Multiple bilateral hip fractures in a patient with dyskeratosis congenita caused by a novel mutation in the PARN gene.

We report a case of a young male patient with clinical signs of dyskeratosis congenita who presented with multiple bilateral low-traumatic hip fractures. Whole exome sequencing (WES) showed a previously unreported mutation in the poly(A)-specific ribonuclease (PARN) gene. Zoledronic acid 5 mg over 3 years was effective at preventing further fractures. A male patient was referred to our clinic at age 24 due to multiple bilateral hip fractures. At the time of admission, the patient's height was 160 cm and weight 40 kg; bone mineral density (BMD) at the lumbar spine was normal (L1-L4 0.0 Z-score). The patient was found to have abnormal skin pigmentation, hyperkeratosis of palms and soles, nail dystrophy, and signs of bone marrow failure (BMF). Bone fragility first presented at 5 years old with a wrist fracture, followed by multiple bilateral low-traumatic hip fractures without falls from 14 to 24 years. WES showed a previously unreported mutation (NM_002582.3: c.1652delA; p.His551fs) in the poly(A)-specific ribonuclease (PARN) gene. Flow fish telomere measurement result was 5.9 (reference range 8.0-12.6), which is consistent with the DC diagnosis. Permanent fixation with internal metal rods and zoledronic acid 5 mg over 3 years was effective at preventing further fractures over 4 years of follow-up. Additionally, BMF did not progress over 4 years of observation. DC associated with PARN gene mutations might predispose to low-traumatic multiple hip fractures in adolescents and young adults. Treatment with zoledronic acid in this case was effective and safe at preventing further fractures.

Clinical, molecular genetics and therapeutic aspects of syndromic obesity.

Obesity has become a major health problem worldwide. To date, more than 25 different syndromic forms of obesity are known in which one (monogenic) or multiple (polygenic) genes are involved. This review gives an overview of these forms and focuses more in detail on 6 syndromes: Prader Willi Syndrome and Prader Willi like phenotype, Bardet Biedl Syndrome, Alström Syndrome, Wilms tumor, Aniridia, Genitourinary malformations and mental Retardation syndrome and 16p11.2 (micro)deletions. Years of research provided plenty of information on the molecular genetics of these disorders and the obesity phenotype leading to a more individualized treatment of the symptoms, however, many questions still remain unanswered. As these obesity syndromes have different signs and symptoms in common, it makes it difficult to accurately diagnose patients which may result in inappropriate treatment of the disease. Therefore, the big challenge for clinicians and scientists is to more clearly differentiate all syndromic forms of obesity to provide conclusive genetic explanations and eventually deliver accurate genetic counseling and treatment. In addition, further delineation of the (functions of the) underlying genes with the use of array- or next-generation sequencing-based technology will be helpful to unravel the mechanisms of energy metabolism in the general population.

Sclerosing bone dysplasias with involvement of the craniofacial skeleton.

In this review we provide a complete overview of the existing sclerosing bone dysplasias with craniofacial involvement. Clinical presentation, disease course, the craniofacial symptoms, genetic transmission pattern and pathophysiology are discussed. There is an emphasis on radiologic features with a large collection of CT and MRI images. In previous reviews the craniofacial area of the sclerosing bone dysplasias was underexposed. However, craniofacial symptoms are often the first symptoms to address a physician. The embryology of the skull and skull base is explained and illustrated for a better understanding of the affected areas.

Wnt signaling and the control of human stem cell fate.

Wnt signaling determines major developmental processes in the embryonic state and regulates maintenance, self-renewal and differentiation of adult mammalian tissue stem cells. Both ß-catenin dependent and independent Wnt pathways exist, and both affect stem cell fate in developing and adult tissues. In this review, we debate the response to Wnt signal activation in embryonic stem cells and human, adult stem cells of mesenchymal, hematopoetic, intestinal, gastric, epidermal, mammary and neural lineages, and discuss the need for Wnt signaling in these cell types. Due to the vital actions of Wnt signaling in developmental and maintenance processes, deregulation of the pathway can culminate into a broad spectrum of developmental and genetic diseases, including cancer. The way in which Wnt signals can feed tumors and maintain cancer stem stells is discussed as well. Manipulation of Wnt signals both in vivo and in vitro thus carries potential for therapeutic approaches such as tissue engineering for regenerative medicine and anti-cancer treatment. Although many questions remain regarding the complete Wnt signal cell-type specific response and interplay of Wnt signaling with pathways such as BMP, Hedgehog and Notch, we hereby provide an overview of current knowledge on Wnt signaling and its control over human stem cell fate.

