Non-lethal Phenotype Research Articles

A syndrome comprising childhood-onset glomerular kidney disease and ocular abnormalities with progressive loss of vision is caused by mutated LAMB2

Pierson syndrome (OMIM 609049) is a severe congenital oculorenal disorder with early lethality. The condition is caused by mutations in the LAMB2 gene leading to complete loss of function of the gene product laminin beta2, an essential component of the glomerular and other basement membranes. We present a non-consanguineous family with seven offspring affected by childhood-onset nephrotic syndrome progressing to end-stage renal failure and ocular abnormalities including cataracts, anterior chamber and iris abnormalities, and progressive blindness due to retinal detachment. The LAMB2 gene was analysed in this family by direct sequencing. The disorder turned out to segregate with compound heterozygosity for two novel LAMB2 mutations, triangle upV79 and Q1728X. Whereas the mutation Q1728X is predicted to confer complete loss of function, triangle upV79 probably represents a hypomorphic allele, thus explaining the substantially milder phenotype in this family. This observation demonstrates that the phenotypic spectrum of LAMB2-associated disorders is broader than previously anticipated, and suggests that milder, non-lethal phenotypes may be associated with mutations retaining some residual function.

The nucleotide exchange factor activity of Grp170 may explain the non-lethal phenotype of loss of Sil1 function in man and mouse

Recent genetic work characterized homozygous mutations in the SIL1 gene as cause for the neurodegeneration that is associated with Marinesco–Sjögren syndrome in man and the woozy mouse mutant. All reported mutations were expected to result in loss of Sil1 function. Sil1 has previously been shown to act as nucleotide exchange factor for the molecular chaperone immunoglobulin heavy chain binding protein (BiP) in the lumen of the endoplasmic reticulum (ER). In the yeast ER Lhs1p was shown to be able to substitute for Sil1p and to represent an alternative nucleotide exchange activity. Therefore, by analogy the mammalian ortholog of Lhs1p, Grp170, was suggested to be able to compensate for the loss of Sil1 function in many mammalian organs. Here we characterize mammalian Grp170 as alternative nucleotide exchange factor for BiP, thus providing a likely explanation for the non-lethal phenotype of the homozygous human and murine SIL1 mutations.

X-chromosome inactivation: role in skin disease expression.

The occurrence of X inactivation in mammals has the consequence that all women are functional mosaics. In X-linked skin disorders, Lyonization usually gives rise to a mosaic pattern, as manifest by the appearance of the lines of Blaschko. This arrangement of lesions is observed in male-lethal X-linked traits, such as incontinentia pigmenti, focal dermal hypoplasia, Conradi-Hünermann-Happle syndrome, oral-facial-digital syndrome type 1 and MIDAS (microphthalmia, dermal aplasia and sclerocornea) syndrome, as well as in various X-linked non-lethal phenotypes, such as hypohidrotic ectodermal dysplasia of Christ-Siemens-Touraine, IFAP (ichthyosis follicularis-alopecia-photophobia) syndrome and X-linked dyskeratosis congenita. Analogous X-inactivation patterns have been documented in human bones, teeth, eyes and, possibly, the brain. Patterns that are distinct from the lines of Blaschko are also seen, such as the lateralization observed in CHILD (congenital hemidysplasia with ichthyosiform nevus and limb defects) syndrome, and the chequerboard pattern seen in women heterozygous for X-linked congenital hypertrichosis. Exceptional cases of either severe or absent involvement in a woman heterozygous for an X-linked trait can be explained by skewing of X inactivation. Some X-linked skin disorders are caused by genes that escape inactivation, which is why heterozygous female 'carriers' of these disorders do not show mosaicism. A well-known example is X-linked recessive ichthyosis due to steroid sulphatase deficiency, the locus for which is situated at the tip of the short arm of the X chromosome and does not undergo Lyonization. On the other hand, in the case of Fabry disease, the gene encoding alpha-galactosidase A is subject to inactivation. Remarkably, however, the skin lesions of women do not show a mosaic pattern. In the various X-linked skin disorders, affected women show quite dissimilar degrees of involvement and forms of manifestation because X inactivation may give rise to different patterns of functional mosaicism. Paradoxically, no such pattern is observed in women with Fabry disease. Like many X-linked diseases, Fabry disease should neither be called recessive nor dominant, because these dichotomous terms are obscured by the mechanism of X inactivation.

