Preimplantation genetic diagnosis for retinoblastoma survivors: a cost-effectiveness study.

This study aimed to investigate the cost-effectiveness of preimplantation genetic diagnosis (PGD) for the reproductive choices of patients with heritable retinoblastoma. The study modelled the costs of three cycles of in-vitro fertilization (IVF) and PGD across all uptake rates of PGD, number of children affected with retinoblastoma at each uptake rate and the estimated quality-adjusted life years (QALYs) gained. Cost-effectiveness analysis was conducted from the Australian public healthcare perspective. The intervention was the use of three cycles (one fresh and two frozen) of IVF and PGD with the aim of live births unaffected by the retinoblastoma phenotype. Compared with the standard care pathway (i.e. natural pregnancy), IVF and PGD resulted in a cost-saving to 18 years of age of AUD$2,747,294 for a base case of 100 couples with an uptake rate of 50%. IVF and PGD resulted in fewer affected (n = 56) and unaffected (n = 78) live births compared with standard care (71 affected and 83 unaffected live births), and an additional 0.03 QALYs per live birth. This modelling suggests that the use of IVF and PGD to achieve an unaffected child for patients with heritable retinoblastoma resulted in an overall cost-saving. There was an increase in QALYs per baby across all uptake rates. However, in total, fewer babies were born following the IVF and PGD pathway.

Phenotype-genotype correlations and emerging pathways in ocular anterior segment dysgenesis.

Disorders of the anterior segment of the eye encompass a variety of clinical presentations including aniridia, Axenfeld and Rieger anomalies, primary congenital glaucoma, Peters anomaly, as well as syndromal associations. These conditions have a significant impact on vision due to disruption of the visual axis, and also secondary glaucoma which occurs in over 50% of patients. Ocular anterior segment disorders occur due to a complex interplay of developmental, embryological and genetic factors, and often have phenotypic overlaps and genetic heterogeneity. Here we present a review of the clinical features and genes associated with aniridia, Axenfeld and Rieger anomalies, primary congenital glaucoma, Peters anomaly, and syndromic forms of these conditions. We also highlight phenotype-genotype correlations, recent discoveries with next-generation sequencing which broaden known phenotypes, and new anterior segment genes and pathways. We provide a guide towards genetic diagnosis for clinicians investigating patients with anterior segment dysgenesis.

New mutations in GJA8 expand the phenotype to include total sclerocornea.

This project expands the disease spectrum for mutations in GJA8 to include total sclerocornea, rudimentary lenses and microphthalmia, in addition to this gene's previously known role in isolated congenital cataracts. Ophthalmic findings revealed bilateral total sclerocornea in 3 probands, with small abnormal lenses in 2 of the cases, and cataracts and microphthalmia in 1 case. Next-generation sequencing revealed de novo heterozygous mutations affecting the same codon of GJA8 : (c.281G>A; p.(Gly94Glu) and c.280G>C; p.(Gly94Arg)) in 2 of the probands, in addition to the c.151G>A; p.(Asp51Asn) mutation we had previously identified in the third case. In silico analysis predicted all of the mutations to be pathogenic. These cases show that deleterious, heterozygous mutations in GJA8 can lead to a severe ocular phenotype of total sclerocornea, abnormal lenses, and/or cataracts with or without microphthalmia, broadening the phenotype associated with this gene. GJA8 should be included when investigating patients with the severe anterior segment abnormality of total sclerocornea.

A puzzle over several decades: eye anomalies with FRAS1 and STRA6 mutations in the same family.

Fraser syndrome (FS) and microphthalmia syndromic 9 (MCOPS9) are autosomal recessive conditions with distinct, and some overlapping features affecting the ocular, respiratory and cardiac systems. Mutations in FRAS1 and FREM2 occur in FS, and mutations in STRA6 occur in MCOPS9. We report two sibships, in the same family, where four deceased offspring had ocular, respiratory and cardiac abnormalities. Two sibs with microphthalmia had syndactyly and laryngeal stenosis, suggesting a clinical diagnosis of FS. Our results indicate that they were compound heterozygotes for novel FRAS1 mutations, p.Cys729Phe and p.Leu3813Pro. The other two sibs, first cousins to the first sib pair, had anophthalmia, lung hypoplasia and cardiac anomalies, suggesting a retrospective diagnosis of MCOPS9. Our results indicate compound heterozygous STRA6 mutations, a novel frameshift leading to p.Tyr18* and a p.Thr644Met mutation. The one surviving individual from these sibships is heterozygous for the p.Tyr18*STRA6 mutation and has bilateral ocular colobomata and microphthalmia. This work emphasises the need for careful phenotypic characterisation to determine genes for assessment in ocular syndromic conditions. It also indicates that heterozygous STRA6 mutations may rarely contribute to microphthalmia and coloboma.

