Screening of CD96 and ASXL1 in 11 patients with Opitz C or Bohring-Opitz syndromes.

Opitz C trigonocephaly (or Opitz C syndrome, OTCS) and Bohring-Opitz syndrome (BOS or C-like syndrome) are two rare genetic disorders with phenotypic overlap. The genetic causes of these diseases are not understood. However, two genes have been associated with OTCS or BOS with dominantly inherited de novo mutations. Whereas CD96 has been related to OTCS (one case) and to BOS (one case), ASXL1 has been related to BOS only (several cases). In this study we analyze CD96 and ASXL1 in a group of 11 affected individuals, including 2 sibs, 10 of them were diagnosed with OTCS, and one had a BOS phenotype. Exome sequences were available on six patients with OTCS and three parent pairs. Thus, we could analyze the CD96 and ASXL1 sequences in these patients bioinformatically. Sanger sequencing of all exons of CD96 and ASXL1 was carried out in the remaining patients. Detailed scrutiny of the sequences and assessment of variants allowed us to exclude putative pathogenic and private mutations in all but one of the patients. In this patient (with BOS) we identified a de novo mutation in ASXL1 (c.2100dupT). By nature and location within the gene, this mutation resembles those previously described in other BOS patients and we conclude that it may be responsible for the condition. Our results indicate that in 10 of 11, the disease (OTCS or BOS) cannot be explained by small changes in CD96 or ASXL1. However, the cohort is too small to make generalizations about the genetic etiology of these diseases.

Genetic determinants of aromatase inhibitor-related arthralgia: the B-ABLE cohort study.

A major side effect of aromatase inhibitor (AI) therapy is AI-related arthralgia (AIA), which often leads to therapy discontinuation. We aimed to identify genetic variants associated with AIA and therapy discontinuation in the first year of AI treatment. Our prospective cohort study included 343 postmenopausal women with early breast cancer starting AI therapy. Single nucleotide polymorphisms (SNPs) in candidate genes involved in estrogen and vitamin D signaling were selected. Univariate and multivariate linear/logistic regressions were fitted in order to asses the association between studied SNPs and AIA intensity (visual analogic scale score) at 3 and 12 months of follow-up, worsening pain, and therapy discontinuation. We also tested for a priori-defined interactions by introducing multiplicative terms in the regression equations. SNPs in CYP17A1 and VDR genes appeared significantly associated with AIA (P = 0.003, P = 0.012, respectively). One SNP in CYP27B1 gene was related to therapy discontinuation [P = 0.02; OR 0.29 (0.09-0.99)]. We revealed interactions between CYP27B1 and both CYP17A1 (P = 0.01) and VDR SNPs (P = 0.06). Furthermore, an additive effect on pain intensity was shown for unfavorable alleles, with two points higher mean absolute pain increase and up to 5.3-fold higher risk of worsening pain compared to favorable genotypes. SNPs in CYP17A1, VDR, and CYP27B1 genes predict the risk of AIA. Their determination would be useful to trigger the monitoring strategies in women at risk of therapy discontinuation.

Identification and functional analyses of CBS alleles in Spanish and Argentinian homocystinuric patients.

Homocystinuria due to CBS deficiency is a rare autosomal recessive disorder characterized by elevated plasma levels of homocysteine (Hcy) and methionine (Met). Here we present the analysis of 22 unrelated patients of different geographical origins, mainly Spanish and Argentinian. Twenty-two different mutations were found, 10 of which were novel. Five new mutations were missense and five were deletions of different sizes, including a 794-bp deletion (c.532-37_736 + 438del794) detected by Southern blot analysis. To assess the pathogenicity of these mutations, seven were expressed heterologously in Escherichia coli and their enzyme activities were assayed in vitro, in the absence and presence of the CBS activators PLP and SAM. The presence of the mutant proteins was confirmed by Western blotting. Mutations p.M173del, p.I278S, p.D281N, and p.D321V showed null activity in all conditions tested, whereas mutations p.49L, p.P200L and p.A446S retained different degrees of activity and response to stimulation. Finally, a minigene strategy allowed us to demonstrate the pathogenicity of an 8-bp intronic deletion, which led to the skipping of exon 6. In general, frameshifting deletions correlated with a more severe phenotype, consistent with the concept that missense mutations may recover enzymatic activity under certain conditions.

