Fenna de Freitas

A new heterozygous mutation (R714C) of the osteopetrosis gene, pleckstrin homolog domain containing family M (with run domain) member 1 (PLEKHM1), impairs vesicular acidification and increases TRACP secretion in osteoclasts.

We studied phenotypic and cellular aspects in a patient with a heterozygous mutation of the PLEKHM1 gene and obtained some indications regarding the role of the protein in bone cell function. Plekhm1 is involved in osteoclast endosomal vesicle acidification and TRACP exocytosis, contributing to events involved in osteoclast–osteoblast cross‐talk.

Identification of Sex-Specific Associations Between Polymorphisms of the Osteoprotegerin Gene, TNFRSF11B, and Paget's Disease of Bone†

We studied the role of TNFRSF11B polymorphisms on the risk to develop Pagets disease of bone in a Belgian study population. We observed no association in men, but a highly significant association was found in women, and this was confirmed in a population from the United Kingdom.

Osteopathia Striata With Cranial Sclerosis Owing to WTX Gene Defect

Osteopathia striata with cranial sclerosis (OSCS) is an X‐linked dominant condition marked by linear striations mainly affecting the metaphyseal region of the long bones and pelvis in combination with cranial sclerosis. Recently, the disease‐causing gene was identified as the WTX gene (FAM123B), an inhibitor of WNT signaling. A correlation was suggested between the position of the mutation and male lethality. We performed genotype and phenotype studies using 18 patients from eight families with possible WTX gene defects and expanded the clinical spectrum of the affected females. All investigated families diagnosed with OSCS had WTX gene defects. One family had a WTX gene deletion; three of four point mutations were novel. The earlier reported WTX c.1072C>T was detected in four sporadic patients and appears to be a hotspot for mutations. Based on the nature of the mutation present in a surviving male patient, our data do not support the hypothesis raised by Jenkins et al. (2009) regarding a genotype‐phenotype correlation for male lethality. The finding of a gene involved in WNT signaling as the cause of this sclerosing bone phenotype is not unexpected, but further functional studies are needed to explain the specific features. The WTX gene is mutated in different types of cancer, and it remains to be explained why osteopathia striata patients appear not to have an increased risk of cancer. Copyright

First missense mutation in the SOST gene causing sclerosteosis by loss of sclerostin function.

Sclerosteosis is a rare bone dysplasia characterized by greatly increased bone mass, especially of the long bones and the skull. Patients are tall, show facial asymmetry and often have syndactyly. Clinical complications are due to entrapment of cranial nerves. The disease is thought to be due to loss‐of‐function mutations in the SOST gene. The SOST gene product, sclerostin, is secreted by osteocytes and transported to the bone surface where it inhibits osteoblastic bone formation by antagonizing Wnt signaling. In a small Turkish family with sclerosteosis, we identified a missense mutation (c.499T>C; p.Cys167Arg) in exon 2 of the SOST gene. This type of mutation has not been previously reported and using different functional approaches, we show that it has a devastating effect on the biological function of sclerostin. The affected cysteine is the last cysteine residue of the cystine‐knot motif and loss of this residue leads to retention of the mutant protein in the ER, possibly as a consequence of impaired folding. Together with a significant reduced ability to bind to LRP5 and inhibit Wnt signaling, the p.Cys167Arg mutation leads to a complete loss of function of sclerostin and thus to the characteristic sclerosteosis phenotype.

