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Despite the essential requirement of telomeric DNA for genome stability, the length of telomere tracts between species substantially differs, raising the question of the minimal length of telomeric DNA necessary for proper function. Here, we address this question using a hypomorphic allele of the telomerase catalytic subunit, TERT. We show that although this construct partially restored telomerase activity to a tert mutant, telomeres continued to shorten over several generations, ultimately stabilizing at a bimodal size distribution. Telomeres on two chromosome arms were maintained at a length of 1 kb, while the remaining telomeres were maintained at 400 bp. The longest telomeres identified in this background were also significantly longer in wild-type populations, suggesting cis-acting elements on these arms either promote telomerase processivity or recruitment. Genetically disrupting telomerase processivity in this background resulted in immediate lethality. Thus, telomeres of 400 bp are both necessary and sufficient for Arabidopsis viability. As this length is the estimated minimal length for t-loop formation, our data suggest that telomeres long enough to form a t-loop constitute the minimal functional length.

Primary ciliary dyskinesia (PCD) is a human condition of dysfunctional motile cilia characterized by recurrent lung infection, infertility, organ laterality defects and partially penetrant hydrocephalus. We recovered a mouse mutant from a forward genetic screen that developed many of the hallmark phenotypes of PCD. Whole-exome sequencing identified this primary ciliary dyskinesia only (Pcdo) allele to be a nonsense mutation (c.5236A>T) in the Spag17 coding sequence creating a premature stop codon (K1746*). The Pcdo variant abolished several isoforms of SPAG17 in the Pcdo mutant testis but not in the brain. Our data indicate differential requirements for SPAG17 in different types of motile cilia. SPAG17 is essential for proper development of the sperm flagellum and is required for either development or stability of the C1 microtubule structure within the central pair apparatus of the respiratory motile cilia, but not the brain ependymal cilia. We identified changes in ependymal ciliary beating frequency, but these did not appear to alter lateral ventricle cerebrospinal fluid flow. Aqueductal stenosis resulted in significantly slower and abnormally directed cerebrospinal fluid flow, and we suggest that this is the root cause of the hydrocephalus. The Spag17Pcdo homozygous mutant mice are generally viable to adulthood but have a significantly shortened lifespan, with chronic morbidity. Our data indicate that the c.5236A>T Pcdo variant is a hypomorphic allele of Spag17 that causes phenotypes related to motile, but not primary, cilia. Spag17Pcdo is a useful new model for elucidating the molecular mechanisms underlying central pair PCD pathogenesis in the mouse. This article has an associated First Person interview with the first author of the paper., Summary: The Spag17pcdo mouse model closely recapitulates the human central pair primary ciliary dyskinesia condition, and the data here reinforce the power of the hypomorphic allele in representing human conditions.

The functional redundancy of the three mammalian Golgi-localized, γ-ear–containing, ADP-ribosylation factor-binding proteins (GGAs) was addressed in a previous study. Using insertional mutagenesis, we found that Gga1 or Gga3 homozygous knockout mice were for the most part normal, whereas mice homozygous for two different Gga2 gene-trap alleles exhibited either embryonic or neonatal lethality in the C57BL/6 background, depending on the source of the vector utilized (Byg vs. Tigm, respectively). We now show that the Byg strain harbors a disrupted Gga2 allele that is hypomorphic, indicating that the Byg lethality is attributable to a mechanism independent of GGA2. This is in contrast to the Tigm Gga2 allele, which is a true knockout and establishes a role for GGA2 during the neonatal period. Placement of the Tigm Gga2 allele into the C57BL6/Ola129Sv mixed background results in a lower incidence of neonatal lethality, showing the importance of genetic background in determining the requirement for GGA2 during this period. The Gga2−/− mice that survive have reduced body weight at birth and this runted phenotype is maintained through adulthood.

