An evolutionary history of the selectin gene cluster in humans.

Molecules involved in leukocyte trafficking have a central role in the development of inflammatory and immune responses. We performed F(ST) analysis of the selectin cluster, as well as of SELPLG, ICAM1 and VCAM1. Peaks of significantly high population genetic differentiation were restricted to two regions in SELP and one in SELPLG. Resequencing data indicated that the region covering SELP exons 11-13 displays high nucleotide diversity in Africans and Europeans (CEU), and a high level of within-species diversity compared with inter-specific divergence. Analysis of inferred haplotypes revealed a complex phylogeny with two deeply separated clades that coalesce at ~3.5 million years (MY) plus a minor clade with a TMRCA (time to the most recent common ancestor) of ~2.2 MY. A splicing assay indicated no haplotype-specific effect on SELP exon 14 inclusion. These data are consistent with a model of multiallelic balancing selection; single-nucleotide polymorphism analysis indicated that the Val640Leu variant represents a likely selection target. In populations of Asian ancestry a distinct haplotype, possibly carrying regulatory variants, has been driven to high frequency by positive selection. No deviation from neutrality was observed for the SELPLG region. Resequencing of SELP in chimpanzees revealed a haplotype phylogeny with extremely deep basal branches, suggesting either long-standing balancing selection or ancestral population structure. Thus, SELP has experienced a complex selective history, possibly as a result of local adaptation. Variants in the gene have been associated with autoimmune and cardiovascular diseases. Association studies would benefit from both taking the complex SELP haplotype structure into account and from analysis of possible regulatory variants in the gene.

A trans-specific polymorphism in ZC3HAV1 is maintained by long-standing balancing selection and may confer susceptibility to multiple sclerosis.

The human ZC3HAV1 gene encodes an antiviral protein. The longest splicing isoform of ZC3HAV1 contains a C-terminal PARP-like domain, which has evolved under positive selection in primates. We analyzed the evolutionary history of this same domain in humans and in Pan troglodytes. We identified two variants that segregate in both humans and chimpanzees; one of them (rs3735007) does not occur at a hypermutable site and accounts for a nonsynonymous substitution (Thr851Ile). The probability that the two trans-specific polymorphisms have occurred independently in the two lineages was estimated to be low (P = 0.0054), suggesting that at least one of them has arisen before speciation and has been maintained by selection. Population genetic analyses in humans indicated that the region surrounding the shared variants displays strong evidences of long-standing balancing selection. Selection signatures were also observed in a chimpanzee population sample. Inspection of 1000 Genomes data confirmed these findings but indicated that search for selection signatures using low-coverage whole-genome data may need masking of repetitive sequences. A case-control study of more than 1,000 individuals from mainland Italy indicated that the Thr851Ile SNP is significantly associated with susceptibility to multiple sclerosis (MS) (odds ratio [OR] = 1.47, 95% confidence intervals [CI]: 1.08-1.99, P = 0.011). This finding was confirmed in a larger sample of 4,416 Sardinians cases/controls (OR = 1.18, 95% CI: 1.037-1.344, P = 0.011), but not in a population from Belgium. We provide one of the first instances of human/chimpanzee trans-specific coding variant located outside the major histocompatibility complex region. The selective pressure is likely to be virus driven; in modern populations, this variant associates with susceptibility to MS, possibly via the interaction with environmental factors.

New molecular findings in congenital myopathies due to selenoprotein N gene mutations.

Selenoprotein N-related myopathy (SEPN1-RM) is an early-onset muscle disorder that can manifest clinically as congenital muscular dystrophy with spinal rigidity and can result in specific pathological entities such as multiminicore disease, desmin-related myopathy with Mallory body-like inclusions, and congenital fiber-type disproportion. Here we describe the clinical, histopathological, muscle magnetic resonance imaging (MRI) and genetic findings of three Italian SEPN1-RM families. Proband 1 is a 31-year-old female who was floppy at birth and developed axial and mild lower limb-girdle weakness. The second proband is a 13-year-old boy with RSMD1. Probands 3 and 4 were brothers showing clinical phenotype of congenital myopathy. Muscle MRI demonstrated selective involvement of sartorius, gluteal muscles and distal gastrocnemius and sparing of rectus femoris and gracilis. Muscle histopathology showed in proband 1 myopathic changes with mild connective tissue increase and some fibres lacking the Z-line, while probands 2 and 3 had multiminicores. SEPN1 gene analysis revealed five mutations, three of which are novel. Proband 1 was a compound heterozygote for a 92-bp (exon 1) and a 1-bp deletion (exon 9); proband 2 had a 99-bp deletion and a 10-bp duplication in exon 1, and proband 3 presented a novel homozygous mutation in intron 10 acceptor splice site.