Screening for genetic variants in BDNF that contribute to childhood obesity.

UNLABELLED: What is already known about this subject BDNF is involved in the regulation of food intake and body weight. BDNF deficient animal models are obese. Chromosomal abnormalities cause obesity in humans. What this study adds Evaluation of point mutations in BDNF. Identification of BDNF mutations in obese children. Point mutations in BDNF are not a common cause of childhood obesity. INTRODUCTION: There is ample evidence that BDNF has a role in the regulation of food intake and body weight. Study of various mouse models gave a clear indication that BDNF deficiency leads to the development of obesity. Functional loss of one copy of the BDNF gene, due to chromosomal rearrangements or microdeletions, can cause an obesity phenotype in humans. Therefore, we wanted to investigate whether point mutations in the gene also result in a comparable phenotype. METHODS: We screened 554 severely overweight and obese children and adolescents and 565 lean adults for mutations in the coding region of BDNF. Mutation screening was performed by high-resolution melting curve analysis and direct sequencing. RESULTS: Screening of obese patients led to the identification of two synonymous variations (V37V and H65H) and two non-synonymous coding mutations (T2I and V46M) in the BDNF gene. When we subsequently screened our control population, we found T2I with comparable frequency and confirmed that this is a rare and non-pathogenic variant. In addition, we found another non-synonymous mutation (N187S) in the control population. CONCLUSIONS: In silico analysis of the V46M variant did not support a clear disease-causing effect and no family data were available in order to determine whether the mutation segregates with obesity. However, we cannot rule out a possible pathogenic effect for this variant. In general, we tend to conclude that mutations in the coding region of BDNF are uncommon in obese patients and are therefore not likely to play an essential role in the pathogenesis of childhood obesity.

Localization of the gene for X-linked calvarial hyperostosis to chromosome Xq27.3-Xqter.

X-linked calvarial hyperostosis is a rare disorder characterized by isolated calvarial thickening. Symptoms are prominent frontoparietal bones, a flat nasal root and a short upturned nose, a high forehead with ridging of the metopic and sagittal sutures, and lateral frontal prominences. The mandible is normal, as are the clavicles, pelvis and long bones. The thickened bone in the skull appears to be softer than normal bone. Despite calvarial hyperostosis, increased intracranial pressure and cranial nerve entrapment do not occur. The major disability seems to be cosmetic. The disease segregates with an X-linked recessive mode of inheritance. Female carriers do not show any clinical symptoms. To date, only one family has been described with X-linked calvarial hyperostosis including three affected individuals. In order to localize the disease causing gene, 31 polymorphic microsatellite markers that spread across the X-chromosome were analyzed. Genotypes were combined in haplotypes to delineate the region. A chromosomal region spanning from Xq27.3 to Xqter cosegregates with the disorder. This region encompasses 23.53cM or 8.2Mb according to the deCODE map and contains 165 genes. CNV-analysis did not show small duplications or deletions in this region. Exome sequencing was performed on a male patient in this family. However, this did not reveal any putative mutation. These results indicate that a non-coding regulatory sequence might be involved in the pathogenesis of this disorder.

Common genetic variation in sFRP5 is associated with fat distribution in men.

Considering the role of sFRP5 in Wnt signalling, an important group of pathways regulating adipogenesis and inflammation, we performed a genetic association study on sFRP5 polymorphisms in a population of obese and lean individuals. Using information from the HapMap, two tagSNPs were identified in the sFRP5 gene region and genotyped on a population of 1,014 obese, non-diabetic individuals and 606 lean controls. We performed logistic and linear regression analysis including a wide variety of obesity parameters (BMI, waist circumference, height, WHR, fat mass, fat mass percentage and visceral, subcutaneous and total abdominal fat), in addition to OGTT and HOMA-IR values. We were able to show a significant association of sFRP5 with both total abdominal and subcutaneous fat. The association signal was only seen in obese males, and in this population, the minor allele of rs7072751 explains 1.8 % of variance in total abdominal fat. In addition, we saw a trend towards an association of rs10748709 with glucose metabolism. Although further research is necessary, we can conclude that sFRP5 is a significant regulator of fat development and distribution in obese males. We postulate that altered transcription factor binding on the rs7072751 surrounding sequence might play a role in the associations we found with both total abdominal and subcutaneous fat. In addition, although no conclusive evidence was found, our results indicate that sFRP5 genetic variation may affect glucose metabolism and it would be interesting to investigate this further.