Effect of IL-1beta, PGE(2), and TGF-beta1 on the expression of OPG and RANKL in normal and osteoporotic primary human osteoblasts.

The RANKL/RANK/OPG pathway is essential for bone remodeling regulation. Many hormones and cytokines are involved in regulating gene expression in most of the pathway components. Moreover, any deregulation of this pathway can alter bone metabolism, resulting in loss or gain of bone mass. Whether osteoblasts from osteoporotic and nonosteoporotic patients respond differently to cytokines is unknown. The aim of this study was to compare the effect of interleukin (IL)-1beta, proftaglandin E(2) (PGE(2)), and transforming growth factor-beta1 (TGF-beta1) treatments on OPG and RANKL gene expression in normal (n = 11) and osteoporotic (n = 8) primary osteoblasts. OPG and RANKL mRNA levels of primary human osteoblastic (hOB) cell cultures were assessed by real-time PCR. In all cultures, OPG mRNA increased significantly in response to IL-1beta treatment and decreased in response to TGF-beta1 whereas PGE(2) treatment had no effect. RANKL mRNA levels were significantly increased by all treatments. Differences in OPG and RANKL responses were observed between osteoporotic and nonosteoporotic hOB: in osteoporotic hOB, the OPG response to IL-1beta treatment was up to three times lower (P = 0.009), whereas that of RANKL response to TGF-beta1 was five times higher (P = 0.002) after 8 h of treatment, as compared with those in nonosteoporotic hOBs. In conclusion, osteoporotic hOB cells showed an anomalous response under cytokine stimulation, consistent with an enhanced osteoclastogenesis resulting in high levels of bone resorption.

Analysis of three functional polymorphisms in relation to osteoporosis phenotypes: replication in a Spanish cohort.

Osteoporosis is a complex disease involving many putative genetic factors. Association analysis of functional SNPs in candidate genes is an important tool for their identification. However, this approach is affected by limited power, population stratification, and other drawbacks that lead to discordant results. Replication in independent cohorts is essential. We performed association analyses of three functional polymorphisms previously associated with bone phenotypes--namely, Ala222Val in MTHFR, Ile1062Val in LRP6, and -13910C>T in LCT--in a cohort of 944 postmenopausal Spanish women, all of them with lumbar spine (LS) bone mineral density (BMD) data and most with femoral neck (FN) BMD and fracture data. We found significant differences between genotypes only for the MTHFR polymorphism and vertebral factures, with an OR of 2.27 (95% CI 1.17-4.38) for the TT vs. CC/CT genotypes, P = 0.018. We present genotype and allele frequency data for LCT -13910C>T for a Spanish population, where the T allele (conferring lactase persistence) has a frequency of 38.6%. Genotype frequencies were consistent with observed clines in Europe and with the prevalence of lactase nonpersistence. The LCT -13910C>T polymorphism was significantly associated with height and weight, such that T allele carriers were 0.88 cm taller (95% CI 0.08-1.59 cm, P = 0.032, adjusted by age) than CC individuals and TT homozygotes were 1.91 kg heavier than CC/CT individuals (95% CI 0.11-3.71 kg, P = 0.038, adjusted by age). In conclusion, no significant association was observed between the studied polymorphisms and LS BMD or FN BMD in postmenopausal Spanish women, and only MTHFR Ala222Val was associated with vertebral fractures.

Case report of a child bearing a novel deleterious splicing variant in PIGT.

RATIONALE: Trio family-based whole exome sequencing (WES) is a powerful tool in the diagnosis of rare neurodevelopmental diseases, even in patients with the unclear diagnosis. There have been previous reports of variants in the phosphatidylinositol glycan anchor biosynthesis class T (PIGT) gene associated with multiple congenital anomalies, with a total of 14 affected individuals across 8 families. PATIENT CONCERNS: An 18-month-old boy of Greek ancestry presented with global developmental delay, generalized tonic-clonic seizures, hypotonia, renal cysts, esotropia, bilateral undescended testes, bilateral vesicoureteric reflux, marked cardiac dextroposition, bilateral talipes equinovarus, and dysmorphic features. DIAGNOSIS: WES revealed 2 compound heterozygous variants in the PIGT gene, c.[494-2A>G]; [547A>C]/p.[Asp122Glyfs*35]; [Thr183Pro]. The splicing mutation was demonstrated to lead to the skipping of exon 4. INTERVENTIONS: Seizures, infections, and other main symptoms were treated. OUTCOMES: The patient died at 2 years of age before the molecular diagnosis was achieved. Genetic counseling has been offered to the family. LESSONS: Most of the clinical features of the patient are in agreement with the previously described PIGT cases corroborating the usefulness of WES as a diagnostic tool.