Long-term balancing selection maintains trans-specific polymorphisms in the human TRIM5 gene.

The human TRIM5 genes encodes a retroviral restriction factor (TRIM5α). Evolutionary analyses of this gene in mammals have revealed a complex and multifaceted scenario, suggesting that TRIM5 has been the target of exceptionally strong selective pressures, possibly exerted by recurrent waves of retroviral infections. TRIM5 displays inter-individual expression variability in humans and high levels of TRIM5 mRNA have been associated with a reduced risk of HIV-1 infection. We resequenced TRIM5 in chimpanzees and identified two polymorphisms in intron 1 that are shared with humans. Analysis of the gene region encompassing the two trans-specific variants in human populations identified exceptional nucleotide diversity levels and an excess of polymorphism compared to fixed divergence. Most tests rejected the null hypothesis of neutral evolution for this region and haplotype analysis revealed the presence of two deeply separated clades. Calculation of the time to the most recent common ancestor (TMRCA) for TRIM5 haplotypes yielded estimates ranging between 4 and 7 million years. Overall, these data indicate that long-term balancing selection, an extremely rare process outside MHC genes, has maintained trans-specific polymorphisms in the first intron of TRIM5. Bioinformatic analyses indicated that variants in intron 1 may affect transcription factor-binding sites and, therefore, TRIM5 transcriptional activity. Data herein confirm an extremely complex evolutionary history of TRIM5 genes in primates and open the possibility that regulatory variants in the gene modulate the susceptibility to HIV-1.

A population genetics study of the familial Mediterranean fever gene: evidence of balancing selection under an overdominance regime.

Familial Mediterranean Fever (FMF) is a recessively inherited systemic autoinflammatory disease caused by mutations in the MEFV gene. The frequency of different disease alleles is extremely high in multiple populations from the Mediterranean region, suggesting heterozygote advantage. Here, we characterize the sequence variation and haplotype structure of the MEFV 3' gene region (from exon 5 to the 3' UTR) in seven human populations. In non-African populations, we observed high levels of nucleotide variation, an excess of intermediate-frequency alleles, reduced population differentiation and a genealogy with two common haplotypes separated by deep branches. These features are suggestive of balancing selection having acted on this region to maintain one or more selected alleles. In line with this finding, an excess of heterozygotes was observed in Europeans and Asians, suggesting an overdominance regime. Our data, together with the earlier demonstration that the MEFV exon 10 has been subjected to episodic positive selection over primate evolution, provide evidence for an adaptive role of nucleotide variation in this gene region. Our data suggest that further studies aimed at clarifying the role of MEFV variants might benefit from the integration of molecular evolutionary and functional analyses.

A CAV3 microdeletion differentially affects skeletal muscle and myocardium.

BACKGROUND: Caveolin-3 is the muscle-specific protein product of the caveolin gene family and an integral membrane component of caveolae. Mutations in the gene encoding caveolin-3 (CAV3) underlie four distinct disorders of skeletal muscle: the autosomal dominant form of limb-girdle muscular dystrophy type 1C (LGMD-1C), rippling muscle disease (RMD), sporadic and familial forms of hyperCKemia, and distal myopathy. OBJECTIVE: To characterize a multigenerational Italian family affected by an autosomal dominant myopathic disorder and to assess the expression of caveolin-3, dystrophin, dystrophin-associated glycoproteins, and neuronal nitric oxide synthase in the myocardium of an affected patient. METHODS: Clinical analysis involved 15 family members. Skeletal muscle expression of sarcolemmal proteins was evaluated by immunohistochemistry and western blot analysis in three affected individuals. Caveolar structures were analyzed through electron microscopy in muscle biopsies and in one heart biopsy. RESULTS: CAV3 genetic analysis showed a heterozygous 3-bp microdeletion (328-330del) in affected individuals, resulting in the loss of a phenylalanine (Phe97del) in the transmembrane domain. In the skeletal muscle, the mutation was associated with severe caveolin-3 deficiency and caveolar disorganization, whereas the expression of the other analyzed muscle proteins was unaltered. Remarkably, caveolin-3 was expressed in myocardium at a level corresponding to about 60% of that of control individuals and was correctly localized at the myocardial cell membranes, with preservation of cardiac myofiber caveolar structures. Clinical analysis revealed the concomitant presence in this family of the following phenotypes: RMD, LGMD, and hyperCKemia. CONCLUSIONS: Intrafamilial phenotypic heterogeneity is associated with caveolin-3 Phe97 microdeletion. The molecular network interacting with caveolin-3 in skeletal muscle and heart may differ.

