We demonstrated that pure isolated hemihyperplasia and hemihypoplasia can occur as BWSp and SRSp, and the methylation difference is valuable for predicting the severity of future LLD. This is the first study to identify an association between the severity of LLD and epigenetic alterations, providing a basis for understanding the development of idiopathic LLD and value of epigenetic tests for patients with isolated hemihyperplasia/hemihypoplasia.
Several previous studies have shown that patients with isolated hemihyperplasia/hemihypoplasia may have epigenetic alterations that are found in BWS/SRS [6, 7, 20–22]. Shuman et al. reported eight patients with paternal UPD 11p15 and three with LOM at DMR2 among 51 patients with isolated hemihyperplasia . Bliek et al. reported a series of eight patients with clinical features ranging from isolated hemihypoplasia to full-spectrum SRS who had LOM at DMR1 . However, all previous studies of “isolated hemihyperplasia/hemihypoplasia” included both patients with pure isolated hemihyperplasia/hemihypoplasia and patients with skin pigmentation or other BWS/SRS clinical features who did not fit the definition of “isolated” [6, 7, 20–22]. Previous studies did not obtain tissue samples from every patient, which may have resulted in false negative results, considering the mosaic distribution of the affected cells. To the best of our knowledge, this is the first study to reveal epigenetic alterations on 11p15 using paired blood-tissue samples in a group of individuals with “pure” isolated hemihyperplasia/hemihypoplasia.
Although CDKN1C mutations have been identified in 8% of patients with BWS , we did not observe these mutations. This may be because the underlying molecular defect of CDKN1C mutations is a germline mutation that affects all cells in the body rather than a somatic mosaicism and because our study population had isolated hemihyperplasia/hemihypoplasia. Previous studies of BWS/SRS reported the extreme rarity of hemihyperplasia/hemihypoplasia in patients with CDKN1C mutations [15, 23]. CDKN1C sequencing is therefore thought to be unnecessary in patients with pure isolated hemihyperplasia/hemihypoplasia.
Because the epigenetic alteration exists in a mosaic form in BWSp/SRSp, it is possible that the proportion of cells with altered methylation is high in hyperplastic/hypoplastic tissues. Therefore, the level of DNA methylation alteration may differ even in patients with the same epigenotype, leading to differences in the phenotype severity. We quantitatively represented this “methylation burden” as the methylation difference between DMR1 and DMR2, which was shown to correlate with the severity of LLD. Particularly, UPD affects methylation at both DMR1 and DMR2 in opposite directions, resulting in a large methylation difference. In this study, two patients showed a UPD pattern. One patient with a maternal UPD pattern had 57 mm of predicted LLD, and another patient with paternal UPD had 112 mm of predicted LLD. A previous case report described a patient with paternal UPD 11p15 who had hemihyperplasia affecting the right leg and showed 50 mm of LLD at an early age of 8.3 years . Therefore, UPD appears to be associated with severe phenotypes. This is the first study to quantitatively analyze the association between the degree of epigenetic alteration and severity of phenotype in patients with BWSp/SRSp. This strategy may be applicable in patients with BWSp to assess tumor development risk.
It may be controversial whether and when to perform epigenetic tests on patients with pure isolated hemihyperplasia/hemihypoplasia. If we identify methylation alterations via epigenetic tests, we can reassure patients that the risk of recurrence of hemihyperplasia/hemihypoplasia in their offspring is negliable . If a UPD pattern is observed, we can inform patients of the possibility of lengthening procedures, rather than an operative procedure that suppresses longitudinal bone growth, or epiphysiodesis, for large LLD. In this study, we confirmed epigenetic alterations using peripheral blood samples in 8/10 patients with methylation defect. Therefore, epigenetic testing using peripheral blood can be performed at an early age and using blood and tissue samples at operation for LLD.
Because there are no clinical features other than a size difference between the sides in pure isolated hemihyperplasia/hemihypoplasia, it is difficult to differentiate these two diseases clinically. Unlike in hemihyperplasia, LLD in hemihypoplasia is rarely more than 25 mm and therefore does not require surgery . However, in this study, the predicted LLDs of two of four patients with the epigenotype of SRS were 57 and 32 mm. Therefore, LLD due to hemihypoplasia in SRSp can exceed 25 mm, and we should not differentiate these two diseases based on the severity of LLD.
In this study, the clinical diagnosis of hemihyperplasia/hemihypoplasia was not compatible with epigenetic alterations in four of 10 patients. Other studies have also found LOM at DMR1, which should cause hemihypoplasia in patients diagnosed with isolated hemihyperplasia [5, 8]. Because screening for embryonal tumors is recommended only for patients with hemihyperplasia, and not for patients with hemihypoplasia [11, 12], the differentiation of isolated hemihyperplasia from hemihypoplasia has serious prognostic implications. However, without epigenetic tests, this discrimination is nearly impossible, except in a few patients with extreme phenotypes. This problem highlights the need to perform epigenetic tests in patients with isolated hemihyperplasia/hemihypoplasia.
LLD is a common orthopedic condition, which frequently occurs for unknown causes . This study provides a basis for understanding the molecular mechanism underlying idiopathic LLD. When epigenetic alterations of BWS/SRS are confined to the thigh and/or leg due to somatic mosaicism, the condition appears as idiopathic LLD. A considerable portion of idiopathic LLD appears to occur as a BWSp/SRSp.
This study has several limitations. First, we did not examine other genetic mechanisms affecting growth [26–28]. These mechanisms can be confounding factors that may block the correlation between the methylation difference and predicted LLD. In the first five consecutive patients, we examined genes involved in the PI3K/AKT/mTOR pathway, which is also known to cause syndromic hemihyperplasia , using tissue-blood paired high-depth exome sequencing. However, we found no meaningful tissue-specific variant in any sample and thus discontinued the examination. Second, tissue samples could be obtained only from the operated legs for ethical reasons. Because the differentiation between hemihyperplasia and hemihypoplasia is confusing, the leg operated on may not be the affected leg. However, we examined peripheral blood and multiple tissues to confirm our results. Third, the predicted LLD calculated by the multiplier method may differ from the true LLD , although this is one of the most frequently used methods for predicting LLD .