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

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 < 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

Epilepsy with auditory features: A heterogeneous clinico-molecular disease

Objective: To identify novel genes implicated in epilepsy with auditory features (EAF) in phenotypically heterogeneous families with unknown molecular basis. Methods: We identified 15 probands with EAF in whom an LGI1 mutation had been excluded. We performed electroclinical phenotyping on all probands and available affected relatives. We used whole-exome sequencing (WES) in 20 individuals with EAF (including all the probands and 5 relatives) to identify single nucleotide variants, small insertions/deletions, and copy number variants. Results: WES revealed likely pathogenic variants in genes that had not been previously associated with EAF: a CNTNAP2 intragenic deletion, 2 truncating mutations of DEPDC5, and a missense SCN1A change. Conclusions: EAF is a clinically and molecularly heterogeneous disease. The association of EAF with CNTNAP2, DEPDC5, and SCN1A mutations widens the phenotypic spectrum related to these genes. CNTNAP2 encodes CASPR2, a member of the voltage-gated potassium channel complex in which LGI1 plays a role. The finding of a CNTNAP2 deletion emphasizes the importance of this complex in EAF and shows biological convergence

PRIMA1 mutation: A new cause of nocturnal frontal lobe epilepsy

Objective Nocturnal frontal lobe epilepsy (NFLE) can be sporadic or autosomal dominant; some families have nicotinic acetylcholine receptor subunit mutations. We report a novel autosomal recessive phenotype in a single family and identify the causative gene. Methods Whole exome sequencing data was used to map the family, thereby narrowing exome search space, and then to identify the mutation. Results Linkage analysis using exome sequence data from two affected and two unaffected subjects showed homozygous linkage peaks on chromosomes 7, 8, 13, and 14 with maximum LOD scores between 1.5 and 1.93. Exome variant filtering under these peaks revealed that the affected siblings were homozygous for a novel splice site mutation (c.93+2T>C) in the PRIMA1 gene on chromosome 14. No additional PRIMA1 mutations were found in 300 other NFLE cases. The c.93+2T>C mutation was shown to lead to skipping of the first coding exon of the PRIMA1 mRNA using a minigene system. Interpretation PRIMA1 is a transmembrane protein that anchors acetylcholinesterase (AChE), an enzyme hydrolyzing acetycholine, to membrane rafts of neurons. PRiMA knockout mice have reduction of AChE and accumulation of acetylcholine at the synapse; our minigene analysis suggests that the c.93+2T>C mutation leads to knockout of PRIMA1. Mutations with gain of function effects in acetylcholine receptor subunits cause autosomal dominant NFLE. Thus, enhanced cholinergic responses are the likely cause of the severe NFLE and intellectual disability segregating in this family, representing the first recessive case to be reported and the first PRIMA1 mutation implicated in disease

Resources and tools for rare disease variant interpretation

Collectively, rare genetic disorders affect a substantial portion of the world’s population. In most cases, those affected face difficulties in receiving a clinical diagnosis and genetic characterization. The understanding of the molecular mechanisms of these diseases and the development of therapeutic treatments for patients are also challenging. However, the application of recent advancements in genome sequencing/analysis technologies and computer-aided tools for predicting phenotype-genotype associations can bring significant benefits to this field. In this review, we highlight the most relevant online resources and computational tools for genome interpretation that can enhance the diagnosis, clinical management, and development of treatments for rare disorders. Our focus is on resources for interpreting single nucleotide variants. Additionally, we present use cases for interpreting genetic variants in clinical settings and review the limitations of these results and prediction tools. Finally, we have compiled a curated set of core resources and tools for analyzing rare disease genomes. Such resources and tools can be utilized to develop standardized protocols that will enhance the accuracy and effectiveness of rare disease diagnosis

Structural Variants at the LMNB1 Locus: Deciphering Pathomechanisms in Autosomal Dominant Adult-Onset Demyelinating Leukodystrophy

