Case Report: Precision genetic diagnosis in a case of Dyggve-Melchior-Clausen syndrome reveals paternal isodisomy and heterodisomy of chromosome 18 with imprinting clinical implications.

A twelve-year-old patient with a previous clinical diagnosis of spondylocostal skeletal dysplasia and moderate intellectual disability was genetically analyzed through next generation sequencing of a targeted gene panel of 179 genes associated to skeletal dysplasia and mucopolysaccharidosis in order to stablish a precision diagnosis. A homozygous nonsense [c.62C>G; p.(Ser21Ter)] mutation in DYM gene was identified in the patient. Null mutations in DYM have been associated to Dyggve-Melchior-Clausen syndrome, which is a rare autosomal-recessive disorder characterized by skeletal dysplasia and mental retardation, compatible with the patient´s phenotype. To confirm the pathogenicity of this mutation, a segregation analysis was carried out, revealing that the mutation p(Ser21Ter) was solely inherited from the father, who is a carrier of the mutation, while the mother does not carry the mutation. With the suspicion that a paternal disomy could be causing the disease, a series of microsatellite markers in chromosome 18, where the DYM gene is harbored, was analyzed in all the members of the family. Haplotype analysis provided strong evidence of paternal isodisomy and heterodisomy in that chromosome, confirming the pathological effect of this mutation. Furthermore, the patient may have a compromised expression of the ELOA3 gene due to modifications in the genomic imprinting that may potentially increase the risk of digestive cancer. All these results highlight the importance of obtaining a precision diagnosis in rare diseases.

Brief Report: Evidence of Autism Spectrum Disorder Caused by a Mutation in ATRX Gene: A Case Report.

ATRX mutations are commonly associated with alpha-thalassaemia mental retardation syndrome (ATR-X syndrome) with a notable variable expressivity. This X-linked disorder is characterized by intellectual disability (ID) in a higher or lesser degree, in which the alpha-thalassaemia feature is not always present. Other phenotypic manifestations like facial dimorphism, hypotonia, microcephaly, skeletal abnormalities or urogenital malformations have been frequently observed in ATR-X syndrome. Herein, we report a missense ATRX mutation (Thr1621Met) in a patient with an autism spectrum disorder (ASD) diagnosis. Except for ID, no typical signs of ATR-X syndrome were found in the patient. These results confirm the extensive phenotypic variability associated to ATRX mutations and show the involvement of this gene in the ASD.

SWATH-MS Quantitative Proteomic Analysis of Deer Antler from Two Regenerating and Mineralizing Sections.

Antlers are the only organ in the mammalian body that regenerates each year. They can reach growth rates of 1-3 cm/day in length and create more than 20 cm2/day of skin in the antler tips (their growth centers). Previous proteomic studies regarding antlers have focused on antler growth centers (tips) compared to the standard bone to detect the proteins involved in tissue growth. However, proteins of cell differentiation and regeneration will be more accurately detected considering more growing tissues. Thus, we set out to compare proteins expressed in antler tips (the highest metabolism rate and cell differentiation) vs. middle sections (moderate cell growth involving bone calcification), using ribs as controls. Samples were obtained in mid-June with antlers' phenology corresponding to the middle of their growth period. Quantitative proteomic analysis identified 259 differentially abundant proteins mainly associated with antioxidant metabolic mechanisms, protein formation and Wnt signalling pathway, meanwhile, the mid antler section was linked to blood proteins. The high metabolic rate and subsequent risk of oxidative stress also seem to have resulted in strong antioxidant mechanisms. These results suggest that redox regulation of proteins is a key factor in the model of deer antler regeneration.

Pedf derived peptides affect colorectal cancer cell lines resistance and tumour re-growth capacity.

