Consanguinity studies and genome research in Mediterranean developing countries

Purpose: Classical studies of consanguinity have taken advantage of the relationship between the gene frequency for a rare autosomal recessive disorder (q) and the proportion of offspring of consanguineous couples who are affected with the same disorder. The Swedish geneticist Gunnar Dahlberg provided the first theoretical formulation of the inverse correlation between q and the increase in frequency of consanguineous marriages among parents of affected children with respect to marriages of the same degree in the general population. Today it is possible to develop a new approach for estimating q using mutation analysis of affected offspring of consanguineous couples. The rationale of this new approach is based on the possibility that the child born of consanguineous parents carries the same mutation in double copy (true homozygosity) or alternatively carries two different mutations in the same gene (compound heterozygosity). In the latter case the two mutations must have been inherited through two different ancestors of the consanguineous parents (in this case the two mutated alleles are not ‘identical by descent’). Patients and methods: Data from the offspring of consanguineous marriages affected with different autosomal recessive disorders were collected by different molecular diagnostic laboratories in Mediterranean countries and in particular in Arab countries, where the frequencies of consanguineous marriages is high, show the validity of this approach. Results: The proportion of compound heterozygotes among children affected with a given autosomal recessive disorder, born of consanguineous parents, can be taken as an indirect indicator of the frequency of the same disorder in the general population. Identification of the responsible gene (and mutations) is the necessary condition to apply this method. Conclusion: The following paper from our group relevant for the present review is being published: Alessandro Gialluisi, Tommaso Pippucci, Yair Anikster, Ugur Ozbek, Myrna Medlej-Hashim, Andre Megarbane and Giovanni Romeo: Estimating the allele frequency of autosomal recessive disorders through mutational records and consanguinity: the homozygosity index (HI) annals of human genetics (in press; acceptance date 1 November 2011) In addition, our experimental data show that the causative mutation for a rare autosomal recessive disorder can be identified by whole exome sequencing of only two affected children of first cousins parents, as described in the following recent paper: Pippucci T, Benelli M, Magi A, Martelli PL, Magini P, Torricelli F, Casadio R, Seri M, Romeo G EX-HOM (EXome HOMozygosity): A Proof of Principle. Hum Hered 2011; 72:45-53

Stress-induced changes to the flora in a geothermal field in central Italy

The vegetation profile and the photosynthetic efficiency, oxidative damage, and stomatal conductance in the evergreen dwarf shrub Calluna vulgaris (L.). Hull was analyzed in a Mediterranean ecosystem characterized by intense geothermal activity. Among the higher plants present in the area, this species appears to be the sole to possess the ability to grow near the geothermal sources. The hot fluid springs strongly alter the environment in their proximity: the emitted water vapor, CO2, H2S, CH4, H3BO3, SO42-, and NH4+ partly condensate and precipitate to the soil, thus leading to its extreme acidification and nutrient depletion. Furthermore, the temperature starts to rise sharply just a few centimeters under the soil surface. Under this multiple stress, the individuals of C. vulgaris growing within a few meters from the springs showed lower photosystem II efficiency, higher oxidative damage to the biomembranes, and lower stomatal conductance than the individuals growing farther away. Drought and high air temperatures occurring in summer exacerbate these harsh conditions, but only the plants closer to the springs did undergo an acute, yet transient crisis, as shown by the analyzed parameters. These results suggest that the main factors of stress are related to the physical and chemical features of the soil, while the adverse climate conditions apparently are of secondary importance. The possible role of reduced stomatal conductivity in enhancing the resistance of C. vulgaris to this hostile environment is discussed

A Novel null homozygous mutation confirms <i>CACNA2D2</i> as a gene mutated in epileptic encephalopathy

Contribution to epileptic encephalopathy (EE) of mutations in CACNA2D2, encoding α2δ-2 subunit of Voltage Dependent Calcium Channels, is unclear. To date only one CACNA2D2 mutation altering channel functionality has been identified in a single family. In the same family, a rare CELSR3 polymorphism also segregated with disease. Involvement of CACNA2D2 in EE is therefore not confirmed, while that of CELSR3 is questionable. In a patient with epilepsy, dyskinesia, cerebellar atrophy, psychomotor delay and dysmorphic features, offspring to consanguineous parents, we performed whole exome sequencing (WES) for homozygosity mapping and mutation detection. WES identified extended autozygosity on chromosome 3, containing two novel homozygous candidate mutations: c.1295delA (p.Asn432fs) in CACNA2D2 and c.G6407A (p.Gly2136Asp) in CELSR3. Gene prioritization pointed to CACNA2D2 as the most prominent candidate gene. The WES finding in CACNA2D2 resulted to be statistically significant (p = 0.032), unlike that in CELSR3. CACNA2D2 homozygous c.1295delA essentially abolished α2δ-2 expression. In summary, we identified a novel null CACNA2D2 mutation associated to a clinical phenotype strikingly similar to the Cacna2d2 null mouse model. Molecular and statistical analyses together argued in favor of a causal contribution of CACNA2D2 mutations to EE, while suggested that finding in CELSR3, although potentially damaging, is likely incidental

