Null Mutation Research Articles

Chromatin architectural alterations due to null mutation of a major CG methylase in rice.

Associations between 3D chromatin architectures and epigenetic modifications have been characterized in animals. However, any impact of DNA methylation on chromatin architecture in plants is understudied, which is confined to Arabidopsis thaliana. Because plant species differ in genome size, composition, and overall chromatin packing, it is unclear to what extent findings from A. thaliana hold in other species. Moreover, the incomplete chromatin architectural profiles and the low-resolution high-throughput chromosome conformation capture (Hi-C) data from A. thaliana have hampered characterizing its subtle chromatin structures and their associations with DNA methylation. We constructed a high-resolution Hi-C interaction map for the null OsMET1-2 (the major CG methyltransferase in rice) mutant (osmet1-2) and isogenic wild-type rice (WT). Chromatin structural changes occurred in osmet1-2, including intra-/inter-chromosomal interactions, compartment transition, and topologically associated domains (TAD) variations. Our findings provide novel insights into the potential function of DNA methylation in TAD formation in rice and confirmed DNA methylation plays similar essential roles in chromatin packing in A. thaliana and rice.

Biallelic variants in PIGN cause Fryns syndrome, multiple congenital anomalies-hypotonia-seizures syndrome, and neurologic phenotypes: A genotype–phenotype correlation study

PurposeBiallelic PIGN variants have been described in Fryns syndrome, multiple congenital anomalies-hypotonia-seizure syndrome (MCAHS), and neurologic phenotypes. The full spectrum of clinical manifestations in relation to the genotypes is yet to be reported. MethodsGenotype and phenotype data were collated and analyzed for 61 biallelic PIGN cases: 21 new and 40 previously published cases. Functional analysis was performed for 2 recurrent variants (c.2679C>G p.Ser893Arg and c.932T>G p.Leu311Trp). ResultsBiallelic-truncating variants were detected in 16 patients—10 with Fryns syndrome, 1 with MCAHS1, 2 with Fryns syndrome/MCAHS1, and 3 with neurologic phenotype. There was an increased risk of prenatal or neonatal death within this group (6 deaths were in utero or within 2 months of life; 6 pregnancies were terminated). Incidence of polyhydramnios, congenital anomalies (eg, diaphragmatic hernia), and dysmorphism was significantly increased.Biallelic missense or mixed genotype were reported in the remaining 45 cases—32 showed a neurologic phenotype and 12 had MCAHS1. No cases of diaphragmatic hernia or abdominal wall defects were seen in this group except patient 1 in which we found the missense variant p.Ser893Arg to result in functionally null alleles, suggesting the possibility of an undescribed functionally important region in the final exon.For all genotypes, there was complete penetrance for developmental delay and near-complete penetrance for seizures and hypotonia in patients surviving the neonatal period. ConclusionWe have expanded the described spectrum of phenotypes and natural history associated with biallelic PIGN variants. Our study shows that biallelic-truncating variants usually result in the more severe Fryns syndrome phenotype, but neurologic problems, such as developmental delay, seizures, and hypotonia, present across all genotypes. Functional analysis should be considered when the genotypes do not correlate with the predicted phenotype because there may be other functionally important regions in PIGN that are yet to be discovered.

Large fragment Sanger sequencing identifies the newly encountered variant that caused null alleles in parentage testing.

Short tandem repeat (STR) is regarded as a crucial tool for personal identification as well as parentage testing. Thus, genotyping errors of STRs could have negative effects on the reliability of forensic identification. A null allele at the combined DNA index system (CODIS) core loci D2S1338 was found in a father-daughter pair with the AGCU Expressmarker 22 kit which was a commonly used commercial kit during our daily laboratory work. This null allele caused the father and daughter to not conform to the laws of inheritance, thus potentially generating erroneous conclusions that excluded parentage. To figure out the reason for this phenomenon, re-amplification with new primers and then large fragment Sanger sequencing was conducted. We found a G to G/T variation at the position which is fifty-nine bases away from the 3' end of the core repeat in both samples. This probably could be considered a novel variant at the primer binding region which had not been reported that resulted in the emergence of the null allele. We also found that there was more than one single-nucleotide polymorphism (SNP) with minor allele frequency (MAF) greater than 0.1 in the upstream and downstream sequences of D2S1338. When designing primers for amplification of D2S1338, the possible adverse results of these SNPs should be taken into account and avoided.

Insulin/IGF signaling regulates presynaptic glutamate release in aversive olfactory learning.

Insulin/insulin-like growth factor (IGF) receptor signaling (IIS) supports context-dependent learning in vertebrates and invertebrates. Here, we identify cell-specific mechanisms of IIS that integrate sensory information with food context to drive synaptic plasticity and learning. In the nematode Caenorhabditis elegans, pairing food deprivation with an odor such as butanone suppresses attraction to that odor. We find that aversive olfactory learning requires the insulin receptor substrate (IRS) protein IST-1 and atypical signaling through the insulin/IGF-1 receptor DAF-2. Cell-specific knockout and rescue demonstrate that DAF-2 acts in the AWCON sensory neuron, which detects butanone, and that learning preferentially depends upon the axonally localized DAF-2c isoform. Acute food deprivation increases DAF-2 levels in AWCON post-transcriptionally through an insulin- and insulin receptor substrate-1 (ist-1)-dependent process. Aversive learning alters the synaptic output of AWCON by suppressing odor-regulated glutamate release in wild-type animals, but not in ist-1 mutants, suggesting that axonal insulin signaling regulates synaptic transmission to support aversive memory.

