Heterozygous rare variants in NR2F2 cause a recognizable multiple congenital anomaly syndrome with developmental delays.

Nuclear receptor subfamily 2 group F member 2 (NR2F2 or COUP-TF2) encodes a transcription factor which is expressed at high levels during mammalian development. Rare heterozygous Mendelian variants in NR2F2 were initially identified in individuals with congenital heart disease (CHD), then subsequently in cohorts of congenital diaphragmatic hernia (CDH) and 46,XX ovotesticular disorders/differences of sexual development (DSD); however, the phenotypic spectrum associated with pathogenic variants in NR2F2 remains poorly characterized. Currently, less than 40 individuals with heterozygous pathogenic variants in NR2F2 have been reported. Here, we review the clinical and molecular details of 17 previously unreported individuals with rare heterozygous NR2F2 variants, the majority of which were de novo. Clinical features were variable, including intrauterine growth restriction (IUGR), CHD, CDH, genital anomalies, DSD, developmental delays, hypotonia, feeding difficulties, failure to thrive, congenital and acquired microcephaly, dysmorphic facial features, renal failure, hearing loss, strabismus, asplenia, and vascular malformations, thus expanding the phenotypic spectrum associated with NR2F2 variants. The variants seen were predicted loss of function, including a nonsense variant inherited from a mildly affected mosaic mother, missense and a large deletion including the NR2F2 gene. Our study presents evidence for rare, heterozygous NR2F2 variants causing a highly variable syndrome of congenital anomalies, commonly associated with heart defects, developmental delays/intellectual disability, dysmorphic features, feeding difficulties, hypotonia, and genital anomalies. Based on the new and previous cases, we provide clinical recommendations for evaluating individuals diagnosed with an NR2F2-associated disorder.

Novel variant in HHAT as a cause of different sex development with partial gonadal dysgenesis associated with microcephaly, eye defects, and distal phalangeal hypoplasia of both thumbs: Case report.

The palmitoylation of the Hedgehog (Hh) family of morphogens, named sonic hedgehog (SHH), desert hedgehog (DHH), and Indian hedgehog (IHH), is crucial for effective short- and long-range signaling. The hedgehog acyltransferase (HHAT) attaches the palmitate molecule to the Hh; therefore, variants in HHAT cause a broad spectrum of phenotypes. A missense HHAT novel variant c.1001T>A/p.(Met334Lys) was described in a patient first referred for a 46,XY different sexual development with partial gonadal dysgenesis but with microcephaly, eye defects, and distal phalangeal hypoplasia of both thumbs. The in silico analysis of the variant predicted an affectation of the nearest splicing site. Thus, in vitro minigene studies were carried out, which demonstrated that the variant does not affect the splicing. Subsequent protein in silico studies supported the pathogenicity of the variant, and, in conclusion, this was considered the cause of the patient's phenotype.

High prevalence of variants in skeletal dysplasia associated genes in individuals with short stature and minor skeletal anomalies.

OBJECTIVE: Next generation sequencing (NGS) has expanded the diagnostic paradigm turning the focus to the growth plate. The aim of the study was to determine the prevalence of variants in genes implicated in skeletal dysplasias in probands with short stature and mild skeletal anomalies. DESIGN: Clinical and radiological data were collected from 108 probands with short stature and mild skeletal anomalies. METHODS: A customized skeletal dysplasia NGS panel was performed. Variants were classified using ACMG recommendations and Sherloc. Anthropometric measurements and skeletal anomalies were subsequently compared in those with or without an identified genetic defect. RESULTS: Heterozygous variants were identified in 21/108 probands (19.4%). Variants were most frequently identified in ACAN (n = 10) and IHH (n = 7) whilst one variant was detected in COL2A1, CREBBP, EXT1, and PTPN11. Statistically significant differences (P < 0.05) were observed for sitting height/height (SH/H) ratio, SH/H ratio standard deviation score (SDS), and the SH/H ratio SDS >1 in those with an identified variant compared to those without. CONCLUSIONS: A molecular defect was elucidated in a fifth of patients. Thus, the prevalence of mild forms of skeletal dysplasias is relatively high in individuals with short stature and mild skeletal anomalies, with variants in ACAN and IHH accounting for 81% of the cases. An elevated SH/H ratio appears to be associated with a greater probability in detecting a variant, but no other clinical or radiological feature has been found determinant to finding a genetic cause. Currently, we cannot perform extensive molecular studies in all short stature individuals so detailed clinical and radiological phenotyping may orientate which are the candidate patients to obtain worthwhile results. In addition, detailed phenotyping of probands and family members will often aid variant classification.