Relapse after chemotherapy treatment depends on the cancer initiating cells (CICs). PEDF (Pigmented Epithelium Derived Factor) is an anti-angiogenic, neurotrophic and self-renewal regulator molecule, also involved in CICs biology. Acute and chronic exposition of colon cancer cell lines to CT/CTE PEDF-derived peptides decreased drug-resistance to conventional colorectal cancer treatments, such as oxaliplatin or irinotecan. We confirmed a reduction in the irinotecan and oxaliplatin IC50 doses for all tested tumour cell lines. After xenograft transplantation, CT/CTE treatments also produced a reduction in resistance to conventional chemotherapy treatments as in culture-assays. Metastatic capacity of these treated cell lines was also depleted. The PEDF signaling pathway could be a future therapeutic tool for use as an adjuvant therapy that decreases IC50 dosis, adverse effects and treatment costs. This pathway could also be involved in an increase of the time relapse in patients, decreased tumourigenicity, and decreased capacity to produce metastasis.

Genotype-phenotype analysis of Bietti crystalline dystrophy in a family with the CYP4V2 Ile111Thr mutation.

PURPOSE: The purposes of this study were to evaluate the genotypic and phenotypic correlations of Bietti crystalline dystrophy and to investigate the utility of in vivo corneal confocal microscopy in diagnosing this disorder. METHODS: A Spanish woman (proband) with a clinical diagnosis of Bietti crystalline dystrophy and 7 members of her family were recruited prospectively for complete clinical ophthalmic examination and genetic study. The medical records of an additional family member were reviewed retrospectively. Genomic DNA was obtained from blood samples, and 11 exons of the CYP4V2 gene were screened for mutations by polymerase chain reaction DNA sequencing. RESULTS: Clinical examination revealed an atypical pattern of corneal dystrophy with central and paracentral distribution not only in the proband but also in 2 elderly heterozygous carriers. Corneal deposits were observed by slit-lamp examination and in vivo corneal confocal microscopy. Genetic analysis revealed the homozygous CYP4V2 Ile111Thr mutation in the proband and identified 5 heterozygous carriers. CONCLUSIONS: The authors identified a case of Bietti crystalline dystrophy with central and paracentral keratopathy and the molecular analysis of the causative gene in a Spanish family. Data suggest a dose-dependent phenotype ranging from subclinical corneal changes in subjects carrying 1 mutant Ile111Thr CYP4V2 allele to the complete manifestation of the disease in homozygous subjects. In vivo corneal confocal microscopy is a useful technique in the diagnosis of this disorder.

Null CYP1B1 genotypes in primary congenital and nondominant juvenile glaucoma.

PURPOSE: To assess the mutation spectrum, enzymatic activity, and phenotypic features associated with CYP1B1 genotypes in primary congenital glaucoma (PCG) and nondominant juvenile glaucoma (ndJG). DESIGN: CYP1B1 genotyping, segregation analysis, and functional evaluation of mutations in a cohort of patients. PARTICIPANTS: A total of 177 probands clinically diagnosed with PCG (161) or ndJG (16). METHODS: Automatic DNA sequencing of the promoter (-1 to -867) and the 3 CYP1B1 exons. CYP1B1 enzymatic activity was evaluated using an ethoxyresorufin O-deethylation assay in transfected HEK-293T cells. MAIN OUTCOME MEASURES: Screening and functional evaluation of CYP1B1 mutations. Glaucoma diagnosis based on slit-lamp examination, measurement of intraocular pressure, gonioscopy, and fundus examination. RESULTS: Thirty-one different mutations were identified in 56 PCG and 7 ndJG index cases. To the best of our knowledge, 3 of the identified mutations were novel (-337G>T, F123L, and I399_P400del). Approximately 56% of all mutation carriers were compound heterozygotes, 25% were homozygotes, and both groups inherited glaucoma as an autosomal recessive trait. Nineteen percent of carriers were heterozygotes and showed non-Mendelian segregation. In vitro and inferred functional analysis showed that no less than approximately 74% of the recessive genotypes result in null enzymatic activity. We detected variable expressivity in relation to age of onset and a possible case of incomplete penetrance in 3 of 6 families (50%), with more than 1 affected child or more than 1 subject carrying 2 CYP1B1 mutant alleles. Altogether, these data support that PCG is not a simple monogenic disease. In addition, most patients with PCG carrying null or putative null genotypes showed severe bilateral phenotypes featured by early disease onset, frequently at birth. The mean number of trabeculectomies per eye was significantly higher in carriers than in noncarriers. CONCLUSIONS: This is the largest analysis of CYP1B1 mutations performed in European patients with PCG to date. Our data show that null CYP1B1 genotypes, and therefore complete absence of CYP1B1 activity, frequently lead to severe phenotypes. Our results support that CYP1B1 glaucoma is not a simple monogenic disease and that CYP1B1 activity levels could influence the phenotype.