Large-scale analysis of association between LRP5 and LRP6 variants and osteoporosis.

CONTEXT: Mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene cause rare syndromes characterized by altered bone mineral density (BMD). More common LRP5 variants may affect osteoporosis risk in the general population. OBJECTIVE: To generate large-scale evidence on whether 2 common variants of LRP5 (Val667Met, Ala1330Val) and 1 variant of LRP6 (Ile1062Val) are associated with BMD and fracture risk. DESIGN AND SETTING: Prospective, multicenter, collaborative study of individual-level data on 37,534 individuals from 18 participating teams in Europe and North America. Data were collected between September 2004 and January 2007; analysis of the collected data was performed between February and May 2007. Bone mineral density was assessed by dual-energy x-ray absorptiometry. Fractures were identified via questionnaire, medical records, or radiographic documentation; incident fracture data were available for some cohorts, ascertained via routine surveillance methods, including radiographic examination for vertebral fractures. MAIN OUTCOME MEASURES: Bone mineral density of the lumbar spine and femoral neck; prevalence of all fractures and vertebral fractures. RESULTS: The Met667 allele of LRP5 was associated with reduced lumbar spine BMD (n = 25,052 [number of participants with available data]; 20-mg/cm2 lower BMD per Met667 allele copy; P = 3.3 x 10(-8)), as was the Val1330 allele (n = 24,812; 14-mg/cm2 lower BMD per Val1330 copy; P = 2.6 x 10(-9)). Similar effects were observed for femoral neck BMD, with a decrease of 11 mg/cm2 (P = 3.8 x 10(-5)) and 8 mg/cm2 (P = 5.0 x 10(-6)) for the Met667 and Val1330 alleles, respectively (n = 25 193). Findings were consistent across studies for both LRP5 alleles. Both alleles were associated with vertebral fractures (odds ratio [OR], 1.26; 95% confidence interval [CI], 1.08-1.47 for Met667 [2001 fractures among 20 488 individuals] and OR, 1.12; 95% CI, 1.01-1.24 for Val1330 [1988 fractures among 20,096 individuals]). Risk of all fractures was also increased with Met667 (OR, 1.14; 95% CI, 1.05-1.24 per allele [7876 fractures among 31,435 individuals)]) and Val1330 (OR, 1.06; 95% CI, 1.01-1.12 per allele [7802 fractures among 31 199 individuals]). Effects were similar when adjustments were made for age, weight, height, menopausal status, and use of hormone therapy. Fracture risks were partly attenuated by adjustment for BMD. Haplotype analysis indicated that Met667 and Val1330 variants both independently affected BMD. The LRP6 Ile1062Val polymorphism was not associated with any osteoporosis phenotype. All aforementioned associations except that between Val1330 and all fractures and vertebral fractures remained significant after multiple-comparison adjustments. CONCLUSIONS: Common LRP5 variants are consistently associated with BMD and fracture risk across different white populations. The magnitude of the effect is modest. LRP5 may be the first gene to reach a genome-wide significance level (a conservative level of significance [herein, unadjusted P < 10(-7)] that accounts for the many possible comparisons in the human genome) for a phenotype related to osteoporosis.

Estudios de asociación de genoma completo (GWAS) versus validación funcional: reto de la era post-GWAS / Genome-wide association studies (GWAS) versus functional validation: challenge of the post-GWAS era

En los últimos años se han dedicado muchos esfuerzos a la determinación de variantes y genes que pueden ser impor-tantes en la determinación de la densidad mineral ósea (DMO) y, a su vez, en diversas patologías óseas. Para conseguiresto, la aproximación que ha presentado mayores éxitos ha sido la de los estudios de asociación de genoma completo(GWAS). En particular, en la investigación sobre la biología ósea, se han publicado más de 50 grandes GWAS o metaa-nálisis de GWAS identificando más de 500 loci genéticos asociados con diferentes parámetros óseos como son la DMO,la resistencia ósea y el riesgo de fractura. Si bien el descubrimiento de las variantes asociadas es un aspecto esencial,es igualmente importante la validación funcional de dichas variantes para dilucidar su efecto y la relación causal quetienen con la enfermedad genética. Al tratarse de un aspecto mucho más lento y tedioso, se ha convertido en el nuevoreto de esta era post-GWAS. Entre los genes que ya se han abordado se incluyen varios de la vía de WNT y en especialel gen SOST, que juega un papel muy importante tanto en la determinación de la DMO poblacional como en enferme-dades monogénicas con elevada masa ósea y que ha dado lugar a un nuevo tratamiento contra la osteoporosis. En estarevisión recogemos los principales estudios GWAS con relación a fenotipos del hueso, así como algunos ejemplos devalidaciones funcionales para analizar las asociaciones encontradas en los mismos.(AU)