Genetic variation in the **TNFRSF11A** gene encoding RANK is associated with susceptibility to Paget's disease of bone

RANK (receptor activator of nuclear factor‐κB), encoded by TNFRSF11A, is a key protein in osteoclastogenesis. TNFRSF11A mutations cause Pagets disease of bone (PDB)–like diseases (ie, familial expansile osteolysis, expansile skeletal hyperphosphatasia, and early‐onset PDB) and an osteoclast‐poor form of osteopetrosis. However, no TNFRSF11A mutations have been found in classic PDB, neither in familial nor in isolated cases. To investigate the possible relationship between TNFRSF11A polymorphisms and sporadic PDB, we conducted an association study including 32 single‐nucleotide polymorphisms (SNPs) in 196 Belgian sporadic PDB patients and 212 control individuals. Thirteen SNPs and 3 multimarker tests (MMTs) turned out to have a p value of between .036 and 3.17 × 10−4, with the major effect coming from females. Moreover, 6 SNPs and 1 MMT withstood the Bonferroni correction (p < .002). Replication studies were performed for 2 nonsynonymous SNPs (rs35211496 and rs1805034) in a Dutch and a British cohort. Interestingly, both SNPs resulted in p values ranging from .013 to 8.38 × 10−5 in both populations. Meta‐analysis over three populations resulted in p = .002 for rs35211496 and p = 1.27 × 10−8 for rs1805034, again mainly coming from the female subgroups. In an attempt to identify the underlying causative SNP, we performed functional studies for the coding SNPs as well as resequencing efforts of a 31‐kb region harboring a risk haplotype within the Belgian females. However, neither approach resulted in significant evidence for the causality of any of the tested genetic variants. Therefore, further studies are needed to identify the real cause of the increased risk to develop PDB shown to be present within TNFRSF11A.

Indications for a genetic association of a VCP polymorphism with the pathogenesis of sporadic Paget's disease of bone, but not for TNFSF11 (RANKL) and IL-6 polymorphisms

Pagets disease of bone (PDB) is, after osteoporosis, the second most common metabolic bone disorder in the elderly Caucasian population. Mutations in the sequestosome 1 gene (SQSTM1) are responsible for the etiology of PDB in a subset of patients, but the disease pathogenesis in the remaining PDB patients is still unknown. Therefore association studies investigating the relationship between genetic polymorphisms and sporadic PDB have been performed in order to find the susceptibility polymorphisms. In this paper, we sought to determine whether polymorphisms in 3 functional candidate genes play a role in the development of sporadic PDB: TNFSF11 (receptor activator of nuclear factor κB ligand, RANKL), VCP (valosin-containing protein) and IL-6 (interleukin 6). Analyzing 9 tag SNPs and 2 multi-marker tests (MMTs) in TNFSF11, 3 tag SNPs and 1 MMT in VCP and 8 tag SNPs in IL-6 in a population of 196 Belgian patients with sporadic PDB and 212 Belgian control individuals revealed that one VCP SNP (rs565070) turned out to be associated with PDB in this Belgian study population (p=5.5×10(-3)). None of the tag SNPs or MMTs selected for TNFSF11 or IL-6 was associated with PDB. Still, replication of our findings in the VCP gene in other populations is important to confirm our results. However, when combining data of VCP with those from other susceptible gene regions from previous association studies (i.e. TNFRSF11A, CSF1, OPTN and TM7SF4), independent effect of each gene region was found and the cumulative population attributable risk is 72.7%.

Identification of three novel genetic variants in the melanocortin-3 receptor of obese children.

The melanocortin‐3 receptor (MC3R), a G‐protein‐coupled receptor expressed in the hypothalamus, is a key component of the leptin‐melanocortin pathway that regulates energy homeostasis. It is suggested that an MC3R defect leads to an increased feed efficiency, by which nutrients are partitioned preferentially into fat. In this study, we hypothesized that early‐onset obesity could be induced by mutations in MC3R. To investigate this hypothesis, we screened the entire coding region of the MC3R gene for mutations in obese subjects. A total of 404 overweight and obese children and adolescents, 86 severely obese adults (BMI ≥40 kg/m2), and 150 normal‐weight control adults were included. Besides three synonymous coding variations in the MC3R gene (S69S, L95L, I226I), we were able to identify three novel heterozygous, nonsynonymous, coding mutations (N128S, V211I, L299V) in three unrelated obese children. None of these mutations were found in any of the control subjects. Functional studies assessing localization and signaling properties of the mutant receptors provided proof for impaired function of the L299V mutated receptor, whereas no conclusive evidence for functional impairment of the N128S and V211I mutated receptors could be established. First, these results provide supporting evidence for a role of the MC3R gene in the pathogenesis of obesity in a small subset of patients. Second, they show that caution is called for the interpretation of newly discovered mutations in MC3R.