Objectives: We aimed to elucidate the pathogenic mechanisms underlying autosomal dominant adult-onset demyelinating leukodystrophy (ADLD), and to understand the genotype/phenotype correlation of structural variants (SVs) in the LMNB1 locus.Background: Since the discovery of 3D genome architectures and topologically associating domains (TADs), new pathomechanisms have been postulated for SVs, regardless of gene dosage changes. ADLD is a rare genetic disease associated with duplications (classical ADLD) or noncoding deletions (atypical ADLD) in the LMNB1 locus.Methods: High-throughput chromosome conformation capture, RNA sequencing, histopathological analyses of postmortem brain tissues, and clinical and neuroradiological investigations were performed.Results: We collected data from >20 families worldwide carrying SVs in the LMNB1 locus and reported strong clinical variability, even among patients carrying duplications of the entire LMNB1 gene, ranging from classical and atypical ADLD to asymptomatic carriers. We showed that patients with classic ADLD always carried intra-TAD duplications, resulting in a simple gene dose gain. Atypical ADLD was caused by LMNB1 forebrain-specific misexpression due to inter-TAD deletions or duplications. The inter-TAD duplication, which extends centromerically and crosses the 2 TAD boundaries, did not cause ADLD. Our results provide evidence that astrocytes are key players in ADLD pathology.Interpretation: Our study sheds light on the 3D genome and TAD structural changes associated with SVs in the LMNB1 locus, and shows that a duplication encompassing LMNB1 is not sufficient per se to diagnose ADLD, thereby strongly affecting genetic counseling. Our study supports breaking TADs as an emerging pathogenic mechanism that should be considered when studying brain diseases. ANN NEUROL 2024.<br

Structural Variants at the LMNB1 Locus: Deciphering Pathomechanisms in Autosomal Dominant Adult-Onset Demyelinating Leukodystrophy

Objectives: We aimed to elucidate the pathogenic mechanisms underlying autosomal dominant adult-onset demyelinating leukodystrophy (ADLD), and to understand the genotype/phenotype correlation of structural variants (SVs) in the LMNB1 locus. Background: Since the discovery of 3D genome architectures and topologically associating domains (TADs), new pathomechanisms have been postulated for SVs, regardless of gene dosage changes. ADLD is a rare genetic disease associated with duplications (classical ADLD) or noncoding deletions (atypical ADLD) in the LMNB1 locus. Methods: High-throughput chromosome conformation capture, RNA sequencing, histopathological analyses of postmortem brain tissues, and clinical and neuroradiological investigations were performed. Results: We collected data from &gt;20 families worldwide carrying SVs in the LMNB1 locus and reported strong clinical variability, even among patients carrying duplications of the entire LMNB1 gene, ranging from classical and atypical ADLD to asymptomatic carriers. We showed that patients with classic ADLD always carried intra-TAD duplications, resulting in a simple gene dose gain. Atypical ADLD was caused by LMNB1 forebrain-specific misexpression due to inter-TAD deletions or duplications. The inter-TAD duplication, which extends centromerically and crosses the 2 TAD boundaries, did not cause ADLD. Our results provide evidence that astrocytes are key players in ADLD pathology. Interpretation: Our study sheds light on the 3D genome and TAD structural changes associated with SVs in the LMNB1 locus, and shows that a duplication encompassing LMNB1 is not sufficient per se to diagnose ADLD, thereby strongly affecting genetic counseling. Our study supports breaking TADs as an emerging pathogenic mechanism that should be considered when studying brain diseases. ANN NEUROL 202

Defective lipid signalling caused by mutations in PIK3C2B underlies focal epilepsy

Epilepsy is one of the most frequent neurological diseases, with focal epilepsy accounting for the largest number of cases. The genetic alterations involved in focal epilepsy are far from being fully elucidated. Here, we show that defective lipid signalling caused by heterozygous ultra-rare variants in PIK3C2B, encoding for the class II phosphatidylinositol 3-kinase PI3K-C2β, underlie focal epilepsy in humans. We demonstrate that patients' variants act as loss-of-function alleles, leading to impaired synthesis of the rare signalling lipid phosphatidylinositol 3,4-bisphosphate, resulting in mTORC1 hyperactivation. In vivo, mutant Pik3c2b alleles caused dose-dependent neuronal hyperexcitability and increased seizure susceptibility, indicating haploinsufficiency as a key driver of disease. Moreover, acute mTORC1 inhibition in mutant mice prevented experimentally induced seizures, providing a potential therapeutic option for a selective group of patients with focal epilepsy. Our findings reveal an unexpected role for class II PI3K-mediated lipid signalling in regulating mTORC1-dependent neuronal excitability in mice and humans