X-chromosome inactivation: role in skin disease expression

The occurrence of X inactivation in mammals has the consequence that all women are functional mosaics. In X-linked skin disorders, Lyonization usually gives rise to a mosaic pattern, as manifest by the appearance of the lines of Blaschko. This arrangement of lesions is observed in male-lethal X-linked traits, such as incontinentia pigmenti, focal dermal hypoplasia, Conradi-Hünermann-Happle syndrome, oral-facial-digital syndrome type 1 and MIDAS (microphthalmia, dermal aplasia and sclerocornea) syndrome, as well as in various X-linked non-lethal phenotypes, such as hypohidrotic ectodermal dysplasia of Christ-Siemens-Touraine, IFAP (ichthyosis follicularis-alopecia-photophobia) syndrome and X-linked dyskeratosis congenita. Analogous X-inactivation patterns have been documented in human bones, teeth, eyes and, possibly, the brain. Patterns that are distinct from the lines of Blaschko are also seen, such as the lateralization observed in CHILD (congenital hemidysplasia with ichthyosiform nevus and limb defects) syndrome, and the chequerboard pattern seen in women heterozygous for X-linked congenital hypertrichosis. Exceptional cases of either severe or absent involvement in a woman heterozygous for an X-linked trait can be explained by skewing of X inactivation. Some X-linked skin disorders are caused by genes that escape inactivation, which is why heterozygous female 'carriers' of these disorders do not show mosaicism. A well-known example is X-linked recessive ichthyosis due to steroid sulphatase deficiency, the locus for which is situated at the tip of the short arm of the X chromosome and does not undergo Lyonization. On the other hand, in the case of Fabry disease, the gene encoding alpha-galactosidase A is subject to inactivation. Remarkably, however, the skin lesions of women do not show a mosaic pattern. In the various X-linked skin disorders, affected women show quite dissimilar degrees of involvement and forms of manifestation because X inactivation may give rise to different patterns of functional mosaicism. Paradoxically, no such pattern is observed in women with Fabry disease. Like many X-linked diseases, Fabry disease should neither be called recessive nor dominant, because these dichotomous terms are obscured by the mechanism of X inactivation.

Genetic interactions between Cdk1-CyclinB and the Separase complex inDrosophila

Cdk1-CycB plays a key role in regulating many aspects of cell-cycle events, such as cytoskeletal dynamics and chromosome behavior during mitosis. To investigate how Cdk1-CycB controls the coordination of these events, we performed a dosage-sensitive genetic screen, which is based on the observations that increased maternal CycB (four extra gene copies) leads to higher Cdk1-CycB activity in early Drosophila embryos, delays anaphase onset, and generates a sensitized non-lethal phenotype at the blastoderm stage (defined as six cycB phenotype). Here, we report that mutations in the gene three rows (thr) enhance, while mutations in pimples (pim, encoding Drosophila Securin) or separase (Sse) suppress, the sensitized phenotype. In Drosophila, both Pim and Thr are known to regulate Sse activity, and activated Sse cleaves a Cohesin subunit to initiate anaphase. Compared with the six cycB embryos, reducing Thr in embryos with more CycB further delays the initiation of anaphase, whereas reducing either Pim or Sse has the opposite effect. Furthermore, nuclei move slower during cortical migration in embryos with higher Cdk1-CycB activity, whereas reducing either Pim or Sse suppresses this phenotype by causing a novel nuclear migration pattern. Therefore, our genetic screen has identified all three components of the complex that regulates sister chromatid separation, and our observations indicate that interactions between Cdk1-CycB and the Pim-Thr-Sse complex are dosage sensitive.

Dilated cardiomyopathy caused by tissue-specific ablation of SC35 in the heart.

Many genetic diseases are caused by mutations in cis-acting splicing signals, but few are triggered by defective trans-acting splicing factors. Here we report that tissue-specific ablation of the splicing factor SC35 in the heart causes dilated cardiomyopathy (DCM). Although SC35 was deleted early in cardiogenesis by using the MLC-2v-Cre transgenic mouse, heart development appeared largely unaffected, with the DCM phenotype developing 3-5 weeks after birth and the mutant animals having a normal life span. This nonlethal phenotype allowed the identification of downregulated genes by microarray, one of which was the cardiac-specific ryanodine receptor 2. We showed that downregulation of this critical Ca2+ release channel preceded disease symptoms and that the mutant cardiomyocytes exhibited frequency-dependent excitation-contraction coupling defects. The implication of SC35 in heart disease agrees with a recently documented link of SC35 expression to heart failure and interference of splicing regulation during infection by myocarditis-causing viruses. These studies raise a new paradigm for the etiology of certain human heart diseases of genetic or environmental origin that may be triggered by dysfunction in RNA processing.

