Mutations of uncertain significance in heterozygous variants as a possible cause of severe short stature: a case report

Linear bone growth is achieved by chondrocyte division at the growth plate. Chondrocyte division is regulated by endocrine and paracrine factors such as growth hormone [1]. Mutations that negatively affect chondrogenesis can potentially be a contributor to short stature [1]. One such mutation occurs in the ACAN gene that encodes Aggrecan, which is a critical proteoglycan component of the extracellular matrix of the growth plate cartilage [2]. Most ACAN mutations are inherited through an autosomal dominant pattern and differ in phenotype, but all of them are characterized by short stature and advanced bone age [3‐5].

Similarly, mutations in the growth hormone receptor (GHR) can lead to Laron syndrome (LS), one of the several disorders that are collectively called growth hormone insensitivity syndrome (GHI) [6‐10]. Studies into heterozygous mutations have found that such mutations may indeed result in shorter-than-average stature, but not to what would be considered a pathological extent; as well, there have been limited studies to suggest that heterozygous mutations may result in GHI [11, 12]. While LS is an autosomal recessive condition, our investigation indicates that if there are simultaneous heterozygous mutations in different genes that normally contribute to linear bone growth, the occurrence of short stature with highly variable phenotypes is quite possible [10, 12‐15].

Our investigations were performed in two phases: we firstly assessed the subject with a physical examination, blood tests, imaging, and genetic testing, and secondly, based on the results, genetic analyses of the subject’s parents were performed.

Blood tests were ordered to assess thyroid function, as well as insulin-like growth factor 1 (IGF1), growth hormone (GH), prolactin levels, and urinary AM cortisol; to rule out autoimmune-related malabsorptive diseases, anti-tissue transglutaminase antibody (anti-tTG) IgG, and IgA levels were evaluated. A GH stimulation test by clonidine was also performed. A urinalysis was conducted, as well as a renal ultrasound, and bone density was assessed. An X-ray was performed of her left hand/wrist to determine bone age. The national growth reference provided by Iran’s Ministry of Health and Medical Education was used in this study.

Subsequently, we ordered conventional G-banding karyotyping to screen for large duplications and deletions, balanced translocations, inversions, and ploidy changes. Then, for a more detailed analysis, we followed this with whole-exome enrichment, performed by the Agilent SureSelect V6 target enrichment kit on Illumina Hiseq 4000 platform with an average depth of 206X performed by Macrogen, South Korea. Nearly all exons and flanking 10 bp (base pairs) were detected and analyzed (detected variants included single-point mutations and small indels (within 20 bp)). The analytical sensitivity and specificity of the next-generation sequencing (NGS) method used in this assay for detection variants mentioned above was more than 95%, and nearly all exons were detected and analyzed.

Based on the results of our analysis of the subject’s genetic testing, we performed a polymerase chain reaction (PCR) followed by Sanger sequencing on the parents to further investigate the validity of the detected variants. This procedure was repeated on available relatives of the subject’s father (his father and his sister). All bioinformatics analysis of sequencing results was performed using international databases (American College of Medical Genetics, The Human Gene Mutation Database and ClinVar) and standard bioinformatics software.

After all evaluations had been completed, growth hormone therapy was initiated. The GH dose was increased to a maximum amount of 67 μg/kg per day for a course of 6 months of treatment.

It is evident that our patient’s mutations in ACAN and SRCAP are sporadic and new, as they did not exist in her parents. Prior to GH therapy, the subject’s GH level was low while a GH stimulation test showed normal results; with GH therapy, the subject’s growth velocity did not increase to the degree expected, suggesting a partial insensitivity to GH.

As mentioned above, mutations in the GHR gene can result in GHI. The subject’s partial GH insensitivity is likely explained by her GHR mutation. Our patient had a heterozygous missense GHR mutation which previously has been reported as a pathogenic variant in The Human Gene Mutation Database (HGMD) and other studies [16], but the ClinVar Database does not support its pathogenicity [9]. Given that her father shares this mutation, it is highly likely that his short stature is also at least in part due to a similar GH insensitivity.