Clinical and Molecular Description of 16 Families With Heterozygous IHH Variants.

CONTEXT: Heterozygous variants in the Indian hedgehog gene (IHH) have been reported to cause brachydactyly type A1 and mild hand and feet skeletal anomalies with short stature. Genetic screening in individuals with short stature and mild skeletal anomalies has been increasing over recent years, allowing us to broaden the clinical spectrum of skeletal dysplasias. OBJECTIVE: The objective of this article is to describe the genotype and phenotype of 16 probands with heterozygous variants in IHH. PATIENTS AND METHODS: Targeted next-generation sequencing or Sanger sequencing was performed in patients with short stature and/or brachydactyly for which the genetic cause was unknown. RESULTS: Fifteen different heterozygous IHH variants were detected, one of which is the first reported complete deletion of IHH. None of the patients showed the classical phenotype of brachydactyly type A1. The most frequently observed clinical characteristics were mild to moderate short stature as well as shortening of the middle phalanx on the fifth finger. The identified IHH variants were demonstrated to cosegregate with the short stature and/or brachydactyly in the 13 probands whose family members were available. However, clinical heterogeneity was observed: Two short-statured probands showed no hand radiological anomalies, whereas another 5 were of normal height but had brachydactyly. CONCLUSIONS: Short stature and/or mild skeletal hand defects can be caused by IHH variants. Defects in this gene should be considered in individuals with these findings, especially when there is an autosomal dominant pattern of inheritance. Although no genotype-phenotype correlation was observed, cosegregation studies should be performed and where possible functional characterization before concluding that a variant is causative.

Molecular Basis of CYP19A1 Deficiency in a 46,XX Patient With R550W Mutation in POR: Expanding the PORD Phenotype.

CONTEXT: Mutations in cytochrome P450 oxidoreductase (POR) cause a form of congenital adrenal hyperplasia (CAH). We report a novel R550W mutation in POR identified in a 46,XX patient with signs of aromatase deficiency. OBJECTIVE: Analysis of aromatase deficiency from the R550W mutation in POR. DESIGN, SETTING, AND PATIENT: Both the child and the mother had signs of virilization. Ultrasound revealed the presence of uterus and ovaries. No defects in CYP19A1 were found, but further analysis with a targeted Disorders of Sexual Development NGS panel (DSDSeq.V1, 111 genes) on a NextSeq (Illumina) platform in Madrid and Barcelona, Spain, revealed compound heterozygous mutations c.73_74delCT/p.L25FfsTer93 and c.1648C > T/p.R550W in POR. Wild-type and R550W POR were produced as recombinant proteins and tested with multiple cytochrome P450 enzymes at University Children's Hospital, Bern, Switzerland. MAIN OUTCOME MEASURE AND RESULTS: POR-R550W showed 41% of the WT activity in cytochrome c and 7.7% activity for reduction of MTT. Assays of CYP19A1 showed a severe loss of activity, and CYP17A1 as well as CYP21A2 activities were also lost by more than 95%. Loss of CYP2C9, CYP2C19, and CYP3A4 activities was observed for the R550W-POR. Predicted adverse effect on aromatase activity as well as a reduction in binding of NADPH was confirmed. CONCLUSIONS: Pathological effects due to POR-R550W were identified, expanding the knowledge of molecular pathways associated with aromatase deficiency. Screening of the POR gene may provide a diagnosis in CAH without defects in genes for steroid metabolizing enzymes.

A Novel Homozygous AMRH2 Gene Mutation in a Patient with Persistent Müllerian Duct Syndrome.

Persistent müllerian duct syndrome (PMDS) is characterized by the presence of müllerian duct derivatives in otherwise phenotypically normal males. Homozygous or compound heterozygous alterations in AMH or AMHR2 have been identified in approximately 88% of PMDS cases. We report on a male patient with bilateral undescended gonads, müllerian derivatives, and normal serum AMH levels. A novel homozygous missense mutation, c.119G>C;p.Gly40Ala, in exon 2 of AMHR2 was detected that supported the clinical diagnosis of PMDS.

Identification of a limb enhancer that is removed by pathogenic deletions downstream of the SHOX gene.