Previous analysis employing chimeric and transgenic rescue experiments has suggested that Otx2 is required in the neuroectoderm for development of the forebrain region. In order to elucidate the precise role of Otx2 in forebrain development, we attempted to generate an allelic series of Otx2 mutations by Flp- and Cre-mediated recombination for the production of conditional knock-out mice. Unexpectedly, the neo-cassette insertion created a hypomorphic Otx2 allele; consequently, the phenotype of compound mutant embryos carrying both a hypomorphic and a null allele (Otx2frt-neo/−) was analyzed. Otx2frt-neo/− mutant mice died at birth, displaying rostral head malformations. Molecular marker analysis demonstrated that Otx2frt-neo/− mutant embryos appeared to undergo anterior–posterior axis generation and induction of anterior neuroectoderm normally; however, these mutants subsequently failed to correctly specify the forebrain region. As the rostral margin of the neural plate, termed the anterior neural ridge (ANR), plays crucial roles with respect to neural plate specification, we examined expression of molecular markers for the ANR and the neural plate; moreover, neural plate explant studies were performed. Analyses revealed that telencephalic gene expression did not occur in mutant embryos due to defects of the neural plate; however, the mutant ANR bore normal induction activity on gene expression. These results further suggest that Otx2 dosage may be crucial in the neural plate with respect to response to inductive signals primarily from the ANR for forebrain specification.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by stereotyped missense mutations in NOTCH3. Whether these mutations lead to the CADASIL phenotype via a neomorphic effect, or rather by a hypomorphic effect, is subject of debate. Here, we report two novel NOTCH3 mutations, both leading to a premature stop codon with predicted loss of NOTCH3 function. The first mutation, c.307C>T, p.Arg103*, was detected in two brothers aged 50 and 55 years, with a brain MRI and skin biopsy incompatible with CADASIL. The other mutation was found in a 40-year-old CADASIL patient compound heterozygous for a pathogenic NOTCH3 mutation (c.2129A>G, p.Tyr710Cys) and an intragenic frameshift deletion. The deletion was inherited from his father, who did not have the skin biopsy abnormalities seen in CADASIL patients. These individuals with rare NOTCH3 mutations indicate that hypomorphic NOTCH3 alleles do not cause CADASIL.

Identifying the piece of DNA culprit of a genetic disease has not been as straightforward as expected with the evolution of sequencing capabilities. This difficulty is exemplified by facioscapulohumeral dystrophy (FSHD) study. Unexpectedly, contributions in completing this picture recently came from mice models carrying a muscle specific knock-out of the protocadherin gene Fat1 or its constitutive hypomorphic allele. Both have been shown to develop muscular and non-muscular defects mimicking human FSHD. In humans, this disease is linked to copy number reduction ( n

Precisely targeted genetic manipulation of the laboratory mouse has been one of the most significant achievements in the field of modeling human genetic diseases. The availability of an ever-increasing number of mutants, and the ability of generating ever-more complex manipulations beyond the “simple” gene knockout, offer unprecedented possibilities for genetic experiments addressing complex questions. Thus, the mouse is getting closer and closer to the most powerful genetic models such as Drosophila.

Autosomal dominant polycystic kidney disease (ADPKD) caused by mutations in PKD1 is significantly more severe than PKD2. Typically, ADPKD presents in adulthood but is rarely diagnosed in utero with enlarged, echogenic kidneys. Somatic mutations are thought crucial for cyst development, but gene dosage is also important since animal models with hypomorphic alleles develop cysts, but are viable as homozygotes. We screened for mutations in PKD1 and PKD2 in two consanguineous families and found PKD1 missense variants predicted to be pathogenic. In one family, two siblings homozygous for R3277C developed end stage renal disease at ages 75 and 62 years, while six heterozygotes had few cysts. In the other family, the father and two children with moderate to severe disease were homozygous for N3188S. In both families homozygous disease was associated with small cysts of relatively uniform size while marked cyst heterogeneity is typical of ADPKD. In another family, one patient diagnosed in childhood was found to be a compound heterozygote for the PKD1 variants R3105W and R2765C. All three families had evidence of developmental defects of the collecting system. Three additional ADPKD families with in utero onset had a truncating mutation in trans with either R3277C or R2765C. These cases suggest the presence of incompletely penetrant PKD1 alleles. The alleles alone may result in mild cystic disease; two such alleles cause typical to severe disease; and, in combination with an inactivating allele, are associated with early onset disease. Our study indicates that the dosage of functional PKD1 protein may be critical for cyst initiation.