Molecular analysis of LGMD-2B and MM patients: identification of novel DYSF mutations and possible founder effect in the Italian population.

Dysferlin, the protein product of the dysferlin gene (DYSF), has been shown to have a role in calcium-induced membrane fusion and repair. Dysferlin is absent or drastically reduced in patients with the following autosomal recessive disorders: limb-girdle muscular dystrophy type 2B (LGMD-2B), Miyoshi myopathy (MM) and distal anterior compartment myopathy. To date, less than 45 mutations have been described in DYSF and a wide inter- and intra-familial variation in clinical phenotype has been associated with the same mutation. This observation underlines the relevance of any new report describing genotype/phenotype correlations in dysferlinopathic patient and families. Here we present the results of clinical, biochemical and genetic analysis performed on one MM and three LGMD Italian families. By screening the entire coding region of DYSF, we identified three novel mutations (two missense substitutions and one frame shift microdeletion). The possible existence of a founder effect for the Arg959Trp mutation in the Italian population is discussed.

Trans-acting factors may cause dystrophin splicing misregulation in BMD skeletal muscles.

We analyzed dystrophin alternative splicing events in a large number of Becker muscular dystrophy (BMD) affected individuals presenting major hot-spot deletions. Evidence is shown that altered splicing patterns in these patients do not directly result from the gene defect but probably derive from modifications in trans- rather than cis-acting factors. Several potential CUG-binding protein 2 (CUG-BP2) binding sites were found to be located in the dystrophin gene region encompassing exons 43-60 and CUG-BP2 transcript analysis indicated that not only expression levels are increased in dystrophic muscles but also that different CUG-BP2 isoforms are expressed. The possibility that CUG-BP2 might have a role in dystrophin splicing regulation is discussed.

Two dystrophin proteins and transcripts in a mild dystrophinopathic patient.

Two muscle dystrophin transcripts and proteins were detected in a 17-year-old boy with a persistently elevated serum creatine kinase level. A decreased amount of full-length dystrophin and a 360 kDa polypeptide lacking the COOH-terminus were detectable in the patient's muscle biopsy; accordingly, transcript analysis revealed the expression of a wild type messenger RNA together with a shorter frameshifted one. No genomic DNA mutation was found and the presence of a somatic mosaicism was excluded. This dystrophinopathy may be caused by a novel dystrophin gene transcriptional defect, namely aberrant intraexonic splicing.

The dystrophin gene is alternatively spliced throughout its coding sequence.

We have analysed splicing patterns in the human dystrophin gene region encoding the rod and cysteine-rich domains in normal skeletal muscle, brain and heart tissues. Sixteen novel alternative transcripts were identified, the majority of them being present in all three tissues. Tissue-specific variants were also identified, suggesting a functional role of transcriptional diversity. Transcript analysis in dystrophinopathic autoptic and bioptic specimens revealed that pre-mRNAs secondary structure formation and relative strength of exon/exon association play little or no role in directing alternative splicing events. This analysis also showed that independent deletion events leading to the loss of the same exons may be associated with transcriptional variability.

Analysis of splicing parameters in the dystrophin gene: relevance for physiological and pathogenetic splicing mechanisms.

