Mitochondrial inheritance and germ line determination in a bivalve species with Doubly Uniparental Inheritance (DUI)

Mitochondria are inherited maternally in most metazoans. However, in some bivalves, two mitochondrial lineages are present: one transmitted through eggs (F), the other through sperm (M). This is called Doubly Uniparental Inheritance (DUI). During male embryo development, spermatozoon mitochondria aggregate and end up in the primordial germ cells, while they are dispersed in female embryos. The molecular mechanisms of segregation patterns are still unknown. In the DUI species Ruditapes philippinarum, I examined sperm mitochondria distribution by MitoTracker, microtubule staining and TEM, and I localized germ line determinants with immunocytochemical analysis. I also analyzed the gonad transcriptome, searching for genes involved in reproduction and sex determination. Moreover, I analyzed an M-type specific open reading frame that could be responsible for maintenance/degradation of M mitochondria during embryo development. These transcripts were also localized in tissues using in situ hybridization. As in Mytilus, two distribution patterns of M mitochondria were detected in R. philippinarum, supporting that they are related to DUI. Moreover, the first division midbody concurs in positioning aggregated M mitochondria on the animal-vegetal axis of the male embryo: in organisms with spiral segmentation this zone is not involved in further cleavages, so aggregation is maintained. Moreover, sperm mitochondria reach the same embryonic area where germ plasm is transferred, suggesting their contribution in male germ line formation. The finding of reproduction and ubiquitination transcripts led to formulate a model in which ubiquitination genes stored in female oocytes during gametogenesis would activate sex-gene expression in the early embryonic developmental stages (preformation). Only gametogenetic cells were labeled by in situ hybridization, proving their specific transcription in developing gametes. Other than having a role in sex determination, some ubiquination factors could also be involved in mitochondrial inheritance, and their differential expression could be responsible for the different fate of sperm mitochondria in the two sexes.

The KIT gene in familial mastocytosis

Familial cutaneous mastocytosis is an exceptional condition of unknown etiology. In this study we report the largest series of patients with familial cutaneous mastocytosis without other manifestations (18 affected subjects from seven unrelated families), and we investigate the role of germ-line KIT mutations in the pathogenesis of the disease. The mean age at onset was 5.4 years (range from birth to 22 years), and the clinical behavior was variable over a mean follow up period of 15.1 years (range 2-36): improvement in seven, stability in eight and worsening in the remaining three patients. The pattern of inheritance was compatible with an autosomal dominant trait with incomplete penetrance; a female preponderance (14 females vs 4 males, ratio 3.5:1) was noted; among the six women who have been pregnant at least once, three experienced important clinical changes during pregnancy. No germ-line mutation was found in the exons 10, 11, and 17 of the KIT proto-oncogene, which are the most commonly mutated exons in sporadic mastocytosis. However, in the majority of affected subjects we found the Met541Leu polymorphic variant of the KIT gene, which seems to confer a growth advantage to mast cells in vitro. This observation further suggests that the Met541Leu may be a predisposing factor of cutaneous mastocytosis, although it seems to be neither necessary nor sufficient for the development of the disease.

Phylogenie und Biogeographie der Linoideae (Linaceae) - Evolution von Heterostylie, Homostylie in Linum