The Role of HIF-1α in Transcriptional Regulation of the Proximal Tubular Epithelial Cell Response to Hypoxia

Epithelial cells of the kidney represent a primary target for hypoxic injury in ischemic acute renal failure (ARF); however, the underlying transcriptional mechanism(s) remain undefined. In this study, human proximal tubular epithelial cells (HK-2) exposed to hypoxia in vitro demonstrated a non-lethal but dysfunctional phenotype, closely reflective of the epithelial pathobiology of ARF. HK-2 cells exposed to hypoxia demonstrated increased paracellular permeability, decreased proliferation, loss of tight junctional integrity, and significant actin disassembly in the absence of cell death. Microarray analysis of transcriptomic changes underlying this response identified a distinct cohort of 48 genes with a closely shared hypoxia-dependent expression profile. Within this hypoxia-sensitive cluster were genes identified previously as hypoxia-inducible factor-1 (HIF-1)-dependent (e.g. vascular endothelial growth factor and adrenomedullin) as well as genes not previously known to be hypoxia-responsive (e.g. stanniocalcin 2). In hypoxia, HIF-1 bound to evolutionarily conserved hypoxia-response elements (HRE) in the promoters of these genes as well as to the HRE consensus motif. A further subset of these genes, not associated with transcriptional regulation by HIF-1, was also present, suggesting alternative HIF-1-independent pathways. Overexpression of HIF-1 alpha in normoxia induced the expression of a significant number of the hypoxia-dependent genes; however, it did not induce the pathophysiologic epithelial response. In summary, hypoxia-elicited alterations in renal proximal tubular epithelial cells in vitro closely resemble the epithelial pathophysiology of ARF. Our data indicate that although this event may rely heavily on HIF-1-dependent gene transcription, it is likely that separate hypoxia-dependent transcriptional regulators also play a role.

Deletion of exon 4 from human surfactant protein C results in aggresome formation and generation of a dominant negative.

Human surfactant protein C (hSP-C) is synthesized by the alveolar type 2 cell as a 197 amino acid integral membrane proprotein and proteolytically processed to a secreted 3.7 kDa mature form. Although the SP-C null mouse possesses a non-lethal phenotype, a heterozygous substitution of A for G in the first base of intron 4 of the human SP-C gene (c.460+1A>G) has been reported in association with familial interstitial lung disease and absence of mature protein. This mutation produces a splice deletion of exon 4 (deltaExon4) resulting in removal of a positionally conserved cysteine in the C-terminal flanking propeptide. Based on a prior study showing that an identical deletion in the rat isoform diverted mutant protein to stable aggregates, we hypothesized that expression of the deltaExon4 mutation would result in disruption of intracellular trafficking of both mutant and wild-type proSP-C. We tested this in vitro using fusion proteins of EGFP conjugated either to wild-type SP-C (EGFP/hSP-C(1-197)) or to SP-C deleted of Exon4 (EGFP/hSP-C(deltaExon4)). Fluorescence microscopy showed that EGFP/hSP-C(1-197) transfected into A549 cells was expressed in a punctuate pattern in CD63 (+) cytoplasmic vesicles, whereas EGFP/hSP-C(deltaExon4) accumulated in ubiquitinated perinuclear inclusions linked to the microtubule organizing center. A similar juxtanuclear pattern was observed following transfection of SP-C cDNA lacking only cysteine residues in the C-terminal propeptide encoded by Exon 4 (EGFP/hSP-C(C120/121G)). To evaluate whether mutant proSP-C could function as a dominant negative, EGFP/hSP-C(deltaExon4) was cotransfected with HA-tagged hSP-C(1-197) and resulted in the restriction of both forms to perinuclear compartments. Addition of Na(+) 4-phenylbutyrate, a facilitator of trafficking of other misfolded proteins, attenuated the aggregation of EGFP/hSP-C(deltaExon4). We conclude that c.460+1A>G mutation of human SP-C results in disruption of disulfide-mediated folding encoded by Exon 4 leading to diversion of unprocessed proSP-C to aggresomes. The heterotypic oligomerization of hSP-C(1-197) and hSP-C(deltaExon4) provides a molecular mechanism for the dominant-negative effect observed in vivo.