Systems Genetics Approaches Uncover Pathway-Level Interactions in Mice Carrying APOL1 G0/G2 Transgene and Gstm1 Null Alleles

Systems Genetics Approaches Uncover Pathway-Level Interactions in Mice Carrying APOL1 G0/G2 Transgene and Gstm1 Null Alleles

Non-human Leukocyte Antigen (Non-HLA) Antibodies Against GSTT1 Are Associated With Homozygote Status for the Null Allele and With Histological Changes of Antibody Mediated Rejection

Non-human Leukocyte Antigen (Non-HLA) Antibodies Against GSTT1 Are Associated With Homozygote Status for the Null Allele and With Histological Changes of Antibody Mediated Rejection

Identification of a novel HLA-A null allele, HLA-A*01:420N allele.

The novel HLA-A*01:420N allele has two changes in exon 4 leading to premature stop codon.

A conditional null allele of Dync1h1 enables targeted analyses of dynein roles in neuronal length sensing.

Size homeostasis is a fundamental process in biology and is particularly important for large cells such as neurons. We previously proposed a motor-dependent length-sensing mechanism wherein reductions in microtubule motor levels would be expected to accelerate neuronal growth, and validated this prediction in dynein heavy chain 1 Loa mutant (Dync1h1Loa) sensory neurons. Here, we describe a new mouse model with a conditional deletion allele of exons 24 and 25 in Dync1h1. Homozygous Islet1-Cre-mediated deletion of Dync1h1 (Isl1-Dync1h1-/-), which deletes protein from the motor and sensory neurons, is embryonic lethal, but heterozygous animals (Isl1-Dync1h1+/-) survive to adulthood with ∼50% dynein expression in targeted cells. Isl1-Dync1h1+/- sensory neurons reveal accelerated growth, as previously reported in Dync1h1Loa neurons. Moreover, Isl1-Dync1h1+/- mice show mild impairments in gait, proprioception and tactile sensation, similar to what is seen in Dync1h1Loa mice, confirming that specific aspects of the Loa phenotype are due to reduced dynein levels. Isl1-Dync1h1+/- mice also show delayed recovery from peripheral nerve injury, likely due to reduced injury signal delivery from axonal lesion sites. Thus, conditional deletion of Dync1h1 exons 24 and 25 enables targeted studies of the role of dynein in neuronal growth.

CVID-Associated B Cell Activating Factor Receptor Variants Change Receptor Oligomerization, Ligand Binding, and Signaling Responses

PurposeBinding of the B cell activating factor (BAFF) to its receptor (BAFFR) activates in mature B cells many essential pro-survival functions. Null mutations in the BAFFR gene result in complete BAFFR deficiency and cause a block in B cell development at the transition from immature to mature B cells leading therefore to B lymphopenia and hypogammaglobulinemia. In addition to complete BAFFR deficiency, single nucleotide variants encoding BAFFR missense mutations were found in patients suffering from common variable immunodeficiency (CVID), autoimmunity, or B cell lymphomas. As it remained unclear to which extent such variants disturb the activity of BAFFR, we performed genetic association studies and developed a cellular system that allows the unbiased analysis of BAFFR variants regarding oligomerization, signaling, and ectodomain shedding.MethodsIn addition to genetic association studies, the BAFFR variants P21R, A52T, G64V, DUP92-95, P146S, and H159Y were expressed by lentiviral gene transfer in DG-75 Burkitt’s lymphoma cells and analyzed for their impacts on BAFFR function.ResultsBinding of BAFF to BAFFR was affected by P21R and A52T. Spontaneous oligomerization of BAFFR was disturbed by P21R, A52T, G64V, and P146S. BAFF-dependent activation of NF-κB2 was reduced by P21R and P146S, while interactions between BAFFR and the B cell antigen receptor component CD79B and AKT phosphorylation were impaired by P21R, A52T, G64V, and DUP92-95. P21R, G64V, and DUP92-95 interfered with phosphorylation of ERK1/2, while BAFF-induced shedding of the BAFFR ectodomain was only impaired by P21R.ConclusionAlthough all variants change BAFFR function and have the potential to contribute as modifiers to the development of primary antibody deficiencies, autoimmunity, and lymphoma, P21R is the only variant that was found to correlate positively with CVID.

OsMADS58 Stabilizes Gene Regulatory Circuits during Rice Stamen Development.