Die Linaceae-Linoideae, vor allem die Gattung Linum, wurden unter Verwendung von zwei molekularen Markern (rbcL und ITS) bzgl. ihrer Phylogenie und Biogeographie untersucht. Die Linaceae entstanden während der mittleren Kreide in den frühen tropischen Regenwäldern, von wo aus sich die monophyletischen Linoideae vor etwa 51-46 Mill. Jahren über die temperaten Gebiete der Nordhemisphäre ausbreiteten. Während die drei basal abspaltenden Gattungen Anisadenia, Reinwardtia und Tirpitzia bzgl. ihrer Verbreitung auf Südostasien beschränkt sind, ist die Gattung Linum heute auf allen Kontinenten vertreten. Der Ursprung von Linum liegt wahrscheinlich in Südwestasien bzw. dem östlichen Mediterraneum, wo es im Oligozän zur Aufspaltung in zwei Entwicklungslinien kam ('Blaue Gruppe' und 'Gelbe Gruppe'). Während die überwiegend blaublühenden Linum-Arten ('Blaue Gruppe') vor allem in Europa und Südwestasien vorkommen, weisen die Vertreter der 'Gelben Gruppe' ein wesentlich größeres Verbreitungsgebiet auf. Gelbblühende Linum Arten findet man auf allen Kontinenten mit Diversitätszentren in Nordostamerika und Südwestasien. Interessanterweise wurde Amerika zweimal unabhängig voneinander besiedelt. Während die gelbblühenden Arten vor etwa 22-20 Mill. Jahren von Westeuropa über den Atlantik den amerikanischen Kontinent erreichten, wanderten Vertreter der 'Blauen Gruppe' im Pliozän (vor 3.78-3.33 Mill Jahren) über die Bering-Landbrücke in die Neue Welt ein. Auch in Südafrika sind einige gelbblühende Linum-Arten zu verzeichnen, die nicht über Nordafrika (wo einige Arten der 'Gelben Gruppe' beheimatet sind) die südliche Spitze des Kontinents erreichten, sondern von Amerika aus. Die molekularphylogenetischen Ergebnisse legen eine Eingliederung der Gattungen Cliococca, Hesperolinon, Radiola und Sclerolinon in Linum nahe, die durch morphologische Merkmale gestützt wird. Linopsis, die artenreichste Sektion der Gattung Linum, bedarf einiger Umstrukturierungen auf der Basis der molekularen und morphologischen Daten. Ein interessantes Phänomen innerhalb der Linaceae ist das Vorkommen von heterostylen und homostylen Arten innerhalb der Familie. Die Kombination der molekular-phylogenetischen Ergebnisse mit morphologischen Beobachtungen des Reproduktionssystems lassen darauf schließen, dass sich Homostylie innerhalb von Linum mehrfach unabhängig voneinander entwickelt hat. Das Modell von Primula wurde als Grundlage verwendet, um Aufschluss über die Entstehung der Homostylie innerhalb von Linum zu erlangen. Aus Primula ist bekannt, dass eine Kopplungsgruppe aus mindestens drei Genen an der Vererbung von Heterostylie beteiligt ist: G/g kodiert hierbei die Griffellänge und die Selbstinkompatibilitäts-reaktion der Narbe, A/a die Länge der Filamente und P/p die Selbst-inkompatibilitätsreaktion des Pollens. Umfangreiche Kreuzungs-experimente einer homostylen und einer heterostylen Linum-Art deuten darauf hin, dass die Genotypen der beiden Blütenformen in heterostylen Linum-Arten denen in Primula entsprechen. Langgriffel sind hiernach homozygot rezessiv (gpa/gpa), während die Kurzgriffel heterozygot sind (GPA/gpa). Selbstkompatible, homostyle Arten können theoretisch durch verschiedene Rekombinations-ereignisse entstehen. Erste Ergebnisse der rasterelektronen-mikroskopischen Betrachtung der Pollenkornoberflächen und Narbenpapillen deuten darauf hin, dass innerhalb von Linum Homostylie durch unterschiedliche Rekombinations-ereignisse mehrfach aus heterostylen Arten entstanden ist. So besitzt die homostyle Linum leonii den Genotyp gPA/gPA, während für die homostylen L. tenuifolium und L. nodiflorum der Genotyp Gpa/Gpa wahrscheinlich ist.

Characterization of mitochondrial genomes in bivalve species with doubly uniparental inheritance of mitochondria

Many bivalve species possess two distinct mtDNA lineages, called F and M, respectively inherited maternally and paternally: this system is called doubly uniparental inheritance (DUI). The main experimental project of my PhD was the quantification of the two mtDNAs during the development of the DUI species Ruditapes philippinarum, from early embryos to sub-adults, using Real-Time qPCR. I identified the time interval in which M mtDNA is lost from female individuals, while it is retained in males (which are heteroplasmic through all of their life cycle). The results also suggested absence of mtDNA replication during early embryogenesis, a process constituting a bottleneck that highly reduces the copy number of mtDNA molecules in cells of developing larvae. In males this bottleneck may produce cells homoplasmic for M mtDNA, and could be considered as a first step of the segregation of M in the male germ line. Another finding was the characterization, in young clams approaching the first reproductive season, of a significant boost in copy number of F mtDNA in females and of M in males. Given the age of animals in which this mtDNA-specific growth was observed, the finding could probably be the outcome of the first round of gonads and gametes production. Other lines of research included the characterization of the unassigned regions in mt genomes of DUI bivalves. These regions can harbor signals involved in the control of replication and/or transcription of the mtDNA molecule, as well as additional open reading frames (ORFs) not related to oxidative phosphorylation. These features in DUI species could be associated to the maintenance of separate inheritance routes for the two mtDNAs. Additional ORFs are also found in other animal mt genomes: I summarized the presence of gene duplications as a co-author in a review focusing on animal mt genomes with unusual gene content.