Haploinsufficiency of the human SHOX gene causes Léri-Weill dyschondrosteosis (LWD), characterized by shortening of the middle segments of the limbs and Madelung deformity of the wrist. As many as 35% of LWD cases are caused by deletions of non-coding sequences downstream of SHOX that presumably remove an enhancer or enhancers necessary for SHOX expression in developing limbs. We searched for these active sequences using a transgenic mouse assay and identified a 563 basepair (bp) enhancer with specific activity in the limb regions where SHOX functions. This enhancer has previously escaped notice because of its poor evolutionary conservation, although it does contain 100 bp that are conserved in non-rodent mammals. A primary cell luciferase assay confirmed the enhancer activity of the conserved core sequence and demonstrated that putative HOX binding sites are required for its activity. This enhancer is removed in most non-coding deletions that cause LWD. However, we did not identify any likely pathogenic variants of the enhancer in a screen of 124 LWD individuals for whom no causative mutation had been found, suggesting that only larger deletions in the region commonly cause LWD. We hypothesize that loss of this enhancer contributes to the pathogenicity of deletions downstream of SHOX.

Heterozygous aggrecan variants are associated with short stature and brachydactyly: Description of 16 probands and a review of the literature.

OBJECTIVE: Mutations in the aggrecan gene (ACAN) have been identified in two autosomal dominant skeletal dysplasias, spondyloepiphyseal dysplasia, Kimberley type (SEDK), and osteochondritis dissecans, as well as in a severe recessive dysplasia, spondyloepimetaphyseal dysplasia, aggrecan type. Next-generation sequencing (NGS) has aided the identification of heterozygous ACAN mutations in individuals with short stature, minor skeletal defects and mild facial dysmorphisms, some of whom have advanced bone age (BA), poor pubertal spurt and early growth cessation as well as precocious osteoarthritis. DESIGN AND METHODS: This study involves clinical and genetic characterization of 16 probands with heterozygous ACAN variants, 14 with short stature and mild skeletal defects (group 1) and two with SEDK (group 2). Subsequently, we reviewed the literature to determine the frequency of the different clinical characteristics in ACAN-positive individuals. RESULTS: A total of 16 ACAN variants were located throughout the gene, six pathogenic mutations and 10 variants of unknown significance (VUS). Interestingly, brachydactyly was observed in all probands. Probands from group 1 with a pathogenic mutation tended to be shorter, and 60% had an advanced BA compared to 0% in those with a VUS. A higher incidence of coxa valga was observed in individuals with a VUS (37% vs 0%). Nevertheless, other features were present at similar frequencies. CONCLUSIONS: ACAN should be considered as a candidate gene in patients with short stature and minor skeletal defects, particularly those with brachydactyly, and in patients with spondyloepiphyseal dysplasia. It is also important to note that advanced BA and osteoarticular complications are not obligatory conditions for aggrecanopathies/aggrecan-associated dysplasias.

Mutations in C-natriuretic peptide (NPPC): a novel cause of autosomal dominant short stature.

PurposeC-type natriuretic peptide (CNP) and its principal receptor, natriuretic peptide receptor B (NPR-B), have been shown to be important in skeletal development. CNP and NPR-B are encoded by natriuretic peptide precursor-C (NPPC) and natriuretic peptide receptor 2 (NPR2) genes, respectively. While NPR2 mutations have been described in patients with skeletal dysplasias and idiopathic short stature (ISS), and several Npr2 and Nppc skeletal dysplasia mouse models exist, no mutations in NPPC have been described in patients to date.MethodsNPPC was screened in 668 patients (357 with disproportionate short stature and 311 with autosomal dominant ISS) and 29 additional ISS families in an ongoing whole-exome sequencing study.ResultsTwo heterozygous NPPC mutations, located in the highly conserved CNP ring, were identified. Both showed significant reductions in cyclic guanosine monophosphate synthesis, confirming their pathogenicity. Interestingly, one has been previously linked to skeletal abnormalities in the spontaneous Nppc mouse long-bone abnormality (lbab) mutant.ConclusionsOur results demonstrate, for the first time, that NPPC mutations cause autosomal dominant short stature in humans. The NPPC mutations cosegregated with a short stature and small hands phenotype. A CNP analog, which is currently in clinical trials for the treatment of achondroplasia, seems a promising therapeutic approach, since it directly replaces the defective protein.