The ASXL1 mutation p.Gly646Trpfs*12 found in a Turkish boy with Bohring-Opitz Syndrome.

In line with a recent study showing that ASXL1 mutations found in the common population cannot be ruled out as pathogenic, we have identified the ASXL1 p.Gly646Trpfs*12 mutation-present in 132 individuals in ExAC-as a very probable cause of the disease in a Bohring-Opitz syndrome patient.

Pro-osteoporotic miR-320a impairs osteoblast function and induces oxidative stress.

MicroRNAs (miRNAs) are important regulators of many cellular processes, including the differentiation and activity of osteoblasts, and therefore, of bone turnover. MiR-320a is overexpressed in osteoporotic bone tissue but its role in osteoblast function is unknown. In the present study, functional assays were performed with the aim to elucidate the mechanism of miR-320a action in osteoblastic cells. MiR-320a was either overexpressed or inhibited in human primary osteoblasts (hOB) and gene expression changes were evaluated through microarray analysis. In addition, the effect of miR-320a on cell proliferation, viability, and oxidative stress in hOB was evaluated. Finally, matrix mineralization and alkaline phosphatase activity were assessed in order to evaluate osteoblast functionality. Microarray results showed miR-320a regulation of a number of key osteoblast genes and of genes involved in oxidative stress. Regulation of osteoblast differentiation and ossification appeared as the best significant biological processes (PANTHER P value = 3.74E-05; and P value = 3.06E-04, respectively). The other enriched pathway was that of the cellular response to cadmium and zinc ions, mostly by the overexpression of metallothioneins. In hOBs, overexpression of miR-320a increased cell proliferation and oxidative stress levels whereas mineralization capacity was reduced. In conclusion, overexpression of miR-320a increased stress oxidation levels and was associated with reduced osteoblast differentiation and functionality, which could trigger an osteoporotic phenotype.

Common and rare variants of WNT16, DKK1 and SOST and their relationship with bone mineral density.

Numerous GWAS and candidate gene studies have highlighted the role of the Wnt pathway in bone biology. Our objective has been to study in detail the allelic architecture of three Wnt pathway genes: WNT16, DKK1 and SOST, in the context of osteoporosis. We have resequenced the coding and some regulatory regions of these three genes in two groups with extreme bone mineral density (BMD) (n = ∼50, each) from the BARCOS cohort. No interesting novel variants were identified. Thirteen predicted functional variants have been genotyped in the full cohort (n = 1490), and for ten of them (with MAF > 0.01), the association with BMD has been studied. We have found six variants nominally associated with BMD, of which 2 WNT16 variants predicted to be eQTLs for FAM3C (rs55710688, in the Kozak sequence and rs142005327, within a putative enhancer) withstood multiple-testing correction. In addition, two rare variants in functional regions (rs190011371 in WNT16b 3'UTR and rs570754792 in the SOST TATA box) were found only present in three women each, all with BMD below the mean of the cohort. Our results reinforce the higher importance of regulatory versus coding variants in these Wnt pathway genes and open new ways for functional studies of the relevant variants.

Expression profiling of microRNAs in human bone tissue from postmenopausal women.