A heterozygous c-Maf transactivation domain mutation causes congenital cataract and enhances target gene activation.

MAF, one of a family of large Maf bZIP transcription factors, is mutated in human developmental ocular disorders that include congenital cataract, microcornea, coloboma and anterior segment dysgenesis. Expressed early in the developing lens vesicle, it is central to regulation of lens crystallin gene expression. We report a semi-dominant mouse c-Maf mutation recovered after ENU mutatgenesis which results in the substitution, D90V, at a highly conserved residue within the N-terminal 35 amino-acid minimal transactivation domain (MTD). Unlike null and loss-of-function c-Maf mutations, which cause severe runting and renal abnormalities, the phenotype caused by the D90V mutation is isolated cataract. In reporter assays, D90V results in increased promoter activation, a situation similar to MTD mutations of NRL that also cause human disease. In contrast to wild-type protein, the c-Maf D90V mutant protein is not inhibited by protein kinase A-dependent pathways. The MTD of large Maf proteins has been shown to interact with the transcriptional co-activator p300 and we demonstrate that c-Maf D90V enhances p300 recruitment in a cell-type dependent manner. We observed the same for the pathogenic human NRL MTD mutation S50T, which suggests a common mechanism of action.

Pulverulent cataract with variably associated microcornea and iris coloboma in a MAF mutation family.

AIMS: To report the detailed clinical findings in a three generation pedigree with autosomal dominant cataract, microcornea, and coloboma resulting from mutation of the lens development gene, MAF. METHODS: Five members of a three generation pedigree with progressive cataracts underwent detailed ophthalmic examination to characterise associated ocular phenotypic features. RESULTS: The cataracts present in all affected individuals were cortical, and/or nuclear, pulverulent opacities. Corneal diameters of 10-10.25 mm were present in two family members. Axial lengths were in the normal range. Bilateral iris coloboma in the 6 o'clock position was present in one patient. Uveal melanoma was present in one patient, with uveal naevi in this and one other patient. CONCLUSION: The bZIP transcription factor MAF is a key lens development gene that regulates the expression of the crystallins. Individuals with a mutation in MAF may have pulverulent cataract alone or cataract in association with microcornea or iris coloboma.

The allocation and differentiation of mouse primordial germ cells.

Analysis of the lineage potency of epiblast cells of the early-streak stage mouse embryo reveals that the developmental fate of the cells is determined by their position in the germ layer. Epiblast cells that are fated to become neuroectoderm can give rise to primordial germ cells (PGCs) and other types of somatic cells when they were transplanted to the proximal region of the epiblast. On the contrary, proximal epiblast cells transplanted to the distal region of the embryo do not form PGCs. Therefore, the germ line in the mouse is unlikely to be derived from a predetermined progenitor population, but may be specified as a result of tissue interactions that take place in the proximal epiblast of the mouse gastrula. The initial phase of the establishment of the PGC population requires, in addition to BMP activity emanating from the extraembryonic ectoderm, normal Lim1 and Hnf3beta activity in the germ layers. The entire PGC population is derived from a finite number of progenitor cells and there is no further cellular recruitment to the germ line after gastrulation. The XX PGCs undergo X-inactivation at the onset of migration from the gut endoderm and re-activate the silenced X-chromosome when they enter the urogenital ridge. Germ cells that are localised ectopically in extragonadal sites do not re-activate the X-chromosome, even when nearly all germ cells in the fetal ovary have restored full activity of both X-chromosomes. XXSxr germ cells can re-activate the X-chromosome in the sex-reversed testis, suggesting that the regulation of X-chromosome activity is independent of ovarian morphogenesis.

Retarded postimplantation development of X0 mouse embryos: impact of the parental origin of the monosomic X chromosome.