Identification of 15 novel partial SHOX deletions and 13 partial duplications, and a review of the literature reveals intron 3 to be a hotspot region.

Short stature homeobox gene (SHOX) is located in the pseudoautosomal region 1 of the sex chromosomes. It encodes a transcription factor implicated in the skeletal growth. Point mutations, deletions or duplications of SHOX or its transcriptional regulatory elements are associated with two skeletal dysplasias, Léri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia (LMD), as well as in a small proportion of idiopathic short stature (ISS) individuals. We have identified a total of 15 partial SHOX deletions and 13 partial SHOX duplications in LWD, LMD and ISS patients referred for routine SHOX diagnostics during a 10 year period (2004-2014). Subsequently, we characterized these alterations using MLPA (multiplex ligation-dependent probe amplification assay), fine-tiling array CGH (comparative genomic hybridation) and breakpoint PCR. Nearly half of the alterations have a distal or proximal breakpoint in intron 3. Evaluation of our data and that in the literature reveals that although partial deletions and duplications only account for a small fraction of SHOX alterations, intron 3 appears to be a breakpoint hotspot, with alterations arising by non-allelic homologous recombination, non-homologous end joining or other complex mechanisms.

Profiling of conserved non-coding elements upstream of SHOX and functional characterisation of the SHOX cis-regulatory landscape.

Genetic defects such as copy number variations (CNVs) in non-coding regions containing conserved non-coding elements (CNEs) outside the transcription unit of their target gene, can underlie genetic disease. An example of this is the short stature homeobox (SHOX) gene, regulated by seven CNEs located downstream and upstream of SHOX, with proven enhancer capacity in chicken limbs. CNVs of the downstream CNEs have been reported in many idiopathic short stature (ISS) cases, however, only recently have a few CNVs of the upstream enhancers been identified. Here, we set out to provide insight into: (i) the cis-regulatory role of these upstream CNEs in human cells, (ii) the prevalence of upstream CNVs in ISS, and (iii) the chromatin architecture of the SHOX cis-regulatory landscape in chicken and human cells. Firstly, luciferase assays in human U2OS cells, and 4C-seq both in chicken limb buds and human U2OS cells, demonstrated cis-regulatory enhancer capacities of the upstream CNEs. Secondly, CNVs of these upstream CNEs were found in three of 501 ISS patients. Finally, our 4C-seq interaction map of the SHOX region reveals a cis-regulatory domain spanning more than 1 Mb and harbouring putative new cis-regulatory elements.

Heterozygous NPR2 Mutations Cause Disproportionate Short Stature, Similar to Léri-Weill Dyschondrosteosis.

CONTEXT: SHOX mutations have been detected in approximately 70% of Léri-Weill dyschondrosteosis (LWD) and approximately 2.5% of idiopathic short stature (ISS) cases, suggesting the implication of other genes or loci. The recent identification of NPR2 mutations in ISS suggested that NPR2 mutations may also be involved in disproportionate short stature. OBJECTIVE: The objective of the study was to investigate whether NPR2 mutations can account for a proportion of the cases referred for LWD and ISS in whom no SHOX mutation was detected. PATIENTS AND METHODS: We undertook NPR2 mutation screening in 173 individuals referred for suspected LWD and 95 for ISS, with no known defect in SHOX or its enhancers. Intracellular localization and natriuretic peptide precursor C-dependent guanylate cyclase activity were determined for the identified NPR2 variants. RESULTS: Eight NPR2 variants were identified in nine individuals, seven referred for suspected LWD and two for ISS. Six were demonstrated to affect NPR-B cell trafficking and/or its ability to synthesize cyclic GMP (cGMP) under response to natriuretic peptide precursor C/brain natriuretic peptide stimulation. All pathogenic mutations were detected in the suspected LWD referral group (∼3%). Interestingly, one of these patients is currently being treated with recombinant human GH and in contrast to previous reports is showing a positive response to the treatment. CONCLUSIONS: NPR2 mutations account for approximately 3% of patients with disproportionate short stature and/or clinical or radiographic indicators of SHOX deficiency and in whom no SHOX defect has been identified. However, no patient has yet presented with Madelung deformity. Thus, NPR2 should be screened in the SHOX-negative LWD referrals.

Expanding the mutation spectrum in 182 Spanish probands with craniosynostosis: identification and characterization of novel TCF12 variants.