Enhanced copy number variants detection from whole-exome sequencing data using EXCAVATOR2

Copy Number Variants (CNVs) are structural rear- rangements contributing to phenotypic variation that have been proved to be associated with many dis- ease states. Over the last years, the identification of CNVs from whole-exome sequencing (WES) data has become a common practice for research and clinical purpose and, consequently, the demand for more and more efficient and accurate methods has increased. In this paper, we demonstrate that more than 30% of WES data map outside the targeted re- gions and that these reads, usually discarded, can be exploited to enhance the identification of CNVs from WES experiments. Here, we present EXCAVATOR2, the first read count based tool that exploits all the reads produced by WES experiments to detect CNVs with a genome-wide resolution. To evaluate the per- formance of our novel tool we use it for analysing two WES data sets, a population data set sequenced by the 1000 Genomes Project and a tumor data set made of bladder cancer samples. The results obtained from these analyses demonstrate that EXCAVATOR2 out- performs other four state-of-the-art methods and that our combined approach enlarge the spectrum of detectable CNVs from WES data with an unprece- dented resolution

EX-HOM (EXome HOMozygosity): A Proof of Principle

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Ultra-Rare Variants Identify Biological Pathways and Candidate Genes in the Pathobiology of Non-Syndromic Cleft Palate Only

: In recent decades, many efforts have been made to elucidate the genetic causes of non-syndromic cleft palate (nsCPO), a complex congenital disease caused by the interaction of several genetic and environmental factors. Since genome-wide association studies have evidenced a minor contribution of common polymorphisms in nsCPO inheritance, we used whole exome sequencing data to explore the role of ultra-rare variants in this study. In a cohort of 35 nsCPO cases and 38 controls, we performed a gene set enrichment analysis (GSEA) and a hypergeometric test for assessing significant overlap between genes implicated in nsCPO pathobiology and genes enriched in ultra-rare variants in our cohort. GSEA highlighted an enrichment of ultra-rare variants in genes principally belonging to cytoskeletal protein binding pathway (Probability Density Function corrected p-value = 1.57 × 10-4); protein-containing complex binding pathway (p-value = 1.06 × 10-2); cell adhesion molecule binding pathway (p-value = 1.24 × 10-2); ECM-receptor interaction pathway (p-value = 1.69 × 10-2); and in the Integrin signaling pathway (p-value = 1.28 × 10-2). Two genes implicated in nsCPO pathobiology, namely COL2A1 and GLI3, ranked among the genes (n = 34) with nominal enrichment in the ultra-rare variant collapsing analysis (Fisher's exact test p-value &lt; 0.05). These genes were also part of an independent list of genes highly relevant to nsCPO biology (n = 25). Significant overlap between the two sets of genes (hypergeometric test p-value = 5.86 × 10-3) indicated that enriched genes are likely to be implicated in physiological palate development and/or the pathological processes of oral clefting. In conclusion, ultra-rare variants collectively impinge on biological pathways crucial to nsCPO pathobiology and point to candidate genes that may contribute to the individual risk of disease. Sequencing can be an effective approach to identify candidate genes and pathways for nsCPO

Characterization and identification of hidden rare variants in the human genome

BackgroundBy examining the genotype calls generated by the 1000 Genomes Project we discovered that the human reference genome GRCh37 contains almost 20,000 loci in which the reference allele has never been observed in healthy individuals and around 70,000 loci in which it has been observed only in the heterozygous state.ResultsWe show that a large fraction of this rare reference allele (RRA) loci belongs to coding, functional and regulatory elements of the genome and could be linked to rare Mendelian disorders as well as cancer. We also demonstrate that classical germline and somatic variant calling tools are not capable to recognize the rare allele when present in these loci. To overcome such limitations, we developed a novel tool, named RAREVATOR, that is able to identify and call the rare allele in these genomic positions. By using a small cancer dataset we compared our tool with two state-of-the-art callers and we found that RAREVATOR identified more than 1,500 germline and 22 somatic RRA variants missed by the two methods and which belong to significantly mutated pathways.ConclusionsThese results show that, to date, the investigation of around 100,000 loci of the human genome has been missed by re-sequencing experiments based on the GRCh37 assembly and that our tool can fill the gap left by other methods. Moreover, the investigation of the latest version of the human reference genome, GRCh38, showed that although the GRC corrected almost all insertions and a small part of SNVs and deletions, a large number of functionally relevant RRAs still remain unchanged. For this reason, also future resequencing experiments, based on GRCh38, will benefit from RAREVATOR analysis results. RAREVATOR is freely available at http://sourceforge.net/projects/rarevator

Is Focal Cortical Dysplasia/Epilepsy Caused by Somatic MTOR Mutations Always a Unilateral Disorder?

To alert about the wide margin of unpredictability that distribution of somatic MTOR mosaicism may have in the brain and the risk for independent epileptogenesis arising from the seemingly healthy contralateral hemisphere after complete removal of epileptogenic focal cortical dysplasia (FCD)