Analysis of Copy Number Variants identifies new candidate genes for Autism Spectrum Disorder and Intellectual Disability

Autism spectrum disorder (ASD) and Intellectual Disability (ID) are complex neuropsychiatric disorders characterized by extensive clinical and genetic heterogeneity and with overlapping risk factors. The aim of my project was to further investigate the role of Copy Numbers Variants (CNVs), identified through genome-wide studies performed by the Autism Geome Project (AGP) and the CHERISH consortium in large cohorts of ASD and ID cases, respectively. Specifically, I focused on four rare genic CNVs, selected on the basis of their impact on interesting ASD/ID candidate genes: a) a compound heterozygous deletion involving CTNNA3, predicted to cause the lack of functional protein; b) a 15q13.3 duplication containing CHRNA7; c) a 2q31.1 microdeletion encompassing KLHL23, SSB and METTL5; d) Lastly, I investigated the putative imprinting regulation of the CADPS2 gene, disrupted by a maternal deletion in two siblings with ASD and ID. This study provides further evidence for the role of CTNNA3, CHRNA7, KLHL23 and CADPS2 as ASD and/or ID susceptibility genes, and highlights that rare genetic variation contributes to disease risk in different ways: some rare mutations, such as those impacting CTNNA3, act in a recessive mode of inheritance, while other CNVs, such as those occurring in the 15q13.3 region, are implicated in multiple developmental and/or neurological disorders possibly interacting with other susceptibility variants elsewhere in the genome. On the other hand, the discovery of a tissue-specific monoallelic expression for the CADPS2 gene, implicates the involvement of epigenetic regulatory mechanisms as risk factors conferring susceptibility to ASD/ID.

Monogenic forms of hypertension

Arterial hypertension in childhood is less frequent as compared to adulthood but is more likely to be secondary to an underlying disorder. After ruling out more obvious causes, some patients still present with strongly suspected secondary hypertension of yet unknown etiology. A number of these children have hypertension due to single gene mutations inherited in an autosomal dominant or recessive fashion. The finding of abnormal potassium levels (low or high) in the presence of suppressed renin secretion, and metabolic alkalosis or acidosis should prompt consideration of these familial diseases. However, mild hypertension and the absence of electrolyte abnormalities do not exclude hereditary conditions. In monogenic hypertensive disorders, three distinct mechanisms leading to the common final pathway of increased sodium reabsorption, volume expansion, and low plasma renin activity are documented. The first mechanism relates to gain-of-function mutations with a subsequent hyperactivity of renal sodium and chloride reabsorption leading to plasma volume expansion (e.g., Liddle's syndrome, Gordon's syndrome). The second mechanism involves deficiencies of enzymes that regulate adrenal steroid hormone synthesis and deactivation (e.g., subtypes of congenital adrenal hyperplasia, apparent mineralocorticoid excess (AME)). The third mechanism is characterized by excessive aldosterone synthesis that escapes normal regulatory mechanisms and leading to volume-dependent hypertension in the presence of suppressed renin release (glucocorticoid remediable aldosteronism). Hormonal studies coupled with genetic testing can help in the early diagnosis of these disorders.

A missense mutation in the skeletal muscle chloride channel 1 (CLCN1) as candidate causal mutation for congenital myotonia in a New Forest pony

A 7-month-old New Forest foal presented for episodes of recumbency and stiffness with myotonic discharges on electromyography. The observed phenotype resembled congenital myotonia caused by CLCN1 mutations in goats and humans. Mutation of the CLCN1 gene was considered as possible cause and mutation analysis was performed. The affected foal was homozygous for a missense mutation (c.1775A>C, p.D592A) located in a well conserved domain of the CLCN1 gene. The mutation showed a recessive mode of inheritance within the reported pony family. Therefore, this CLCN1 polymorphism is considered to be a possible cause of congenital myotonia.

Isolated autosomal dominant growth hormone deficiency: stimulating mutant GH-1 gene expression drives GH-1 splice-site selection, cell proliferation, and apoptosis