Mutation screen of the SIM1 gene in pediatric patients with early-onset obesity.

BACKGROUND: The transcription factor SIM1 (Single-minded 1) is involved in the control of food intake and in the pathogenesis of obesity. In mice, Sim1 is involved in the development of the paraventricular nucleus, and Sim1 deficiency leads to severe obesity and hyperphagia. In humans, chromosomal abnormalities in the SIM1 gene region have been reported in obese individuals. Furthermore, recent data also suggest that loss-of-function point mutations in SIM1 are responsible for SIM1 haplo-insufficiency that is involved in causing human obesity. In this study, we therefore wanted to expand the evidence regarding the involvement of SIM1 mutations in the pathogenesis of severe early-onset obesity. METHODS: We screened 561 severely overweight and obese children and adolescents and 453 lean adults for mutations in the coding region of the SIM1 gene. Mutation screening in all patients and lean individuals was performed by high-resolution melting curve analysis combined with direct sequencing. To evaluate the effect of the mutations on SIM1 transcriptional activity, luciferase reporter assays were performed. RESULTS: Mutation analysis identified four novel nonsynonymous coding variants in SIM1 in four unrelated obese individuals: p.L242V, p.T481K, p.A517V and p.D590E. Five synonymous variants, p.P57P, p.F93F, p.I183I, p.V208V and p.T653T, were also identified. Screening of the lean control population revealed the occurrence of four other rare SIM1 variants: p.G408R, p.R471P, p.S492P and p.S622F. For variants p.T481K and p.A517V, which were found in obese individuals, a decrease in SIM1 transcriptional activity was observed, whereas the transcriptional activity of all variants found in lean individuals resembled wild type. CONCLUSIONS: In this study, we have demonstrated the presence of rare SIM1 variants in both an obese pediatric population and a population of lean adult controls. Further, we have shown that functional in vitro analysis of SIM1 variants may help in distinguishing benign variants of no pathogenic significance from variants which contribute to the obesity phenotype.

Localization of the gene for hyperostosis cranialis interna to chromosome 8p21 with analysis of three candidate genes.

Hyperostosis cranialis interna (HCI) is a rare autosomal dominant disorder characterized by intracranial hyperostosis and osteosclerosis, which is confined to the skull, especially the calvarium and the skull base. The rest of the skeleton is not affected. Progressive bone overgrowth causes nerve entrapment that leads to recurrent facial nerve palsy, disturbance of the sense of smell, hearing and vision impairments, impairment of facial sensibility, and disturbance of balance due to vestibular areflexia. The treatment is symptomatic. Histomorphological investigations showed increased bone formation with a normal tissue structure. Biochemical parameters were normal. Until today the disease has been described in only three related Dutch families with common progenitors and which consist of 32 individuals over five generations. HCI was observed in 12 family members over four generations. Patients are mildly to severely affected. Besides HCI, several bone dysplasias with hyperostosis and sclerosis of the craniofacial bones are known. Examples are Van Buchem disease, sclerosteosis, craniometaphyseal dysplasia, and Camurati-Engelmann disease. However, in these cases the long bones are affected as well. Linkage analysis in a family with HCI resulted in the localization of the disease-causing gene to a region on chromosome 8p21 delineated by markers D8S282 and D8S382. Interesting candidate genes in this region are BMP1, LOXL2, and ADAM28. Sequence analysis of these genes did not reveal any putative mutations. This suggests that a gene not previously involved in a sclerosing bone dysplasia is responsible for the abnormal growth in the skull of these patients.

A gene variant of PNPLA3, but not of APOC3, is associated with histological parameters of NAFLD in an obese population.