Association study of MC4R with complex obesity and replication of the rs17782313 association signal.

Recently, genome-wide association studies have discovered several single nucleotide polymorphisms (SNPs) involved in the etiology of complex obesity. A variant downstream from the melanocortin-4 receptor gene (MC4R), a gene known to be involved in monogenic obesity, was reported to be highly associated with BMI. In the present study, we performed a replication study with the previously reported SNP rs17782313. We also included 3 tagSNPs (rs8087522, rs11872992, and rs1943226) for the MC4R gene region in our study to understand the role of this gene in complex obesity. We genotyped all 4 SNPs in a population of 1049 obese cases (mean BMI=38.2±6.2) and 312 healthy lean individuals (mean BMI 22.0±1.7). We could confirm that rs17782313 is highly associated with complex obesity in our population (odds ratio=1.42, 95% CI 1.14-1.77, P=0.002). Furthermore, we found this SNP to be associated with BMI (B=0.92, 95% CI 0.19-1.65, P=0.01) and body weight (B=2.44, 95% CI 0.28-4.60, P=0.03). In addition, we could also detect an association between rs11872992 and complex obesity (odds ratio=0.74, 95% CI 0.57-0.98, P=0.03). Through conditional analysis, we demonstrate that this effect is independent from the rs17782313 association signal. No associations with obesity could be found for rs8087522 and rs1943226. In conclusion, we could replicate the previously reported association between rs17782313 and complex obesity. Furthermore, our data do not support the hypothesis that a SNP in MC4R causes the rs17782313 association signal.

Identification and Functional Characterization of Novel Mutations in the Melanocortin-4 Receptor

Objective: Melanocortin-4 receptor (MC4R) deficiency is the most common cause of monogenic obesity. In the present study, we screened the MC4R gene for mutations in a population of overweight and obese children and adolescents. Method: Cross-sectional mutation analysis of 112 overweight/obese children and adolescents and 121 lean individuals. Results: We identified 11 sequence variations, 5 of which were present in our control population or had been previously reported as polymorphisms. The remaining 6 variations are disease-causing mutations including 2 novel ones: a I186V mutation and a F280L mutation. The 4 previously described mutations (D90N, M200V, P260Q, Q307X) were identified in single probands. Using confocal imaging, we demonstrated that F280L and P260Q cause intracellular retention of the mutant receptor. No difference in cell surface expression could be detected for the I186V mutation. Using a cAMP responsive luciferase vector, we demonstrated that the receptor with I186V is unable to activate its intracellular signaling pathway while the P260Q mutation causes reduced activation of the receptor. Conclusion: We detected MC4R deficiency in 6 patients from our cohort, amounting to a prevalence of 5.3%. Two novel mutations were identified. We also confirmed that intracellular retention is a common pathogenic effect of MC4R mutations.

Analysis of genetic variations in the resistin gene shows no associations with obesity in women.

Resistin is thought to be involved in the development of obesity and insulin resistance. Polymorphisms in the gene encoding resistin could contribute to this link, but different studies have yielded contradictory results. In this study, we investigated whether polymorphisms in resistin are involved in the development of obesity in a Belgian female population. We selected three single‐nucleotide polymorphisms (SNPs; rs1862513, rs3745367, and rs3745369) and compared their genotype and allele frequencies between female obese patients (N = 541) and control individuals (N = 235). This analysis showed association with neither obesity for any of the variants nor with the haplotypes of these SNPs. Furthermore, we also investigated whether these variants have an influence on BMI. After Kruskal–Wallis analysis, we found that there was no difference in BMI between the genotypes for all variants. Together, these results suggest that these variants in resistin are not associated with obesity in the female population.