About 12-17% of the embryos obtained by mating mice carrying the In(X)1H or Paf mutations are of the 39,X (X0) genotype. Depending on the mutant mice used for mating, the monosomic X chromosome can be inherited from the paternal (XP) or the maternal (XM) parent. The XP0 embryos display developmental retardation at gastrulation and early organogenesis. XP0 embryos also display poor development of the ectoplacental cone, which is significantly smaller in size and contains fewer trophoblasts than XX siblings. In contrast, XM0 embryos develop normally and are indistinguishable from XX littermates. In both types of X0 embryos, an X-linked lacZ transgene is expressed in nearly all cells in both the embryonic and the extraembryonic tissues, suggesting that X inactivation does not occur when only one X is present. Of particular significance is the maintenance of an active XP chromosome in the extraembryonic tissues where normally the paternal X chromosome is preferentially inactivated in XX embryos. The differential impact of the inheritance of X chromosomes from different parents on the development of the X0 embryos raises the possibility that the XP is less capable than the XM in providing the appropriate dosage of X-linked activity that is necessary to support normal development of the embryo and the ectoplacental cone. Alternatively, the development of the XP0 embryo may be compromised by the lack of activity of one or several X-linked genes which are expressed only from the maternal X chromosome. Without the activity of these genes, embryonic development may be curtailed even though all other loci on the XP chromosome are actively transcribed.

Sertoli cell differentiation and Y-chromosome activity: a developmental study of X-linked transgene activity in sex-reversed X/XSxra mouse embryos.

The requirement of Y-chromosome activity for the differentiation of somatic cells and germ cells was studied in the fetal gonads of X/XSxra mouse embryos where the activity of the Sxra fragment of the Y chromosome is influenced by the inactivation and reactivation of the X chromosome. In the interstitial somatic cells, random inactivation of the X and the XSxra chromosomes took place which was revealed by the mosaic expression of an X-linked lacZ transgene. The Sertoli cells, however, displayed a preferentially active XSxra chromosome and the presence of Sxra-active Sertoli cells was associated with the morphogenesis of testicular tubules in the sex-reversed gonads. The activity of the Y-chromosome fragment is therefore necessary for the differentiation of the Sertoli cells which may direct the development of the testis. The expression pattern of the X-linked transgene in X/XSxra germ cells suggests that both the X and the XSxra chromosomes are active. This finding suggests that the presence of Sxra has no impact on the reactivation of the X chromosome in the germ cells and that the X chromosome can be reactivated even though the germ cells are found in the testicular environment. Our results are consistent with the concept that the activity of genes on the XSxra fragment is essential for the differentiation of Sertoli cells and the morphogenesis of the testis, but not for premeiotic differentiation of germ cells in sex-reversed mice.

X-chromosome inactivation during the development of the male urogenital ridge of the mouse.

In the mouse, the activity of Sry (sex-determining gene on the Y chromosome) initiates the transformation of the indifferent gonad into a testis. In humans, a partial Xp21 duplication leads to the development of ovaries instead of testes in XY individuals. This observation indicates that sex determination might also be influenced by a gene-dosage compensation mechanism, in addition to a dominant action of the Sry gene. In female mammals, the regulation of X-linked gene dosage at early embryogenesis is achieved through the inactivation of one of the two X chromosomes. Here we have investigated the possibility that inactivation of the X chromosome may play a role in male sex determination. We have shown, using an X-linked lacZ transgenic mouse line, that loss of beta-galactosidase activity occurs in certain somatic cells of the developing male urogenital ridge. When changes associated with apoptosis of mesonephric tubules in the developing urogenital ridges are taken into account, expression of the Xist (X inactive specific transcript) gene correlates with X inactivation revealed by loss of beta-galactosidase activity in very early mesonephric tubule epithelial cells, gonadal interstitial mesenchymal cells and coelomic epithelial cells.

X-chromosome activity: impact of imprinting and chromatin structure.

The analysis of the imprinting of the X chromosome has provided insight into factors that affect the initiation and the choice of the chromosome for inactivation in the early mammalian embryo (Lyon, 1996). There are significant differences in the chromatin configuration, methylation and gene expression between Xi and Xa in somatic cells. Preferential paternal X inactivation that is concomitant with widespread heterochromatinization first occurs in the trophectoderm in the blastocyst. It is now clear that the activity of some paternal X-linked genes are suppressed before this stage. In the epiblast there may be early preferential paternal X inactivation before a random pattern supersedes. These observations suggest that parent-specific modification of the chromosome may determine the choice of which X chromosome is to be inactivated (Lyon, 1996). Differential methylation within the Xist gene or the XIC may lead to imprinted X-chromosome behavior. Alternatively, we postulate that imprinting of the X chromosome may be related to differences in chromatin configuration of the X chromosome in male and female germ cells which may then influence X-linked gene expression in the early embryo (Fig. 4). This may occur with a gene by gene effect leading to suppression of paternal alleles. An overall chromatin difference in the chromosomes may influence imprinted paternal Xist expression in early embryos and in the trophectoderm and primary endoderm populations that segregate early from the totipotent progenitors. Alternatively more specific differences in the chromatin architecture of the Xist gene or other gene loci in the Xic may constitute the signature of the imprint.