A total of 12 families with multiple affected LWD and LMD individuals were found to carry the A170P SHOX
mutation in heterozygosity and homozygosity, respectively. A common haplotype around SHOX
was observed in all 12 families, suggesting the presence of a common ancestor. The probands originate from different Spanish provinces, 11 were of gypsy origin, thus suggesting that the common ancestor belonged to this ethnic group. The probability of this SHOX haplotype in the unaffected Spanish Gypsy population was low and even lower in the Spanish population. The haplotype block spanned between 77 and 165
kb. Multiple recombination events had occurred at increased distances. A study of linkage disequilibrium around SHOX
showed that in Northern-European populations, disequilibrium decays rapidly with physical distance and that the recombination rate of the PAR1 is approximately 20-fold higher than the genome average.21
Therefore, it is not unexpected to observe multiple recombinational events in these families.
As the A170P mutation was identified in 11 Spanish Gypsy families, we investigated its history and incidence of this mutation in the European Gypsy population. A panel of population controls from Bulgaria, representing Gypsy groups that remained in the Balkans during the early Gypsy diaspora across Europe, was screened for the A170P mutation. No mutation was identified, but failure to identify any mutation carrier does not rule out its presence in the Eastern Europe, where it could be confined to specific sub-isolate(s) not represented in the panel or could occur at very low frequency. The A170P SHOX mutation should therefore be the first choice in diagnostic analyses of LWD/LMD patients from this ethnic group. The mutation is more prevalent in the Spanish Gypsy population, in which six out of the seven prenatal diagnoses that we have undertaken in recent years belonged to high-risk Gypsy families.
Interestingly, the A170P mutation was highly penetrant, with all carriers presenting with mesomelic shortening of the limbs and the Madelung deformity, characteristics of LWD, in contrast to other SHOX
mutations, which may present as either LWD or idiopathic short stature (ISS, MIM 300582). Only one individual did not fulfill the criteria of short stature (family 12, individual III.1). The high penetrance is likely to be due to the functional importance of alanine 170, which resides within a five non-classic basic amino acid nuclear localization signal (AKCRK), located in the homeodomain of SHOX.16, 18
Alanine 170 is one of the four amino acids which are invariably conserved in the homeodomain of all paired related homeodomain proteins. The A170P mutation has been shown to result in the incorrect subcellular localization of the SHOX protein, affecting the translocation from the cytosol to the nucleus, which would lead to the functional impairment of the transcription factor.16
Indeed, these amino acids have been shown to not only be essential for SHOX nuclear localization, but may also participate in other processes such as DNA binding, dimerization22
and interaction with its cofactors.20
We have investigated, for the first time, the effects of the A170P mutation on the growth plate of the radius and ulna of a LMD fetus. The chondrocytes in the reserve zone appeared enlarged and in pairs, whereas the proliferative zone was disorganized, with the columns appearing shortened and stacked side by side rather than in an orderly stacking. Despite this, the pattern of SHOX expression was unchanged between the normal growth plate and that of the LMD fetus. The histology of the growth plate in LMD fetuses has been previously described,23
but at this time point, the implicated gene had not been discovered. Later, comparison of the histology and SHOX expression in the growth plate of normal and four LWD fetuses, two with SHOX
deletions, one with a splice site mutation and one with a deletion of the SHOX
enhancer region was studied.24
In all cases of LWD and LMD, disorganization of the chondrocyte columnar stacking has been observed.
We have also identified two independent LWD probands with a previously unreported mutation (c.509C>A) affecting the same amino acid, p.A170D. We expressed the mutant protein in U2OS osteosarcoma cells and showed that the A170D mutant protein, as shown with the A170P mutation, failed to translocate to the nucleus, thus leading to the malfunction of the transcription factor.
Including the cases reported in this study and identified by our group, six different mutations within the five amino acids of the SHOX nuclear localization signal have now been reported in 28 LWD/LMD/ISS probands (unpublished data and SHOX database: http://hyg-serv-01.hyg.uni-heidelberg.de/lovd/index.php?select_db=SHOX
). Five additional mutations affecting the adjacent amino acids arginine 168 and 169 have been reported in 14 further LWD/ISS cases. Although it has been experimentally shown that these two amino acids are not included within the minimal nuclear localization signal, it has been postulated that they may support the minimal signal by their basicity.18
Thus, the nuclear localization signal and adjacent basic amino acids appear to be a frequently mutated region associated with LWD and ISS. A note of caution must be mentioned in the detection of mutations in the nuclear localization signal, as both the A170P and A170D mutations result in a false positive deletion of SHOX
exon 4 using the commercial MLPA assay.
In conclusion, we have identified A170P as the first common SHOX mutation in Spanish LWD individuals, which appears to have arisen from a common ancestor, most likely of Gypsy origin. Thus, it is advisable to screen the A170P mutation as a first step in the genetic screening cascade of SHOX/PAR1 alterations in Spanish gypsies with LWD or LMD. Moreover, we have also identified a novel mutation, A170D, altering the same conserved alanine 170 residue, which also impairs the nuclear localization of the protein.