Bone tissue is composed of several cell types, which express their own microRNAs (miRNAs) that will play a role in cell function. The set of total miRNAs expressed in all cell types configures the specific signature of the bone tissue in one physiological condition. The aim of this study was to explore the miRNA expression profile of bone tissue from postmenopausal women. Tissue was obtained from trabecular bone and was analyzed in fresh conditions (n = 6). Primary osteoblasts were also obtained from trabecular bone (n = 4) and human osteoclasts were obtained from monocyte precursors after in vitro differentiation (n = 5). MicroRNA expression profiling was obtained for each sample by microarray and a global miRNA analysis was performed combining the data acquired in all the microarray experiments. From the 641 miRNAs detected in bone tissue samples, 346 (54%) were present in osteoblasts and/or osteoclasts. The other 46% were not identified in any of the bone cells analyzed. Intersection of osteoblast and osteoclast arrays identified 101 miRNAs shared by both cell types, which accounts for 30-40% of miRNAs detected in these cells. In osteoblasts, 266 miRNAs were detected, of which 243 (91%) were also present in the total bone array, representing 38% of all bone miRNAs. In osteoclasts, 340 miRNAs were detected, of which 196 (58%) were also present in the bone tissue array, representing 31% of all miRNAs detected in total bone. These analyses provide an overview of miRNAs expressed in bone tissue, broadening our knowledge in the microRNA field.

A De Novo FOXP1 Truncating Mutation in a Patient Originally Diagnosed as C Syndrome.

De novo FOXP1 mutations have been associated with intellectual disability (ID), motor delay, autistic features and a wide spectrum of speech difficulties. C syndrome (Opitz C trigonocephaly syndrome) is a rare and genetically heterogeneous condition, characterized by trigonocephaly, craniofacial anomalies and ID. Several different chromosome deletions and and point mutations in distinct genes have been associated with the disease in patients originally diagnosed as Opitz C. By whole exome sequencing we identified a de novo splicing mutation in FOXP1 in a patient, initially diagnosed as C syndrome, who suffers from syndromic intellectual disability with trigonocephaly. The mutation (c.1428 + 1 G > A) promotes the skipping of exon 16, a frameshift and a premature STOP codon (p.Ala450GLyfs*13), as assessed by a minigene strategy. The patient reported here shares speech difficulties, intellectual disability and autistic features with other FOXP1 syndrome patients, and thus the diagnosis for this patient should be changed. Finally, since trigonocephaly has not been previously reported in FOXP1 syndrome, it remains to be proved whether it may be associated with the FOXP1 mutation.

Functional Characterization of a GGPPS Variant Identified in Atypical Femoral Fracture Patients and Delineation of the Role of GGPPS in Bone-Relevant Cell Types.

Atypical femoral fractures (AFFs) are a rare but potentially devastating event, often but not always linked to bisphosphonate (BP) therapy. The pathogenic mechanisms underlying AFFs remain obscure, and there are no tests available that might assist in identifying those at high risk of AFF. We previously used exome sequencing to explore the genetic background of three sisters with AFFs and three additional unrelated AFF cases, all previously treated with BPs. We detected 37 rare mutations (in 34 genes) shared by the three sisters. Notably, we found a p.Asp188Tyr mutation in the enzyme geranylgeranyl pyrophosphate synthase, a component of the mevalonate pathway, which is critical to osteoclast function and is inhibited by N-BPs. In addition, the CYP1A1 gene, responsible for the hydroxylation of 17ß-estradiol, estrone, and vitamin D, was also mutated in all three sisters and one unrelated patient. Here we present a detailed list of the variants found and report functional analyses of the GGPS1 p.Asp188Tyr mutation, which showed a severe reduction in enzyme activity together with oligomerization defects. Unlike BP treatment, this genetic mutation will affect all cells in the carriers. RNAi knockdown of GGPS1 in osteoblasts produced a strong mineralization reduction and a reduced expression of osteocalcin, osterix, and RANKL, whereas in osteoclasts, it led to a lower resorption activity. Taken together, the impact of the mutated GGPPS and the relevance of the downstream effects in bone cells make it a strong candidate for AFF susceptibility. We speculate that other genes such as CYP1A1 might be involved in AFF pathogenesis, which remains to be functionally proved. The identification of the genetic background for AFFs provides new insights for future development of novel risk assessment tools. © 2018 American Society for Bone and Mineral Research.

Erratum to: The p.T191M mutation of the CBS gene is highly prevalent among homocystinuric patients from Spain, Portugal and South America.

In this article, one of the novel mutations, c.208_209+ 8del10, was incorrectly given as c.69_70+8del10. It corresponds to patient 64 in Table 4.

Bone mass of a 113-year-old man.

