm. The patient was heterozygous for CLCN5 gene (NM_001127898 exon14: c.2333T>G) which led to amino acid change p.L778R. This gene provides instructions for the synthesis CLC-5 protein which transports chloride ions across cell membranes and has an integral role in proximal tubule cells of the kidney19. CLCN5 gene mutations are inherited through an X-linked recessive pattern19. Pathologic mutations of this gene are a known cause of Dent disease which is a chronic renal disorder and is almost exclusively observed in males19. This mutation affects serum calcium and vitamin D levels as well20. Also, it has been observed that some female carriers could manifest the symptoms due to random X-chromosome inactivation21,22. Short stature is commonly seen among patients with Dent disease22. In our case report, the child and her mother both had evidence of CLCN5 gene mutation. With respect to the mother’s short height, we concluded that the mutation in CLCN5 gene is the cause of short stature in both the child and her mother. Despite the normal GH levels of the child, we opted to initiate hormonal therapy. However, her response to the intervention was poor. Thus, we concluded that hormonal treatment may not be indicated for this condition.
The third patient was a two-year-old girl with short stature, microcephaly and hearing loss that was homozygous for c.395T>G in exon 4 of the CLPP gene (NM_006012). The mutation in CLPP gene is inherited through AR-pattern and is associated with Perrault syndrome type 323. There are no formal studies conducted on the functional effects of this mutation, yet with respect to the patient’s phenotype and symptoms that are mainly observed in pathologic mutations, we concluded that this mutation is in fact pathologic in nature. Perrault syndrome type 3 is a rare condition that may represent with different signs and symptoms and affects both male and females23. A key feature of the disease is hearing loss and females with this condition may have ovarian dysgenesis with normal external genitalia23. Patients may also suffer from neurological conditions such as ataxia, peripheral neuropathy, and intellectual disability23. Lastly, patients with Perrault syndrome type 3 have low levels of GH and evidence of short stature23,24. It is highly likely that this individual’s short stature with hearing loss and microcephaly resulted from Perrault syndrome type 3. Due to our patient's low levels of GH, we initiated hormonal interventions, but no significant difference was observed after the treatment. Thus, we concluded that hormonal therapy may not be indicated in this condition.
The fourth case is a 10 and a half-year-old boy with a height of 126 cm and an SD of -2.2 for his age with normal height parents. He had hearing loss and three different mutations. The first mutation was a heterozygous variant in TMPRSS3 (NM_032404.2 exon5: c.266G>A) which led to amino acid change p.R89H. The second one was a heterozygous variant in HOMER2 gene (NM_199330 exon3: c.188C>T) that resulted in amino acid change p.P63L. The third one was a heterozygous variant in the FGFR3 gene (NM_001163213 exon8: c.992G>A). TMPRSS3 gene encodes a protein of serine protease family that is required for ear’s saccular hair cell survival, and pathologic mutations of this gene could result in autosomal recessive deafness (DFNB8)25,26. However, since the mutation has an AR pattern of inheritance and our patient was heterozygous for this gene, we concluded that the hearing loss is not attributed to the first mutation. The HOMER2 gene encodes a dendritic protein from the Homer family and mutations in this gene may lead to autosomal dominant deafness (DFNA68)26. Although the mutation in this child was of uncertain significance due to its AD pattern inheritance, it was likely the cause of his hearing loss. The third gene, FGFR3 provides instructions for synthesizing fibroblast growth factor receptor 327. Mutations in this gene may result in hypochondroplasia, which is a form of short-limbed dwarfism27.
In our case report, the patient presented with mildly short limbs. Although this mutation was of uncertain significance and no formal studies were conducted on the effects of this mutation, with respect to our patient’s phenotype and its AD pattern of inheritance, we concluded that the mutation in FGFR3 gene is probably the cause of his short stature. The patient's GH levels were normal, however we opted for a trial of hormonal therapy, but the results were not promising, and the treatment was discontinued. It is questionable whether continuance of hormonal therapy for this patient is effective and indicated.