Role of CYP1B1 gene polymorphisms in bladder cancer susceptibility.

PURPOSE: CYP1B1 activates procarcinogens in some human tissues, including the urinary tract. Changes related to genetic polymorphisms are a known risk factor for cancer. We analyzed the association between CYP1B1 sequence variations and bladder cancer. MATERIALS AND METHODS: Sequence variations in the coding region (exons 2 and 3) and the neighboring introns of CYP1B1 were analyzed by direct polymerase chain reaction and DNA sequencing in 208 unrelated patients with bladder cancer and 208 healthy controls. RESULTS: We identified 6 known single nucleotide polymorphisms organized into 2 linkage disequilibrium blocks. The Ala/Ala and Leu/Val genotypes of the Ala119Ser and Leu432Val polymorphisms were significantly more common in patients than in controls (55.3% vs 42.8% and 54.8% vs 42.3%, respectively). The strongest individual single nucleotide polymorphism risk was found under an over dominant model for Leu432Val (OR 1.65, CI 95% 1.12-2.44). The 2 susceptibility single nucleotide polymorphisms were predicted to be structured into 4 haplotypes and more than 10 diplotypes. No individual haplotype was associated with bladder cancer but the diplotype Ala-Leu/Ala-Val was significantly overrepresented in cases compared to controls (31.73% vs 17.31%, OR 2.22, 95% CI 1.36-3.62, p = 0.001). The OR was approximately 1.6 for the individual genotypes Ala/Ala and Leu/Val, which increased to 2.2 for the Ala-Leu/Ala-Val diplotype. A risk occupation had a modifying effect, increasing the crude OR of the combined genotype Ala/Ala + Leu/Val from 2.2 to 8.3. CONCLUSIONS: This study provides strong evidence for the role of common CYP1B1 variants as risk factors for bladder cancer, which increases with occupational exposure.

WDR36 and P53 gene variants and susceptibility to primary open-angle glaucoma: analysis of gene-gene interactions.

PURPOSE: To investigate the role of WDR36 and P53 sequence variations in POAG susceptibility. METHODS: The authors performed a case-control genetic association study in 268 unrelated Spanish patients (POAG1) and 380 control subjects matched for sex, age, and ethnicity. WDR36 sequence variations were screened by either direct DNA sequencing or denaturing high-performance liquid chromatography. P53 polymorphisms p.R72P and c.97-147ins16bp were analyzed by single-nucleotide polymorphism (SNP) genotyping and PCR, respectively. Positive SNP and haplotype associations were reanalyzed in a second sample of 211 patients and in combined cases (n = 479). RESULTS: The authors identified almost 50 WDR36 sequence variations, of which approximately two-thirds were rare and one-third were polymorphisms. Approximately half the variants were novel. Eight patients (2.9%) carried rare mutations that were not identified in the control group (P = 0.001). Six Tag SNPs were expected to be structured in three common haplotypes. Haplotype H2 was consistently associated with the disease (P = 0.0024 in combined cases). According to a dominant model, genotypes containing allele P of the P53 p.R72P SNP slightly increased glaucoma risk. Glaucoma susceptibility associated with different WDR36 genotypes also increased significantly in combination with the P53 RP risk genotype, indicating the existence of a genetic interaction. For instance, the OR of the H2 diplotype estimated for POAG1 and combined cases rose approximately 1.6 times in the two-locus genotype H2/RP. CONCLUSIONS: Rare WDR36 variants and the P53 p.R72P polymorphism behaved as moderate glaucoma risk factors in Spanish patients. The authors provide evidence for a genetic interaction between WDR36 and P53 variants in POAG susceptibility, although this finding must be confirmed in other populations.