Osteoporosis is a common disease that affects elderly people. Aging induces loss of bone density and quality resulting in a progressive incidence of fragility fractures. In this study, we report the bone density of one of the oldest men in the world and of several of his first-degree relatives, as well as a genetic screen of these cases. No fractures have been suffered by any of them, and their bone mineral density (BMD) values in terms of z score were normal or lightly decreased. Neither mutations at the longevity-related gene KLOTHO nor the Gly171Val mutation of LRP5 associated with high bone mass was detected in the two centenarian stepbrothers.

A CBS haplotype and a polymorphism at the MSR gene are associated with cardiovascular disease in a Spanish case-control study.

OBJECTIVES: The aim of this study was to evaluate the association of polymorphisms present in genes related to homocysteine (Hcy) metabolism with coronary artery disease (CAD). DESIGN AND METHODS: We examined 8 polymorphisms in the cystathionine beta-synthase (CBS), glutamate carboxypeptidase II (GCPII), methionine synthase (MS), methionine synthase reductase (MSR) and methylenetetrahydrofolate reductase (MTHFR) genes in 140 CAD patients and 113 controls, by means of Chi-square, logistic regression, ANOVA and the Mann-Whitney U test. RESULTS: The c.66 G allele of MSR conferred an odds-ratio for CAD of 1.76 (95% CI 1.12-2.77), while a CBS haplotype [c.699C-c.844wt-c.1080C] was found over-represented in CAD [OR of 2.16 (1.29-3.63)]. CONCLUSIONS: Our results not only highlight the involvement of the MSR and CBS genes in the etiology of cardiovascular disease, but also emphasize the strength of haplotype analyses in association studies.

A De Novo Nonsense Mutation in MAGEL2 in a Patient Initially Diagnosed as Opitz-C: Similarities Between Schaaf-Yang and Opitz-C Syndromes.

Opitz trigonocephaly C syndrome (OTCS) is a rare genetic disorder characterized by craniofacial anomalies, variable intellectual and psychomotor disability, and variable cardiac defects with a high mortality rate. Different patterns of inheritance and genetic heterogeneity are known in this syndrome. Whole exome and genome sequencing of a 19-year-old girl (P7), initially diagnosed with OTCS, revealed a de novo nonsense mutation, p.Q638*, in the MAGEL2 gene. MAGEL2 is an imprinted, maternally silenced, gene located at 15q11-13, within the Prader-Willi region. Patient P7 carried the mutation in the paternal chromosome. Recently, mutations in MAGEL2 have been described in Schaaf-Yang syndrome (SHFYNG) and in severe arthrogryposis. Patient P7 bears resemblances with SHFYNG cases but has other findings not described in this syndrome and common in OTCS. We sequenced MAGEL2 in nine additional OTCS patients and no mutations were found. This study provides the first clear molecular genetic basis for an OTCS case, indicates that there is overlap between OTCS and SHFYNG syndromes, and confirms that OTCS is genetically heterogeneous. Genes encoding MAGEL2 partners, either in the retrograde transport or in the ubiquitination-deubiquitination complexes, are promising candidates as OTCS disease-causing genes.

Functional assays testing pathogenicity of 14 cystathionine-beta synthase mutations.

In this study, 14 CBS alleles from homocystinuric patients were expressed heterologously in E. coli and their enzyme activities were assayed in vitro. Additionally, mutant CBS proteins were visualized by Western blot from denaturing and non-denaturing polyacrylamide gels. The 14 mutations characterized were: p.R125W (c.373C>T), p.G148R (c.442G>A), p.M173V (c.517A>G), p.T191M (c.572C>T), p.A226T (c.676G>A), p.C275Y (c.824G>A), p.R336C (c.1006C>T), p.R336H (c.1007G>A), p.L338P (c.1013T>C), p.S349N (c.1046G>A), p.R379Q (c.1136G>A), p.L456P (c.1367T>C), p.G522fsX540 (c.1566delG), and p.R548Q (c.1643G>A). Eleven of the mutant alleles exhibited an activity lower than 4% of the wild-type protein. In contrast, mutations p.A226T and p.M173V presented 20% and 40% of the wild-type activity, respectively, whereas the activity of p.R548Q was up to 60% of the wild-type. This suggests that it is a new rare variant rather than a pathogenic mutation. Most of the mutated proteins exhibited a decreased signal in Western blot analyses. The non-denaturing PAGE revealed that the wild-type protein retained the capacity to form a multimeric quaternary structure, whereas in the mutations p.M173V, p.A226T, and p.G548Q, this structure grade was dramatically reduced and was completely absent in the rest of the mutations.