OBJECTIVE: Mechanisms explaining the relationship in non-alcoholic fatty liver disease (NAFLD), obesity, and insulin resistance are poorly understood. A genetic basis has been suggested. We studied the association between the genes patatin-like phospholipase domain-containing protein 3 (PNPLA3) and apolipoprotein C3 (APOC3) and metabolic and histological parameters of NAFLD in obese patients. DESIGN AND METHODS: Overweight and obese patients underwent a metabolic and liver assessment. If NAFLD was suspected, liver biopsy was proposed. APOC3 variant rs2854117 and PNPLA3 variant rs738409 were genotyped. RESULTS: Four hundred seventy patients were included (61.1% had liver biopsy). The percentage of patients with non-alcoholic steatohepatitis (NASH) was significantly different according to the PNPLA3 variant. After adjustment for age and body mass index, the PNPLA3 variant was associated with alanine aminotransferase (P < 0.001) and aspartate aminotransferase (P < 0.001). The PNPLA3 variant was associated with more severe features of steatohepatitis: steatosis (P < 0.001), lobular inflammation (P < 0.001), and ballooning (P = 0.002), but not with liver fibrosis, anthropometry, or insulin resistance. No significant difference in liver histology, anthropometric, or metabolic parameters was found between carriers and non-carriers of the APOC3 variant. CONCLUSIONS: PNPLA3 polymorphism rs738409 was associated with NASH and the severity of necroinflammatory changes independently of metabolic factors. No association between APOC3 gene variant rs2854117 and histological or metabolic parameters of NAFLD was found.

No conclusive evidence for association of polymorphisms in the adiponectin receptor 1 gene, AdipoR1, with common obesity.

AdipoR1 is one of the adiponectin receptors which are important for adiponectin signaling. Because adiponectin is a candidate gene for common obesity, it is also hypothesized that variations in AdipoR1 may be involved in the development of complex obesity. Therefore, we designed an association study for the AdipoR1 gene. We performed a case-control association study including 1,021 obese subjects (mean age 42 ± 12 years; mean BMI 38.2 ± 6.2 kg/m²) and 226 lean, healthy individuals (mean age 36 ± 7 years; mean BMI 22.1 ± 1.7 kg/m²). Nine tagSNPs were selected to cover the entire AdipoR1 gene and surrounding 7 kb region (based on HapMap data). TagSNPs were genotyped using AcycloPrime-Fluorescence Polarization (FP) SNP Detection kits and TaqMan Pre-Designed SNP Genotyping assays according to manufacturer's protocols. We found that the rs1075399 non-reference allele decreases obesity risk by 45 % in men only [odds ratio (OR) = 0.55, 95 % CI 0.35-0.87, nominal P = 0.010]. However, after Bonferroni correction for multiple testing, this association is lost. None of the other tagSNPs were associated with obesity when studying the entire population, nor when looking at men and women separately. Quantitative analysis of the effect of each SNP on height, weight, and BMI revealed that none of the tagSNPs are associated with weight or BMI. We report here that we found no decisive evidence for association between AdipoR1 tagSNPs and complex obesity in our Belgian Caucasian population.

Genetic association between WNT10B polymorphisms and obesity in a Belgian case-control population is restricted to males.

The Wnt pathway has been shown to play an important role in maintenance of stem cells and cell fate decisions in embryonic and adult stem cell populations. Activation of the Wnt pathway in mesenchymal stem cells and 3 T3-L1 cells inhibits adipogenesis and can lead to osteoblastogenesis. To evaluate the role of the Wnt pathway in adipogenesis and obesity further, we analysed the genetic association between polymorphisms in WNT10B, an activator of the Wnt pathway, and various obesity parameters in a Belgian population. Four tagSNPs that captured variation of ten SNPs (MAF>5%) in a 15.2 kb region spanning the WNT10B gene and its 3' and 5' flanking regions were genotyped. Our population consisted of 1013 obese patients (BMI≥30 kg/m(2); 468 males) and 531 lean healthy individuals (18.5 kg/m(2)≤BMI≤24.9 kg/m(2); 194 males). We found a significant association with body mass index (BMI) for three of the genotyped tagSNPs (rs4018511, rs10875902, rs833841) in the male population as analysed by logistic regression. Allelic heterogeneity testing demonstrated that these associations all represent the same significant signal. Two of the three significant SNPs were also found to be associated with BMI and weight in the male population as analysed by linear regression. In conclusion, common variation in WNT10B was shown to be associated with BMI and weight in a case-control population of Belgian males. Nonetheless, replication of this result and elucidation of the molecular actions of WNT10B remain necessary.

Two novel WTX mutations underscore the unpredictability of male survival in osteopathia striata with cranial sclerosis.