Craniosynostosis, caused by the premature fusion of one or more of the cranial sutures, can be classified into non-syndromic or syndromic and by which sutures are affected. Clinical assignment is a difficult challenge due to the high phenotypic variability observed between syndromes. During routine diagnostics, we screened 182 Spanish craniosynostosis probands, implementing a four-tiered cascade screening of FGFR2, FGFR3, FGFR1, TWIST1 and EFNB1. A total of 43 variants, eight novel, were identified in 113 (62%) patients: 104 (92%) detected in level 1; eight (7%) in level 2 and one (1%) in level 3. We subsequently screened additional genes in the probands with no detected mutation: one duplication of the IHH regulatory region was identified in a patient with craniosynostosis Philadelphia type and five variants, four novel, were identified in the recently described TCF12, in probands with coronal or multisuture affectation. In the 19 Saethre-Chotzen syndrome (SCS) individuals in whom a variant was detected, 15 (79%) carried a TWIST1 variant, whereas four (21%) had a TCF12 variant. Thus, we propose that TCF12 screening should be included for TWIST1 negative SCS patients and in patients where the coronal suture is affected. In summary, a molecular diagnosis was obtained in a total of 119/182 patients (65%), allowing the correct craniosynostosis syndrome classification, aiding genetic counselling and in some cases provided a better planning on how and when surgical intervention should take place and, subsequently the appropriate clinical follow up.

A new overgrowth syndrome is due to mutations in RNF125.

Overgrowth syndromes (OGS) are a group of disorders in which all parameters of growth and physical development are above the mean for age and sex. We evaluated a series of 270 families from the Spanish Overgrowth Syndrome Registry with no known OGS. We identified one de novo deletion and three missense mutations in RNF125 in six patients from four families with overgrowth, macrocephaly, intellectual disability, mild hydrocephaly, hypoglycemia, and inflammatory diseases resembling Sjögren syndrome. RNF125 encodes an E3 ubiquitin ligase and is a novel gene of OGS. Our studies of the RNF125 pathway point to upregulation of RIG-I-IPS1-MDA5 and/or disruption of the PI3K-AKT and interferon signaling pathways as the putative final effectors.

NPPB and ACAN, two novel SHOX2 transcription targets implicated in skeletal development.

SHOX and SHOX2 transcription factors are highly homologous, with even identical homeodomains. Genetic alterations in SHOX result in two skeletal dysplasias; Léri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia (LMD), while no human genetic disease has been linked to date with SHOX2. SHOX2 is, though, involved in skeletal development, as shown by different knockout mice models. Due to the high homology between SHOX and SHOX2, and their functional redundancy during heart development, we postulated that SHOX2 might have the same transcriptional targets and cofactors as SHOX in limb development. We selected two SHOX transcription targets regulated by different mechanisms: 1) the natriuretic peptide precursor B gene (NPPB) involved in the endochondral ossification signalling and directly activated by SHOX; and 2) Aggrecan (ACAN), a major component of cartilage extracellular matrix, regulated by the cooperation of SHOX with the SOX trio (SOX5, SOX6 and SOX9) via the protein interaction between SOX5/SOX6 and SHOX. Using the luciferase assay we have demonstrated that SHOX2, like SHOX, regulates NPPB directly whilst activates ACAN via its cooperation with the SOX trio. Subsequently, we have identified and characterized the protein domains implicated in the SHOX2 dimerization and also its protein interaction with SOX5/SOX6 and SHOX using the yeast-two hybrid and co-immunoprecipitation assays. Immunohistochemistry of human fetal growth plates from different time points demonstrated that SHOX2 is coexpressed with SHOX and the members of the SOX trio. Despite these findings, no mutation was identified in SHOX2 in a cohort of 83 LWD patients with no known molecular defect, suggesting that SHOX2 alterations do not cause LWD. In conclusion, our work has identified the first cofactors and two new transcription targets of SHOX2 in limb development, and we hypothesize a time- and tissue-specific functional redundancy between SHOX and SHOX2.

Identification of the first recurrent PAR1 deletion in Léri-Weill dyschondrosteosis and idiopathic short stature reveals the presence of a novel SHOX enhancer.