The majority of mutations that cause isolated GH deficiency type II (IGHD II) affect splicing of GH-1 transcripts and produce a dominant-negative GH isoform lacking exon 3 resulting in a 17.5-kDa isoform, which further leads to disruption of the GH secretory pathway. A clinical variability in the severity of the IGHD II phenotype depending on the GH-1 gene alteration has been reported, and in vitro and transgenic animal data suggest that the onset and severity of the phenotype relates to the proportion of 17.5-kDa produced. The removal of GH in IGHD creates a positive feedback loop driving more GH expression, which may itself increase 17.5-kDa isoform productions from alternate splice sites in the mutated GH-1 allele. In this study, we aimed to test this idea by comparing the impact of stimulated expression by glucocorticoids on the production of different GH isoforms from wild-type (wt) and mutant GH-1 genes, relying on the glucocorticoid regulatory element within intron 1 in the GH-1 gene. AtT-20 cells were transfected with wt-GH or mutated GH-1 variants (5'IVS-3 + 2-bp T->C; 5'IVS-3 + 6 bp T->C; ISEm1: IVS-3 + 28 G->A) known to cause clinical IGHD II of varying severity. Cells were stimulated with 1 and 10 mum dexamethasone (DEX) for 24 h, after which the relative amounts of GH-1 splice variants were determined by semiquantitative and quantitative (TaqMan) RT-PCR. In the absence of DEX, only around 1% wt-GH-1 transcripts were the 17.5-kDa isoform, whereas the three mutant GH-1 variants produced 29, 39, and 78% of the 17.5-kDa isoform. DEX stimulated total GH-1 gene transcription from all constructs. Notably, however, DEX increased the amount of 17.5-kDa GH isoform relative to the 22- and 20-kDa isoforms produced from the mutated GH-1 variants, but not from wt-GH-1. This DEX-induced enhancement of 17.5-kDa GH isoform production, up to 100% in the most severe case, was completely blocked by the addition of RU486. In other studies, we measured cell proliferation rates, annexin V staining, and DNA fragmentation in cells transfected with the same GH-1 constructs. The results showed that that the 5'IVS-3 + 2-bp GH-1 gene mutation had a more severe impact on those measures than the splice site mutations within 5'IVS-3 + 6 bp or ISE +28, in line with the clinical severity observed with these mutations. Our findings that the proportion of 17.5-kDa produced from mutant GH-1 alleles increases with increased drive for gene expression may help to explain the variable onset progression, and severity observed in IGHD II.

Autosomal-dominant isolated growth hormone deficiency (IGHD type II) with normal GH-1 gene

BACKGROUND: Autosomal-dominant isolated GH deficiency (IGHD) is a rare disorder that is commonly believed to be due to heterozygous mutations in the GH-1 gene (GH-1). These mutations cause the production of a protein that affects the release of the product of the normal allele. Rarely, heterozygous mutations in the gene encoding for HESX-1 gene (HESX-1) may cause autosomal-dominant IGHD, with penetrance that has been shown to be variable in both humans and mice. SUBJECTS AND METHODS: We have sequenced the whole GH-1 in the index cases of 30 families with autosomal-dominant IGHD. In all the families other possible causes of GH deficiency and other pituitary hormones deficits were excluded. We here describe the clinical, biochemical and radiological picture of the families without GH-1 mutations. In these families, we also sequenced the HESX-1. RESULTS: The index cases of the five families with autosomal-dominant IGHD had normal GH-1, including the intronic sequences. They had no HESX-1 mutations. CONCLUSION: This study shows that GH-1 mutations are absent in 5/30 (16.6%) of the families with autosomal-dominant IGHD and raises the possibility that mutations in other gene(s) may be involved in IGHD with this mode of transmission.

Analysis of gene order data supports vertical inheritance of the leukotoxin operon and genome rearrangements in the 5' flanking region in genus Mannheimia

BACKGROUND: The Mannheimia subclades belong to the same bacterial genus, but have taken divergent paths toward their distinct lifestyles. For example, M. haemolytica + M. glucosida are potential pathogens of the respiratory tract in the mammalian suborder Ruminantia, whereas M. ruminalis, the supposed sister group, lives as a commensal in the ovine rumen. We have tested the hypothesis that vertical inheritance of the leukotoxin (lktCABD) operon has occurred from the last common ancestor of genus Mannheimia to any ancestor of the diverging subclades by exploring gene order data. RESULTS: We examined the gene order in the 5' flanking region of the leukotoxin operon and found that the 5' flanking gene strings, hslVU-lapB-artJ-lktC and xylAB-lktC, are peculiar to M. haemolytica + M. glucosida and M. granulomatis, respectively, whereas the gene string hslVU-lapB-lktC is present in M. ruminalis, the supposed sister group of M. haemolytica + M. glucosida, and in the most ancient subclade M. varigena. In M. granulomatis, we found remnants of the gene string hslVU-lapB-lktC in the xylB-lktC intergenic region. CONCLUSION: These observations indicate that the gene string hslVU-lapB-lktC is more ancient than the hslVU-lapB-artJ-lktC and xylAB-lktC gene strings. The presence of (remnants of) the ancient gene string hslVU-lapB-lktC among any subclades within genus Mannheimia supports that it has been vertically inherited from the last common ancestor of genus Mannheimia to any ancestor of the diverging subclades, thus reaffirming the hypothesis of vertical inheritance of the leukotoxin operon. The presence of individual 5' flanking regions in M. haemolytica + M. glucosida and M. granulomatis reflects later genome rearrangements within each subclade. The evolution of the novel 5' flanking region in M. haemolytica + M. glucosida resulted in transcriptional coupling between the divergently arranged artJ and lkt promoters. We propose that the chimeric promoter have led to high level expression of the leukotoxin operon which could explain the increased potential of certain M. haemolytica + M. glucosida strains to cause a particular type of infection.