Osteopathia striata with cranial sclerosis (OMIM ##300373) is an X-linked dominant sclerosing bone dysplasia that presents in females with macrocephaly, cleft palate, mild learning disabilities, sclerosis of the long bones and skull, and longitudinal striations visible on radiographs of the long bones, pelvis, and scapulae. In males this entity is usually associated with foetal or neonatal lethality, because of severe heart defects and/or gastrointestinal malformations, and is often accompanied by bilateral fibula aplasia. Recently, the disease-causing gene was identified as the WTX gene (FAM123B). Initially it was suggested that the mutations in the 5' region of the WTX gene are associated with male lethality. Mutation analysis in individuals of two families diagnosed with OSCS revealed two novel WTX mutations. In one family, the affected male is still alive in his teens. These mutations underline the unpredictability of male survival and suggest that WTX mutations should be considered in cases of male cranial sclerosis, even if striations are not present. An overview of all known mutations and their associated characteristics provide a valuable resource for the molecular analysis of OSCS.

Polymorphisms in the endocannabinoid receptor 1 in relation to fat mass distribution.

OBJECTIVE: Both animal and human studies have associated the endocannabinoid system with obesity and markers of metabolic dysfunction. Blockade of the cannabinoid receptor 1 (CB1) caused weight loss and reduction in waist size in both obese and type II diabetics. Recent studies on common variants of the CB1 receptor gene (CNR1) and the link to obesity have been conflicting. The aim of the present study was to evaluate whether selected common variants of the CNR1 are associated with measures of obesity and fat distribution. DESIGN AND METHODS: The single nucleotide polymorphisms (SNPs) rs806381, rs10485179 and rs1049353 were genotyped, and body fat and fat distribution were assessed by the use of dual-energy X-ray absorptiometry and magnetic resonance imaging in a population-based study comprising of 783 Danish men, aged 20-29 years. RESULTS: The rs806381 polymorphism was significantly associated with visceral fat mass (FM) only, whereas the rs1049353 was significantly and directly associated with visceral and intermuscular FM. None of the SNPs analysed were associated with total body FM or subcutaneous FM. CONCLUSION: The results point towards a link between common variants of the CNR1 and fat distribution in young men.

Sclerostin in mineralized matrices and van Buchem disease.

Sclerostin is an inhibitor of bone formation expressed by osteocytes. We hypothesized that sclerostin is expressed by cells of the same origin and also embedded within mineralized matrices. In this study, we analyzed (a) sclerostin expression using immunohistochemistry, (b) whether the genomic defect in individuals with van Buchem disease (VBD) was associated with the absence of sclerostin expression, and (c) whether this was associated with hypercementosis. Sclerostin was expressed by cementocytes in mouse and human teeth and by mineralized hypertrophic chondrocytes in the human growth plate. In individuals with VBD, sclerostin expression was absent or strongly decreased in osteocytes and cementocytes. This was associated with increased bone formation, but no overt changes in cementum thickness. In conclusion, sclerostin is expressed by all 3 terminally differentiated cell types embedded within mineralized matrices: osteocytes, cementocytes, and hypertrophic chondrocytes.

Refined genomic localization of the genetic lesion in the osteopetrosis (op) rat and exclusion of three positional and functional candidate genes, Clcn7, Atp6v0c, and Slc9a3r2.

Osteopetrosis is a disease characterised by a generalized skeletal sclerosis resulting from a reduced osteoclast-mediated bone resorption. Several spontaneous mutations lead to osteopetrotic phenotypes in animals. Moutier et al. (1974) discovered the osteopetrosis (op) rat as a spontaneous, lethal, autosomal recessive mutant. op rats have large nonfunctioning osteoclasts and severe osteopetrosis. Dobbins et al. (2002) localized the disease-causing gene to a 1.5-cM genetic interval on rat chromosome 10, which we confirm in the present report. We also refined the genomic localization of the disease gene and provide statistical evidence for a disease-causing gene in a small region of rat chromosome 10. Three strong functional candidate genes are within the delineated region. Clcn7 was previously shown to underlie different forms of osteopetrosis, in both human and mice. ATP6v0c encodes a subunit of the vacuolar H(+)-ATPase or proton pump. Mutations in TCIRG1, another subunit of the proton pump, are known to cause a severe form of osteopetrosis. Given the critical role of proton pumping in bone resorption, the Slc9a3r2 gene, a sodium/hydrogen exchanger, was also considered as a candidate for the op mutation. RT-PCR showed that all 3 genes are expressed in osteoclasts, but sequencing found no mutations either in the coding regions or in intron splice junctions. Our ongoing mutation analysis of other genes in the candidate region will lead to the discovery of a novel osteopetrosis gene and further insights into osteoclast functioning.