The aryl hydrocarbon receptor-associated protein-9 (ARA9) is a chaperone of the aryl hydrocarbon receptor (AHR). The AHR has been shown to play a late developmental role in the normal closure of a fetal hepatovascular shunt known as the ductus venosus (DV). Given that Ara9-null mice display early embryonic lethality, we generated a hypomorphic Ara9 allele (designated Ara9(fxneo)) that displays reduced ARA9 protein expression. In an effort to demonstrate the role of ARA9 protein in AHR-mediated DV closure, we used combinations of Ara9 wild-type [Ara9(+/+)], null [Ara9(-/-)], and hypomorphic [Ara9(fxneo/fxneo)] alleles to produce mice with a graded expression of the ARA9 protein. Liver perfusion studies demonstrated that although none of the Ara9(+/+) mice displayed a patent DV, the shunt was observed in 10% of the Ara9(+/fxneo) mice, 55% of the Ara9(+/-) mice, and 83% of the Ara9(fxneo/fxneo) mice. That expression level of ARA9 correlates with the frequency of a phenocopy of the Ahr-null allele supports the conclusion that the ARA9 protein is essential for AHR signaling during development.

Mahoganoid ( Mgrn1md) is a mutation of the mahogunin ( Mgrn1) gene. The hypomorphic allele suppresses the yellow pigmentation and obesity of the Aymouse that ubiquitously overexpresses agouti signaling protein (ASP). To assess the physiological effects of MGRN1 on energy and glucose homeostasis, we generated animals doubly mutant for Mgrn1mdand Ay, Lepob, or a null allele of Mc4r, and diet-induced obesity (DIO) mice segregating for Mgrn1md. Mgrn1mdsuppressed the obesity, hyperglycemia, and hyperinsulinemia of Aymice. Mgrn1mdsuppressed Ay-induced obesity by reducing food intake, and reduced adiposity in Lepob/Lepobfemales, but did not alter the body weight or body composition of mice fed a high-fat diet. There was no effect of Mgrn1mdon weight gain, body composition, energy intake, or energy expenditure in Mc4r-null animals. Mgrn1mdreduced circulating insulin concentrations in DIO, Ay, and Mc4r-null but not Lepob/Lepobmice. The effect of Mgrn1mdon circulating insulin concentrations was not due primarily to reductions in fat mass, since the plasma insulin concentrations of Mgrn1mdmice segregating for either Ayor Mc4r-null alleles, adjusted for fat mass and plasma glucose, were reduced compared with Ayand Mc4r mice, respectively. The effect of Mgrn1mdon insulin sensitivity of Mc4r-null mice suggests that Mgrn1mdmay be increasing insulin sensitivity via the hypothalamic melanocortin-3 receptor pathway.

9687 Background: TGFBR1*6A is a hypomorphic allele of the type I transforming growth factor beta receptor (TGFBR1) that has been proposed as a cancer susceptibility allele for various malignancies....

Mutations of PKD1 and PKD2 account for most cases of autosomal dominant polycystic kidney disease (ADPKD). Compared to PKD2, patients with PKD1 typically have more severe renal disease. Here, we report a follow-up study of a unique multi-generation family with bilineal ADPKD (NFL10) in which a PKD1 disease haplotype and a PKD2 (L736X) mutation co-segregated with 18 and 14 affected individuals, respectively. In our updated genotype-phenotype analysis of NFL10, we found that PKD1-affected individuals had uniformly mild renal disease similar to PKD2-affected individuals. By sequencing all the exons and splice junctions of PKD1, we identified two missense mutations (Y528C and R1942H) from a PKD1-affected individual. Although both variants were predicted to be damaging to the mutant protein, only Y528C co-segregated with all the PKD1-affected individuals in NFL10. To further establish the pathogenicity of Y528C, we performed in-vitro studies in stable MDCK cell lines expressing wild-type and mutant forms of PKD1. We found that MDCK cell lines expressing the Y528C variant formed cysts in culture and demonstrated increased rates of growth and apoptosis. Taken together, our data suggest that Y528C functions as a hypomorphic PKD1 allele. These findings have important implications for pathogenic mechanisms and molecular diagnostics of ADPKD.