Novel ITGB4 Mutations in Lethal and Nonlethal Variants of Epidermolysis Bullosa with Pyloric Atresia: Missense versus Nonsense

Epidermolysis bullosa with pyloric atresia (EB-PA), an autosomal recessive genodermatosis, manifests with neonatal cutaneous blistering associated with congenital pyloric atresia. The disease is frequently lethal, but nonlethal cases have also been reported. Expression of the alpha6 beta4 integrin is altered at the dermal-epidermal basement-membrane zone; recently, mutations in the corresponding genes (ITGA6 and ITGB4) have been disclosed in a limited number of patients, premature termination codons in both alleles being characteristic of lethal variants. In this study, we have examined the molecular basis of EB-PA in five families, two of them with lethal and three of them with nonlethal variants of the disease. Mutation analysis disclosed novel lesions in both ITGB4 alleles of each proband. One of the patients with lethal EB-PA was a compound heterozygote for premature termination-codon mutations (C738X/4791delCA), whereas the other patient with a lethal variant was homozygous for a missense mutation involving a cysteine residue (C61Y). The three nonlethal cases had missense mutations in both alleles (C562R/C562R, R1281W/R252C, and R1281W/R1281W). Immunofluorescence staining of skin in two of the nonlethal patients and in one of the lethal cases was positive, yet attenuated, for alpha6 and beta4 integrins. These results confirm that ITGB4 mutations underlie EB-PA and show that missense mutations may lead to nonlethal phenotypes.

Recurrence of osteogenesis imperfecta because of paternal mosaicism: Gly862-->Ser substitution in a type I collagen gene (COL1A1).

We determined that two siblings with type III osteogenesis imperfecta (OI) had the same single base substitution that converted the codon for glycine (Gly) 862 to a codon for serine (Ser) in exon 44 of the alpha 1 chain of the type I (alpha 1(I)) collagen gene (COL1A1). The mutation was also detected in various paternal tissues; the mutant allele accounted for approximately 11% of the COL1A1 alleles in blood, 24% of those in fibroblasts, and 43% of those in sperm determined by allele-specific colony hybridization using amplified genomic sequences. These findings demonstrate that germ-line mosaicism in the phenotypically normal father is responsible for the recurrence. There is a cluster of serine substitutions for Gly (Gly832, Gly844 and Gly901) which is associated with nonlethal phenotypes and which is located between two lethal clusters. In the cases studied here, a Gly862-->Ser mutation was identified that is located inside the nonlethal cluster.

Two additional cases of osteogenesis imperfecta with substitutions for glycine in the alpha 2(I) collagen chain. A regional model relating mutation location with phenotype.

The relationship between the clinical severity of osteogenesis imperfecta (OI) and the location and type of amino acid substitution in type I collagen is not identical for mutations in the alpha 1(I) and alpha 2(I) chains. Furthermore, the alpha 2(I) chain, once thought to be associated with moderate forms of OI, has now been associated with approximately as many lethal as non-lethal cases. We describe two novel substitutions for glycine in the alpha 2(I) chain, one associated with a lethal phenotype in twins and the other with a moderate non-lethal phenotype. The type I collagen of all probands was characterized electrophoretically by two populations of alpha chains, one normal and one with delayed migration. Cyanogen bromide peptides of the overmodified alpha 1(I) chains revealed delayed migration of all peptides except CB6. The indicated target region of alpha 1(I) and alpha 2(I) cDNA of the probands was analyzed by RNA-DNA hybrid analysis with RNase A digestion. All probands had mismatches in the region of alpha 2(I) coding for amino acids 642-912. The lethal phenotype was associated with a G-->A mutation, resulting in Gly706-->serine; the non-lethal mutation was a G-->T change resulting in Gly676-->valine. Both mutations occurred de novo in the probands; parental leukocyte DNA was normal. In conjunction with the previously described exon deletions and point mutations in alpha 2(I), these mutations define five alternating non-lethal/lethal regions along the chain and support a regional, as opposed to a gradient, model of OI pathophysiology. These mutations in particular help to define a lethal/non-lethal junction at about alpha 2(I) amino acid 700.

HSP78 encodes a yeast mitochondrial heat shock protein in the Clp family of ATP-dependent proteases.