BACKGROUND: SHOX, located in the pseudoautosomal region 1 (PAR1) of the sexual chromosomes, encodes a transcription factor implicated in human growth. Defects in SHOX or its enhancers have been observed in ∼60% of Leri-Weill dyschondrosteosis (LWD) patients, a skeletal dysplasia characterised by short stature and/or the characteristic Madelung deformity, and in 2-5% of idiopathic short stature (ISS). To identify the molecular defect in the remaining genetically undiagnosed LWD and ISS patients, this study screened previously unanalysed PAR1 regions in 124 LWD and 576 ISS probands. METHODS: PAR1 screening was undertaken by multiplex ligation dependent probe amplification (MLPA). Copy number alterations were subsequently confirmed and delimited by locus-specific custom-designed MLPA, array comparative genomic hybridisation (CGH) and breakpoint junction PCR/sequencing. RESULTS: A recurrent PAR1 deletion downstream of SHOX spanning 47543 bp with identical breakpoints was identified in 19 LWD (15.3%) and 11 ISS (1.9%) probands, from 30 unrelated families. Eight evolutionarily conserved regions (ECRs 1-8) identified within the deleted sequence were evaluated for SHOX regulatory activity by means of chromosome conformation capture (3C) in chicken embryo limbs and luciferase reporter assays in human U2OS osteosarcoma cells. The 3C assay indicated potential SHOX regulatory activity by ECR1, which was subsequently confirmed to act as a SHOX enhancer, operating in an orientation and position independent manner, in human U2OS cells. CONCLUSIONS: This study has identified the first recurrent PAR1 deletion in LWD and ISS, which results in the loss of a previously uncharacterised SHOX enhancer. The loss of this enhancer may decrease SHOX transcription, resulting in LWD or ISS due to SHOX haploinsufficiency.

Identification of the first PAR1 deletion encompassing upstream SHOX enhancers in a family with idiopathic short stature.

Short stature homeobox-containing gene, MIM 312865 (SHOX) is located within the pseudoautosomal region 1 (PAR1) of the sex chromosomes. Mutations in SHOX or its downstream transcriptional regulatory elements represent the underlying molecular defect in ~60% of Léri-Weill dyschondrosteosis (LWD) and ~5-15% of idiopathic short stature (ISS) patients. Recently, three novel enhancer elements have been identified upstream of SHOX but to date, no PAR1 deletions upstream of SHOX have been observed that only encompass these enhancers in LWD or ISS patients. We set out to search for genetic alterations of the upstream SHOX regulatory elements in 63 LWD and 100 ISS patients with no known alteration in SHOX or the downstream enhancer regions using a specifically designed MLPA assay, which covers the PAR1 upstream of SHOX. An upstream SHOX deletion was identified in an ISS proband and her affected father. The deletion was confirmed and delimited by array-CGH, to extend ~286 kb. The deletion included two of the upstream SHOX enhancers without affecting SHOX. The 13.3-year-old proband had proportionate short stature with normal GH and IGF-I levels. In conclusion, we have identified the first PAR1 deletion encompassing only the upstream SHOX transcription regulatory elements in a family with ISS. The loss of these elements may result in SHOX haploinsufficiency because of decreased SHOX transcription. Therefore, this upstream region should be included in the routine analysis of PAR1 in patients with LWD, LMD and ISS.

Identification of a Gypsy SHOX mutation (p.A170P) in Léri-Weill dyschondrosteosis and Langer mesomelic dysplasia.

We report the clinical and molecular characteristics of 12 Spanish families with multiple members affected with Léri-Weill dyschondrosteosis (LWD) or Langer mesomelic dysplasia (LMD), who present the SHOX (short stature homeobox gene) mutation p.A170P (c.508G>C) in heterozygosity or homozygosity, respectively. In all studied families, the A170P mutation co-segregated with the fully penetrant phenotype of mesomelic limb shortening and Madelung deformity. A shared haplotype around SHOX was observed by microsatellite analysis, confirming the presence of a common ancestor, probably of Gypsy origin, as 11 of the families were of this ethnic group. Mutation screening in 359 Eastern-European Gypsies failed to identify any carriers. For the first time, we have shown SHOX expression in the human growth plate of a 22-week LMD fetus, homozygous for the A170P mutation. Although the mutant SHOX protein was expressed in all zones of the growth plate, the chondrocyte columns in the proliferative zone were disorganized with the chondrocytes occurring in smaller columnal clusters. We have also identified a novel mutation at the same residue, c. 509C>A (p.A170D), in two unrelated Spanish LWD families, which similar to A170P mutation impedes nuclear localization of SHOX. In conclusion, we have identified A170P as the first frequent SHOX mutation in Gypsy LWD and LMD individuals.