CYP1B1 mutations in Spanish patients with primary congenital glaucoma: phenotypic and functional variability.

PURPOSE: To analyze the contributions of cytochrome P4501B1 (CYP1B1) mutations to primary congenital glaucoma (PCG) in Spanish patients. METHODS: We analyzed, by polymerase chain reaction (PCR) DNA sequencing, the presence of promoter (-1 to -867) and exon CYP1B1 mutations in 38 unrelated Spanish probands affected by PCG. Functional analysis of nine identified mutations was performed measuring ethoxyresorufin O-deethylation activity and CYP1B1 stability in transiently transfected human embryonic kidney 293T (HEK-293-T) cells. RESULTS: We found a total of 16 different mutations in 13 (34.2%) index cases. The identified mutations included nine missense and three nonsense nucleotide changes, three small deletions, and a short duplication. Eleven probands were compound heterozygotes and two were heterozygotes. Six of the identified mutations were novel (A106D, E173X, F261L, E262X, W341X, and P513_K514del). Mutations T404fsX30 and R355fsX69 were the most prevalent among index cases and were detected in six (23.0%) and three (11.5%) patients, respectively. Functional analysis showed that the three nonsense mutants assayed (E173X, E262X, and W341X) and F261L were null alleles. Of the remaining mutants, four (P52L, G61E, Y81N, and E229K) showed catalytic activities ranging from 20% to 40% of wild-type CYP1B1 and high protein instability. Mutation P400S showed normal catalytic activity and moderate instability. These five mutants were classified as hypomorphic alleles. Patients carrying two null alleles showed severe phenotypes featured by very early PCG onset usually at birth or in the first month of life (0.6+/-0.9 months). Incomplete penetrance was detected in patients carrying hypomorphic alleles. CONCLUSIONS: Our data indicate that approximately one-third of Spanish patients with PCG carry loss-of-function CYP1B1 and show that null alleles are associated with the most severe phenotypes. Hypomorphic alleles may contribute to some cases of incomplete penetrance.

MYOC gene mutations in Spanish patients with autosomal dominant primary open-angle glaucoma: a founder effect in southeast Spain.

PURPOSE: Primary open angle glaucoma (POAG) is a genetically heterogeneous disease resulting in optic disc cupping and visual impairment. It can be inherited as either a complex or a monogenic trait. Autosomal dominant POAG is the most frequent type of monogenic glaucoma. In this study, we investigated the role of myocilin MYOC in Spanish patients with autosomal dominant POAG. METHODS: We retrospectively analyzed the MYOC gene by PCR-DNA sequencing in five Southeast Spanish families and one Colombian family of Hispanic origin affected by autosomal dominant juvenile-onset open angle glaucoma (JOAG). We also analyzed two families with adult-onset POAG (AOAG). RESULTS: MYOC mutations D380A and P370L segregated with the disease in the five JOAG Spanish families and the Colombian family, respectively. Neither MYOC mutations nor cytochrome P4501B1 CYP1B1 mutations were detected in the AOAG families. The disease showed an insidious onset in D380A carriers, making early diagnosis difficult. A delay in diagnosis resulted in severe visual impairment. Topical medications were effective in controlling intraocular pressure (IOP) in D380A carriers, but 72.2% of them required surgery for long-term IOP control. Conversely, only 30% of AOAG patients required surgery. Mutation P370L was associated with a severe phenotype unresponsive to medical treatment. Analysis of the four MYOC-linked polymorphic microsatellite markers in the JOAG Spanish families revealed a common disease haplotype, indicating that the D380A mutation was inherited from the same founder. CONCLUSIONS: This is the first evidence of a founder effect for a MYOC mutation in Spanish JOAG patients. Analysis of the MYOC gene in Spanish patients with JOAG is useful to identify at-risk individuals thus help prevent visual impairment through early treatment.