The Saccharomyces cerevisiae nuclear gene for a 78-kDa mitochondrial heat shock protein (hsp78) was identified in a lambda gt11 expression library through immunological screening with an hsp78-specific monoclonal antibody. Sequencing of HSP78 revealed a long open reading frame capable of encoding an 811-amino-acid, 91.3-kDa basic protein with a putative mitochondrial leader sequence and two potential nucleotide-binding sites. Sequence comparisons revealed that hsp78 is a member of the highly conserved family of Clp proteins and is most closely related to the Escherichia coli ClpB protein, which is thought to be an ATPase subunit of an intracellular ATP-dependent protease. The steady-state levels of HSP78 transcripts and protein varied in response to both thermal stress and carbon source with an approximately 30-fold difference between repressed levels in cells growing fermentatively on glucose at 30 degrees C and derepressed levels in heat-shocked cells growing on a nonfermentable carbon source. The response to heat shock is consistent with the presence of a characteristic heat shock regulatory element in the 5'-flanking DNA. Submitochondrial fractionation showed that hsp78 is a soluble protein located in the mitochondrial matrix. Cells carrying disrupted copies of HSP78 lacked hsp78 but were not impaired in respiratory growth at normal and elevated temperatures or in the ability to survive and retain mitochondrial function after thermal stress. The absence of a strong mitochondrial phenotype in hsp78 mutants is comparable to the nonlethal phenotypes of mutations in other Clp genes in bacteria and yeast. HSP78 is the third gene, with SSC1 and HSP60, known to encode a yeast mitochondrial heat shock protein and the second gene, with HSP104, for a yeast ClpB homolog.

HSP78 Encodes a Yeast Mitochondrial Heat Shock Protein in the Clp Family of ATP-Dependent Proteases

The Saccharomyces cerevisiae nuclear gene for a 78-kDa mitochondrial heat shock protein (hsp78) was identified in a lambda gt11 expression library through immunological screening with an hsp78-specific monoclonal antibody. Sequencing of HSP78 revealed a long open reading frame capable of encoding an 811-amino-acid, 91.3-kDa basic protein with a putative mitochondrial leader sequence and two potential nucleotide-binding sites. Sequence comparisons revealed that hsp78 is a member of the highly conserved family of Clp proteins and is most closely related to the Escherichia coli ClpB protein, which is thought to be an ATPase subunit of an intracellular ATP-dependent protease. The steady-state levels of HSP78 transcripts and protein varied in response to both thermal stress and carbon source with an approximately 30-fold difference between repressed levels in cells growing fermentatively on glucose at 30 degrees C and derepressed levels in heat-shocked cells growing on a nonfermentable carbon source. The response to heat shock is consistent with the presence of a characteristic heat shock regulatory element in the 5'-flanking DNA. Submitochondrial fractionation showed that hsp78 is a soluble protein located in the mitochondrial matrix. Cells carrying disrupted copies of HSP78 lacked hsp78 but were not impaired in respiratory growth at normal and elevated temperatures or in the ability to survive and retain mitochondrial function after thermal stress. The absence of a strong mitochondrial phenotype in hsp78 mutants is comparable to the nonlethal phenotypes of mutations in other Clp genes in bacteria and yeast. HSP78 is the third gene, with SSC1 and HSP60, known to encode a yeast mitochondrial heat shock protein and the second gene, with HSP104, for a yeast ClpB homolog.

Modelling neutral and selective evolution of protein folding

We examine a model evolutionary space consisting of genotypes mapped to their corresponding phenotypes. This mapping is derived from a lattice model for proteins which, despite its highly idealized nature, has been shown to share general properties with real proteins. Large evolutionary networks are observed, with genotypes corresponding to non-lethal phenotypes linked by unit mutational steps. Neutral mutations are necessary for traversing the evolutionary networks, and even one neutral mutation in a genotype can change the phenotypes attainable by a unit mutational step.

A rehabilitation of the genetic map of the 84B-D region in Drosophila melanogaster.

A reanalysis of the 84B3 to 84D3,5 region of the polytene chromosomes of Drosophila melanogaster has led to the identification and localization of 16 genes. These genes include 11 vital loci, four genes exhibiting nonlethal visible mutant phenotypes and one gene encoding a nonessential enzyme. The identity of the gene products of two of the vital genes has been determined to be alpha-tubulin and glucose dehydrogenase (Gld). Three newly identified genes, sticking (stk), half out (hat) and trapped (ted), as well as Gld are required for eclosion. Among the nonessential genes are roughened eye (roe) and ruffed eye (rue), which affect eye texture. The roe phenotype is greatly enhanced by deletions that simultaneously remove roe and an unidentified locus in 84E. Mutations in another nonessential gene, rotund (rn), are characterized by pattern deletions of most adult appendages.