The molecular mechanisms that direct splice-site selection and assure orderly exon juxtaposition have not been fully clarified. The extraordinary nature of the dystrophin gene points to several hurdles in the processing of transcripts. In this study, dystrophin statistical and thermodynamic splicing parameters have been evaluated providing the first comprehensive description for a single human gene. We show that concomitant use of consensus values (CV) and DeltaDG degrees 37 values for U1snRNA annealing better discriminates between real donor sites and donor-like sequences. Evidence is also provided that, on average, out-of-frame dystrophin exons have significantly stronger CVs and more favorable DeltaDG degrees 37 values; this feature has never been reported and might reflect evolutionary-driven minimization of out-of-frame exon misplicing. Dystrophin splicing mutations have been reported to determine either Duchenne or Becker Muscular Dystrophy, but no comprehensive genotypic/phenotypic correlation has ever been investigated. We have analyzed splicing affecting single base-pair substitutions in the dystrophin gene with respect to their effect on splicing parameters; functional and clinical consequences are also reported. We have found 5'-splice-site mutation occurrence to be statistically related to mutability quotients and propose the use of DeltaDG degrees 37 values as a more effective tool than CV alone to describe donor site mutation consequences. Our analysis also indicates a nearly 100% correlation between clinical phenotype and the reading-frame rule determined at the RNA level. We consider that elucidation of the relative importance of splicing determinants might help to clarify the molecular mechanisms that direct correct splicing in complex genes and might be useful in the validation of predictive models.

Transcriptional activation of the non-muscle, full-length dystrophin isoforms in Duchenne muscular dystrophy skeletal muscle.

Despite promoter tissue specificity, up-regulation of the brain and Purkinje cell type dystrophin isoforms was described in skeletal muscle of X-linked dilated cardiomyopathy (XLDCM) and BMD affected individuals. An extended population of 11 Duchenne muscular dystrophy (DMD) and 11 Becker muscular dystrophy (BMD) patients was investigated to determine whether ectopic muscle expression of the two full-length non-muscular isoforms is a common event in dystrophinopathies and if it has functional significance. Up-regulation of the two non-muscle-specific isoforms was detected in four DMD patients but in none of the BMD affected individuals or non-dystrophic controls. This is the first report of an expression of these two isoforms in DMD skeletal muscle. Ectopic expression is not confined to regenerating or revertant fibers and does not correlate with age at biopsy, clinical phenotype, cardiac involvement, deletion size or location. We consider that muscle ectopic expression of the brain and Purkinje cell-type isoforms has no favorable prognostic significance in DMD and BMD patients.

Primary beta-sarcoglycanopathy manifesting as recurrent exercise-induced myoglobinuria.

We report an unusual presentation of a primary beta-sarcoglycanopathy (LGMD type 2E). A 12- year-old boy came to our attention after six episodes of exercise-induced myoglobinuria. Electromyogram showed mild myopathic features of the proximal lower limb muscles. Electrocardiogram was normal. Neurological examination revealed normal muscle strength and reduced deep tendon reflexes. A muscle biopsy showed rare regenerating fibers; the immunohistochemistry was normal for dystrophin, while all the sarcoglycans were diffusely decreased. Western blot analysis showed a relevant decrease of all sarcoglycan proteins and a mild dystrophin reduction. beta-Sarcoglycan gene analysis demonstrated a compound heterozygous status for these mutations: a novel A-T base pair substitution at nucleotide 85 in exon 2, changing the codon Arg to a stop codon; a C-T base pair substitution at nucleotide 272 in exon 3 changing a Arg to a Cys residue. We consider that exercise-induced myoglobinuria may be the presenting sign of primary beta-sarcoglycanopathy.

Xotx1 maternal transcripts are vegetally localized in Xenopus laevis oocytes.

Xotx1 is a Xenopus homeobox gene related to the Drosophila gene orthodenticle (otd). We previously reported that Xotx1 transcripts are already present in unfertilized egg. Here we report that maternal Xotx1 mRNA is vegetally localized during oogenesis. In stage II oocytes Xotx1 transcripts are localized within the mitochondrial cloud, in a perinuclear position; later on, they are translocated to the vegetal cortex within the mitochondrial cloud. We also observed that in stage III oocytes the expression domain of Wnt11 is contained within the one of Xotx1 while, at stage IV, the Xotx1 expression domain is contained within the one of Vg1.