Polymorphisms in the low-density lipoprotein receptor-related protein 5 (LRP5) gene are associated with peak bone mass in non-sedentary men: results from the Odense androgen study.

PURPOSE: To investigate the impact of the Ala1330Val (rs3736228, exon 18) and Val667Met (rs4988321, exon 9) polymorphisms of the low-density lipoprotein receptor-related protein 5 (LRP5) gene on peak bone mass in young men. METHODS: The Odense Androgen Study (OAS) is a population-based study comprising 783 Caucasian men aged 20-30 years. Genotyping was performed using real-time polymerase chain reaction (PCR) or fluorescence polarization. Bone mineral density (BMD) measurements were performed using dual-energy X-ray absorptiometry. RESULTS: The CC, CT, and TT genotypes in Ala1330Val were found in 75.6%, 21.8%, and 2.6% of the participants, respectively. Similarly, the GG, GA, and AA genotypes of Val667Met were found in 89.7%, 9.8%, and 0.5%, respectively. For the Ala1330Val polymorphism, no significant differences between the genotypes were found regarding BMD in the overall study population. However, when analysis was restricted to non-sedentary men (n = 589), a significant association between the number of T-alleles and BMD in the spine and whole body were found. Each copy of the T-allele changed the Z-score of the spine by (median and 95% confidence interval) -0.21 [95% CI: -0.40; -0.03] (p < 0.02). Analysis suggested an association between the AA genotype in the Val667Met polymorphism and increased body height and decreased BMD of the femoral neck; however, no significant gene-dose effect of the A-allele could be demonstrated in the whole population. When the analysis was restricted to non-sedentary subjects, however, each number of A-alleles was associated with a change in Z-score of -0.26 [95% CI: -0.51; -0.01] (p = 0.04). No further significant results emerged with haplotype analysis. CONCLUSION: The Ala1330Val and Val667Met polymorphisms in the LRP5 gene are significantly associated with peak bone mass in physically active men.

Identification and molecular characterization of a novel splice-site mutation (G1205C) in the SQSTM1 gene causing Paget's disease of bone in an extended American family.

Paget's disease of bone (PDB) is a common late-onset bone disorder characterized by focal areas of abnormal bone remodeling. Positional cloning efforts resulted in the identification of seven genetic loci (PDB1-7) with putative involvement in the pathogenesis of PDB. Meanwhile, the PDB-causing gene from the PDB3 region on chromosome 5q35 has been identified as the SQSTM1 gene. All mutations identified in this gene so far are located in or close to the ubiquitin-associated (UBA) domain of the protein. In 2001, we reported genotyping results of genetic markers located in the PDB3 region in an extended American family, indicating the involvement of the PDB3 locus. Here, we report the identification of a novel mutation (G1205C) in the SQSTM1 gene in this family. The G1205C mutation is located in the splice donor site of intron 7 and reverse-transcription polymerase chain reaction experiments showed that the presence of the C allele results in the production of two abnormal mRNA transcripts. Translation of the first transcript would result in a protein that lacks amino acids 351-388, including 26 amino acids of the second PEST domain in addition to two amino acids of the UBA domain. The second mutant mRNA transcript could result in a truncated protein (390X) that lacks almost the complete UBA domain. PDB mutations that disrupt the function of the PEST domain of SQSTM1 have not been reported before, so probably the pathogenic effect of both transcripts resides in the disruption of the ubiquitin-binding properties of the protein.

Adams-Oliver syndrome: clinical description of a four-generation family and exclusion of five candidate genes.

We present a Belgian Adams-Oliver syndrome (AOS) family with 10 affected individuals over four generations, of which six were available for this study. Clinical symptoms observed in these patients were very variable as previously reported in other families and included large areas of alopecia on the vertex of the skull and serious limb reduction defects with agenesis of all toes of one foot. To identify the disease-causing gene, we sequenced the MSX1, CART1, P63 (P73L), RUNX2, and HOXD13 genes in this family and nine previously reported families, but no disease-causing mutations were found. Further investigation is ongoing in these families in order to identify the genetic cause of AOS.