Pathologic mutations
The first case of pathologic mutations was a five-year-old boy with a height of 99 cm and a SD of -2.2 for his age with a short father who had a height of 150 cm. He was heterozygous for UROD gene (NM_000347 exon9: c. 912C>A) with an AD or AR pattern of inheritance that led to amino acid change p.N304K. This gene provides instruction for the synthesis of uroporphyrinogen decarboxylase enzyme29. Pathologic mutations in this gene disrupt chemical steps that lead to heme production and results in porphyria30. There are several types of porphyria distinguished by the genome sequences and the symptoms. In our case, the patient’s genome sequence showed evidence of hepatoerythropoietic porphyria (HEP). HEP results in fragile and blistered skin in exposure to the direct sunlight and makes the individual more vulnerable to infection scaring and pigmentation30,31. HEP is characterized by osteolysis, i.e. shortening of distal phalanges, sclerodactyly, and progressive joint deformities32. Furthermore, short stature has been noted in some types of porphyria, mainly, congenital erythropoietic porphyria33. There is one case report of a patient with HEP who had a noticeable short stature34. Due to the rare nature of this disease, the correlation between HEP and short stature remains questionable. However, in our case, the child’s father had evidence of the mutation and short stature. Given that the child and the father both have evidence of short stature and the mutation, we concluded that there is, in fact, a correlation between HEP and short stature. Yet, given the rarity of the disease, further studies and case reports are required to confirm our hypothesis. Despite the patient’s normal GH levels, we decided to initiate hormonal therapy. The response was feeble thus we discontinued the treatment and concluded that GH therapy may not be indicated for this medical condition.
The second patient with a pathologic mutation was a two and a half-year-old boy with a height of 84 cm and an SD of -2.2 for his age with normal height parents. He was heterozygous for RYR1 gene (NM_000540 exon15: c. 1589G>A) that led to amino acid change p.R530H and it was inherited through AD pattern35,36. RYR1 provides instructions for synthesizing ryanodine receptor 1 protein that transports calcium ions and is critical in muscle contraction and the movement35,37. Mutations in this gene make patients prone to malignant hyperthermia susceptibility 1 especially in an event of invasive procedure or general anesthetics38,39. It has been observed that RYR1 gene mutation results in King-Denborough syndrome and short stature 40,41. To conclude that this mutation is the reason for our patient's short stature, further studies are indicated but it cannot be ruled out as a probable cause. Other coexisting conditions with this patient were seizures. As reported in previous studies, we presume his seizures could be due to stress-induced hyperpyrexia42. Despite the patient’s normal GH levels, we initiated hormonal therapy and observed a good response.
The third patient was an eleven-months-old girl with short stature and an SD of -3 for her age. Her parents were of normal height. She was heterozygous for SMAD4 gene (NM_005359 exon 12: c. 1498 A>G) that led to amino acid change p.1500V and was inherited through AD pattern43. SMAD4 provides instructions for the synthesis SMAD4 protein which is part of transforming growth factor betas (TGF-β) signalling pathway and acts as a transcription factor and a tumour suppressor44,45. The mutation in our patient is associated with Myhre syndrome. Myhre syndrome has a characteristic facial feature and may result in short stature43,46. Affected patients may also present with hearing loss, joint stiffness, limited joint mobility fibrosis, cardiovascular problems, respiratory complications, and muscular and skeletal problems43,47. The patient’s phenotype and development of the aforementioned conditions vary and depend on the mutation types and the domains 43. In our case, besides mild facial features and short stature, abnormal TSH changes and lower normal limits of IGF-1 were noticeable.
After GH therapy initiation at a proper age, the expected difference in her height and velocity of growth was not observed, although due to her age the response observed to treatment can not determine if hormonal therapy is indicated or not.
The third patient was a two-year-old girl with short stature and an SD of -2.2 with normal height parents. She was heterozygous, for COL9A3 gene (NM_001853: exon 18:c. 920G>A) that led to amino acid change p.G307D and was associated with multiple epiphyseal dysplasia (OMIM 600969)48,49. The mode of inheritance was of AD pattern50. Multiple epiphyseal dysplasia is a mild and variable condition in which irregular ossification of the epiphyseal cartilage occurs50. Common signs and symptoms include early-onset arthritis, knee and hip cartilage anomalies and short stature 49,50. In our case, the patient's phenotype correlated with the known characteristics of the disease, thus we concluded that the mutation in COL9A3 is the cause of her short stature. Although the patient had an average levels of GH, we decided to initiate GH therapy, but despite our best efforts, we observed a weak response. Thus, we concluded that hormonal therapy is not indicated in this patient.
The last patient, was a seven-year-old boy with a height of 99 cm and a SD of -4.2 for his age with normal height parents. He was heterozygous for CFTR gene (NM_000492 exon11: c.1397C>G) that led to amino acid change p.S466. The mode of inheritance was of an AR pattern. Mutations in this gene may cause cystic fibrosis51. In our case, the patient had no evidence of cystic fibrosis and was heterozygous, hence it is unlikely that his short stature is attributed to the mutation in CFTR gene. Despite the patient’s normal GH levels, we initiated hormonal therapy and observed an excellent response. The summary of treatment results is shown in Table 2.