That said, the severity of our patient’s short stature is most likely the result of not only her GHR mutation, but the additive effects of her mutations in ACAN or SRCAP or indeed both these genes.

As explained earlier, pathogenic ACAN mutations can result in short stature and advanced bone age similar to those seen in our patient. However, to our knowledge, her specific mutation has not been reported as pathogenic in previous publications. Since her phenotype resembles those seen in pathogenic mutations of the ACAN gene, here we would like to suggest that her specific variation is likely pathogenic and a contributor to her advanced bone age and short stature.

Mutations in the SRCAP gene can result in Floating-Harbor syndrome (FHS), a rare autosomal dominant mutation in the SRCAP gene that can also result in short stature and other phenotypes like a triangular face, low hanging columella, short philtrum, wide mouth, and an elongated nose with a narrow bridge [17]. To our knowledge, the pathogenicity of our patient’s specific SRCAP mutation was not reported previously, and its significance is unknown; however, her long eyelashes, her seemingly wide columella, and her slightly short philtrum all resemble those futures seen in FHS, and could be the result of this mutation [18]. Since other work-up did not explain her mildly low levels of GH and IGF1, here we would like to report her SRCAP mutation as the cause; other studies have reported similar findings [19]. Mutations of the AGBL1 gene are associated with late-onset of Fuchs’ corneal dystrophy, an autosomal dominant condition that results in corneal epithelial destruction [20]. In our patient, her mutation is likely pathogenic based on the American College of Medical Genetics and Genome (ACMG) and as reported in other studies [20]. Therefore, we referred our patient to an ophthalmologist to be further evaluated for this condition and to avoid corneal epithelial destruction and loss of eyesight.

The frequency in the general population, pathogenicity, signs, and symptoms of the aforementioned variants are summarized in Table 4.

To our knowledge, the concomitance of these mutations as they relate to the resultant phenotype in our patient is unique and lead to a new hypothesis: Based on our clinical investigations and our review of the latest relevant literature, the highly unusual phenotype seen in our patient can most likely be attributed to her particular variations.

One explanation for her phenotype is that these variants are pathogenic and of the autosomal dominant pattern of inheritance, despite being categorized in the VUS section. The evidence that we have to support our claim are the signs and symptoms of these variants that are observed only in pathogenic mutations of the genes, mentioned above. Regardless of the pattern of inheritance of these variations, the attributed signs and symptoms were mild, so we could assume that if they do cause short stature, it would be to a mild degree, in correlation with other signs and symptoms. That said, with short stature being one of the signs of all of these mutations, and given that its severity does not correlate directly with the other signs and symptoms, we can assume that ultimately the impact on stature is a result of the synergistic effects of all the abovementioned variations. One possible mechanism is that, along with her partial GHI, the mutation in her SRCAP gene caused reduced plasma levels of GH and IGF1, resulting in synergistic effects with the GHI. Likewise, the synergistic effects of ACAN and SRCAP mutations coinciding with GHI could explain our patient’s lack of response to treatment, in spite of her heterozygous GHR mutation. Thus, we can presume that while these particular variations may have an insignificant effect individually on a person’s height, their usual phenotypes could be altered due to the synergistic effects they impart on one another, influencing a patient’s height dramatically. Also, many patients are labeled as having idiopathic or familial short stature (the father of our patient is an example) because no genetic studies are conducted and they do not show signs or symptoms that could lead the physician to the correct diagnosis. Therefore, we think that many of these mutations are not as rare as previously believed, but frequently go undiagnosed due to their variable phenotypes. We suggest that a complete genetic study and WES may be beneficial in certain patients who are suspected to carry the aforementioned genes prior to assigning a diagnosis of idiopathic or familial short stature.