Role of MYOC and OPTN sequence variations in Spanish patients with primary open-angle glaucoma.

PURPOSE: To retrospectively investigate the contribution of myocilin (MYOC) and optineurin (OPTN) sequence variations to adult-onset ocular hypertension (OHT) and primary open-angle glaucoma (POAG) in Spanish patients. METHODS: The promoter region and the three exons of MYOC were analyzed by direct PCR DNA sequencing in 40 OHT and 110 POAG unrelated patients. We used 98 subjects in whom OHT or glaucoma had been ruled out as controls. We also screened the complete coding region of the OPTN gene (exons 4-16) in all subjects by single-stranded conformational polymorphisms (SSCPs). RESULTS: We identified six common single nucleotide polymorphisms (SNPs) in the promoter region of MYOC (-1000C>G, -387C>T, -306G>A, -224T>C, -126T>C and -83G>A) and a polymorphic GT microsatellite (-339(GT)11-19). In addition, we detected four novel, rare DNA polymorphisms. None of these DNA sequence variations were associated with either OHT or POAG. We also found three (2.7%) POAG patients with MYOC pathogenic mutations. Two of these pathogenic mutations (Gln368Stop and Ala445Val) were previously described whereas the third (Tyr479His) was novel. Transient expression of the novel mutation in 293T cells supported its pathogenicity. Only two OPTN polymorphisms, which are not associated with the disease, were detected. CONCLUSIONS: Overall, our data show that in Spain a minority of adult-onset high-pressure POAG patients carry heterozygous disease-causing mutations in the MYOC gene and that OPTN is not involved in either OHT or POAG.

Heterozygous CYP1B1 gene mutations in Spanish patients with primary open-angle glaucoma.

PURPOSE: To investigate CYP1B1 gene mutations in Spanish patients with ocular hypertension (OHT) or primary open angle glaucoma (POAG). METHODS: The two coding exons of CYP1B1 were screened for sequence alterations by direct PCR DNA sequencing in 37 and 82 unrelated Spanish subjects diagnosed with OHT and POAG, respectively. As a control we used a group of 93 subjects from whom OHT or glaucoma were ruled out. RESULTS: We found three different predicted amino acid substitutions (Ala189Pro, Ala330Ser, and Ala443Gly) in three (8.1%) OHT subjects, and seven different mutations (Ser28Trp, Gly61Glu, Tyr81Asn, Gln144His, Arg145Trp, Glu229Lys, and Val409Phe) in nine (10.9%) glaucoma patients. These sequence variations showed higher frequencies in cases than in controls (as recently reported in French patients). They are predicted to produce a significant change in the amino acid sequence and affect conserved regions of the protein. All these missense mutations were found as heterozygots. In addition, four of them have been previously found in PCG and/or POAG patients, whereas the other six mutations (Ser28Trp, Gln144His, Arg145Trp, Ala189Pro, Ala330Ser, and Val409Phe) have not been previously described. Clinically, these mutations are associated with an age at diagnosis ranging from 12 to 58 years (mean 34.3 years) and from 48 to 77 years (mean 59.9 years) among OHT and glaucoma patients, respectively. CONCLUSIONS: Heterozygous CYP1B1 mutations could confer increased susceptibility to the development of POAG in the Spanish population.