Exon splice enhancer mutation (GH-E32A) causes autosomal dominant growth hormone deficiency

CONTEXT AND OBJECTIVE: Alteration of exon splice enhancers (ESE) may cause autosomal dominant GH deficiency (IGHD II). Disruption analysis of a (GAA) (n) ESE motif within exon 3 by introducing single-base mutations has shown that single nucleotide mutations within ESE1 affect pre-mRNA splicing. DESIGN, SETTING, AND PATIENTS: Confirming the laboratory-derived data, a heterozygous splice enhancer mutation in exon 3 (exon 3 + 2 A-->C) coding for GH-E32A mutation of the GH-1 gene was found in two independent pedigrees, causing familial IGHD II. Because different ESE mutations have a variable impact on splicing of exon 3 of GH and therefore on the expression of the 17.5-kDa GH mutant form, the GH-E32A was studied at the cellular level. INTERVENTIONS AND RESULTS: The splicing of GH-E32A, assessed at the protein level, produced significantly increased amounts of 17.5-kDa GH isoform (55% of total GH protein) when compared with the wt-GH. AtT-20 cells coexpressing both wt-GH and GH-E32A presented a significant reduction in cell proliferation as well as GH production after forskolin stimulation when compared with the cells expressing wt-GH. These results were complemented with confocal microscopy analysis, which revealed a significant reduction of the GH-E32A-derived isoform colocalized with secretory granules, compared with wt-GH. CONCLUSION: GH-E32A mutation found within ESE1 weakens recognition of exon 3 directly, and therefore, an increased production of the exon 3-skipped 17.5-kDa GH isoform in relation to the 22-kDa, wt-GH isoform was found. The GH-E32A mutant altered stimulated GH production as well as cell proliferation, causing IGHD II.

Catch-up growth in autosomal dominant isolated growth hormone deficiency (IGHD type II)

OBJECTIVE: Data on the GH-induced catch-up growth of severely GH-deficient children affected by monogenetic defects are missing. PATIENTS: Catch-up growth of 21 prepubertal children (6 females, 15 males) affected with IGHD type II was analyzed in a retrospective chart review. At start of therapy, mean age was 6.2 years (range, 1.6-15.0), mean height SDS was -4.7 (-7.6 to -2.2), mean IGF-I SDS was -6.2 (-10.1 to -2.2). GH was substituted using a mean dose of 30.5microg/kg*d. RESULTS: Catch-up growth was characterized by a mean height gain of +0.92, +0.82, and +0.61 SDS after 1, 2, and 3 years of GH therapy, respectively. Mean height velocities were 10.7, 9.2 and 7.7cm/year during the first three years. Mean duration of complete catch-up growth was 6 years (3-9). Mean height SDS reached was -0.97 (-2.3 to +1.1), which was within the range of the estimated target height of -0.60 SDS (-1.20 to -0.15). The younger and shorter the children were at start of therapy the better they grew during the first year independent of the dose. Mean bone age was delayed at start by 2.1 years and progressed by 2.5 years during the first two years of therapy. Incomplete catch-up growth was caused by late initiation or irregular administration of GH in four cases. CONCLUSIONS: Our data suggest that GH-treated children with severe IGHD show a sustained catch-up growth over 6 years (mean) and reach their target height range. This response to GH is considered to be characteristic for young children with severe growth retardation due to IGHD.

[Hypophyseal incidentaloma in a patient with autosomal dominant polycystic kidney disease]

The prevalence of incidentally discovered lesions within the pituitary (pituitary incidentalomas) is about 10%. The most common form of sellar mass are clinically nonfunctioning adenomas (less than 10 mm); functioning adenomas, however, are rare. Incidentally discovered pituitary microadenomas causing growth hormone hypersecretion are uncommon. In addition, the association of autosomal dominant polycystic kidney disease with acromegaly is exceptional and has not yet been reported to our knowledge.