Emmanouil Kanavakis

Diagnostic exome sequencing to elucidate the genetic basis of likely recessive disorders in consanguineous families.

Rare, atypical, and undiagnosed autosomal‐recessive disorders frequently occur in the offspring of consanguineous couples. Current routine diagnostic genetic tests fail to establish a diagnosis in many cases. We employed exome sequencing to identify the underlying molecular defects in patients with unresolved but putatively autosomal‐recessive disorders in consanguineous families and postulated that the pathogenic variants would reside within homozygous regions. Fifty consanguineous families participated in the study, with a wide spectrum of clinical phenotypes suggestive of autosomal‐recessive inheritance, but with no definitive molecular diagnosis. DNA samples from the patient(s), unaffected sibling(s), and the parents were genotyped with a 720K SNP array. Exome sequencing and array CGH (comparative genomic hybridization) were then performed on one affected individual per family. High‐confidence pathogenic variants were found in homozygosity in known disease‐causing genes in 18 families (36%) (one by array CGH and 17 by exome sequencing), accounting for the clinical phenotype in whole or in part. In the remainder of the families, no causative variant in a known pathogenic gene was identified. Our study shows that exome sequencing, in addition to being a powerful diagnostic tool, promises to rapidly expand our knowledge of rare genetic Mendelian disorders and can be used to establish more detailed causative links between mutant genotypes and clinical phenotypes.

p53 codon 72 Pro homozygosity increases the risk of cutaneous melanoma in individuals with dark skin complexion and among noncarriers of melanocortin 1 receptor red hair variants

Background  p53 has a common polymorphism at amino acid 72, encoding either arginine or proline. p53Arg and p53Pro exhibit differences in various biological activities, such as cell‐cycle arrest and induction of apoptosis. Numerous epidemiological studies have examined the role of this polymorphism in several human malignancies, including cutaneous cancers, with contradictory results.

Association of MMP-1 -1607 1G/2G (rs1799750) polymorphism with primary knee osteoarthritis in the Greek population

Osteoarthritis is the most common form of arthritis with still unknown pathogenic etiology and considerable contribution of genetic factors. One of the mechanisms of cartilage degradation in osteoarthritis is enzymatic proteolysis of the extracellular matrix by metalloproteinases. MMP‐1, produced by chondrocytes and synovial cells, is a major proteinase of the MMPs family. The present study aims at evaluating the association of MMP1 gene ‐1607 1G/2G (rs1799750) polymorphism with primary knee osteoarthritis in the Greek population. One hundred fifty five patients with primary symptomatic knee osteoarthritis participated in the study along with 139 controls. Genotypes were determined using PCR—RLFP technique. Allelic and genotypic frequencies were compared between both study groups. There was no significant association between MMP1 ‐1607 1G/2G polymorphism and knee osteoarthritis, in crude analysis; however, after multiple logistic regression analysis, 1G/2G was associated with reduced odds of knee osteoarthritis by 75% in males, compared to genotypes 1G/1G + 2G/2G, adjusting for age and BMI (adjusted OR: 0.25, 95% CI: 0.069, 0.910, p = 0.035). The present study shows that MMP1 ‐1607 1G/2G (rs1799750) polymorphism might be a risk factor for knee osteoarthritis susceptibility in the Greek population. Further investigations are needed to confirm this association in the pathogenesis of osteoarthritis.

Application of high-resolution array comparative genomic hybridization in children with unknown syndromic microcephaly

BackroundMicrocephaly can either be isolated or it may coexist with other neurological entities and/or multiple congenital anomalies, known as syndromic microcephaly. Although many syndromic cases can be classified based on the characteristic phenotype, some others remain uncertain and require further investigation. The present study describes the application of array-comparative genomic hybridization (array-CGH) as a diagnostic tool for the study of patients with clinically unknown syndromic microcephaly.MethodsFrom a cohort of 210 unrelated patients referred with syndromic microcephaly, we applied array-CGH analysis in 53 undiagnosed cases. In all the 53 cases except one, previous standard karyotype was negative. High-resolution 4 × 180K and 1 × 244K Agilent arrays were used in this study.ResultsIn 25 out of the 53 patients with microcephaly among other phenotypic anomalies, array-CGH revealed copy number variations (CNVs) ranging in size between 15 kb and 31.6 Mb. The identified CNVs were definitely causal for microcephaly in 11/53, probably causal in 7/53, and not causal for microcephaly in 7/53 patients. Genes potentially contributing to brain deficit were revealed in 16/53 patients.ConclusionsArray-CGH contributes to the elucidation of undefined syndromic microcephalic cases by permitting the discovery of novel microdeletions and/or microduplications. It also allows a more precise genotype–phenotype correlation by the accurate definition of the breakpoints in the deleted/duplicated regions.

412 Combination of Genomic Technologies and Consanguinity in Order to Identify Pathogenic Variants in Recessive Disorders

Consanguinity and inbreeding increase the sharing of alleles among individuals; thus a considerable number of autosomal recessive phenotypes occur in offspring(s) of consanguineous couples. We have collected samples from consanguineous families with different phenotypes of unknown etiology that are compatible with autosomal recessive transmission, in order to identify the responsible functional genomic variation. 42 families of different ethnic background have been collected so far. From each family, DNA from the patient(s), unaffected siblings and the parents is extracted. Samples are i/analyzed by array-CGH for the detection of homozygous deletions; ii/genotyped with a 720K SNP-array in order to identify Runs of Homozygosity and the areas of the genome that could include the causative variant; iii/exome sequenced (one affected individual/family). Mean coverage is 130x and 98.2% of the coding region of RefSeq is covered at least 8x. By comparing the genotyping and sequencing data, we found that Single Nucleotide Variants (that passed the quality threshold) were detected with a specificity of 99.95%, sensitivity of 97.7%, Positive Predictive Value 99.2% and Negative Predictive Value 98.6%. On average we identified 21901 variants/exome. So far we analysed 26 families and identified the causative variation in known genes in 3 of them:VLDLR, FKTN and DMP1. In 12 families 23 candidate genes/variants have been identified(more than 1 candidate genes/family). In 11 families the likely molecular defect has not been identified. Consanguineous families provide an opportunity to identify pathogenic variants responsible for recessive phenotypes and rapidly fill in the gap between genotype and phenotype.

Molecular investigation of Angelman syndrome in Greece. Screening for UBE3A mutations: preliminary results

Background Angelman syndrome (AS) is a severe neurodevelopmental disorder characterized by mental retardation, absence of speech, ataxia, seizures and hyperactivity. Individuals with AS lack a normal active maternal copy of the UBE3A gene, encoding ubiquitin protein ligase (E6AP). In 80% of patients the clinical diagnosis is verified by molecular detection of one of the typical 15q11-q13 abnormalities, including chromosomal deletions (70%), paternal uniparental disomy (35%) or imprinting centre mutations (7%). Heterozygous lossoffunction mutations of E6AP have been identified in approximately 8% of cases. UBE3A gene is imprinted in human brain, with the paternal allele being normally silenced. E6AP is a member of E3 ubiquitin ligase protein family which plays a role in defining substrate specificity of the ubiquitinproteasome degradation system. The exact mechanism by which the defective E6-AP gene causes AS remains unknown. Clinical findings seem to be due to failure to degrade various proteins, accumulation of which may be harmful for an individual.