Rice (Oryza sativa) OsMADS58 is a C-class MADS box protein, and characterization of a transposon insertion mutant osmads58 suggested that OsMADS58 plays a role in stamen development. However, as no null mutation has been obtained, its role has remained unclear. Here, we report that the CRISPR knockout mutant osmads58 exhibits complex altered phenotypes, including anomalous diploid germ cells, aberrant meiosis, and delayed tapetum degeneration. This CRISPR mutant line exhibited stronger changes in expression of OsMADS58 target genes compared with the osmads58 dSpm (transposon insertion) line, along with changes in multiple pathways related to early stamen development. Notably, transcriptional regulatory circuits in young panicles covering the stamen at stages 4-6 were substantially altered in the CRISPR line compared to the dSpm line. These findings strongly suggest that the pleiotropic effects of OsMADS58 on stamen development derive from a potential role in stabilizing gene regulatory circuits during early stamen development. Thus, this work opens new avenues for viewing and deciphering the regulatory mechanisms of early stamen development from a network perspective.

Generation of myostatin gene-edited blotched snakehead (Channa maculata) using CRISPR/Cas9 system

Blotched snakehead (Channa maculata) is one of the most important freshwater fish in China. Genome editing is of great potential in generating new snakehead strains with valuable economic traits without integration of exogenous genes. Myostatin (Mstn) inhibits the development and growth of skeletal muscle in vertebrates, including teleosts. Mstn mutations induced by genome editing can stimulate muscle growth and development, therefore Mstn is an appropriate candidate to breed the fast-growing snakehead strain. For this study, one orthologous Mstn gene was characterized in C. maculata, designated as CmMstn, which possessed the relatively conserved gene and protein structure. CmMstn had the highest expression level and sexual dimorphism in muscles. Two CRISPR/Cas9 target sites were designed to generate knockout mutants within the mature mRNA sequence of CmMstn located in Exon I and Exon II. 1 nL mixture solution of 200 pg gRNA and 300 pg Cas9 protein was directly microinjected into each snakehead embryonic yolk at 1⁓4 cell stage. Mstn was efficiently edited by CRISPR/Cas9 system in shakehead, and all designed gRNAs effectively worked at their target sites with high mutagenesis efficiencies, from 40.0% to 53.3%. The majority of mutations were frame-shift and resulted in premature termination of transcription and disruption of the molecular function of CmMstn protein. Furthermore, we obtained four founders (F0) of snakehead with the mutated Mstn gene in their primordial germ cells, and nineteen F1 offspring of the F0 mutants mated with wild-type individuals were displayed to carry Mstn null alleles. It is the first application of genome editing technology in blotched snakehead, and CRISPR/Cas9 system successfully edit the primordial germ cells. Our findings will accelerate our understanding of the role of Mstn gene in snakehead growth, and speed up the cultivation of the fast-growing snakehead strain.

Analysis of ABO subgroups which result in ABO discrepancies in Iranian blood donors

The ABO blood group system plays a vital role in the blood transfusion and transplantation medicine [ [1] Hosseini‐Maaf B. Hellberg Å. Chester M.A. Olsson M.L. An extensive polymerase chain reaction–allele‐specific polymorphism strategy for clinical ABO blood group genotyping that avoids potential errors caused by null, subgroup, and hybrid alleles. Transfusion. 2007; 47: 2110-2125 Crossref PubMed Scopus (43) Google Scholar ]. It consists of the major A, B, O alleles and many of subtypes with weak expression of A or B antigens on red blood cells. The results of both forward and reverse ABO typing are necessary for ABO blood group typing, as they are both necessary for interpreting the results [ [2] Arumugam P. Hamsavardhini S. Ravishankar J. Bharath R. Resolving ABO discrepancies by serological workup‐an analysis of few cases. Int J Res Med Sci. 2017; 5: 893-900 Crossref Google Scholar ]. ABO discrepancies may be due to technical or human errors, weak subgroups, the transient expression of antigens in some disease like carcinomas and mixed-filled agglutination after massive or chronic transfusion [ 3 Kaur G. Kaur P. Basu S. Kaur R. Blood group discrepancies at a tertiary care centre–analysis and resolution. Int J Lab Hematol. 2014; 36: 481-487 Crossref PubMed Scopus (18) Google Scholar , 4 Meny G. Recognizing and resolving ABO discrepancies. Immunohematology. 2017; 33: 76-81 Crossref PubMed Google Scholar , 5 Storry J.R. Condon J. Hult A.K. Harrison A. Jørgensen R. Olsson M.L. An age‐dependent ABO discrepancy between mother and baby reveals a novel A weak allele. Transfusion. 2015; 55: 422-426 Crossref PubMed Scopus (7) Google Scholar ]. The incidence of ABO discrepancies among blood donors was reported from 0.02 % to 0.06 %, Also the ABO subgroups are considered the most common cause of ABO discrepancies [ 3 Kaur G. Kaur P. Basu S. Kaur R. Blood group discrepancies at a tertiary care centre–analysis and resolution. Int J Lab Hematol. 2014; 36: 481-487 Crossref PubMed Scopus (18) Google Scholar , 6 Sharma T. Garg N. Singh B. ABO blood group discrepancies among blood donors in Regional Blood Transfusion Centre GTB Hospital, Delhi, India. Transfus Apher Sci. 2014; 50: 75-80 Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar , 7 Makroo R.N. Kakkar B. Agrawal S. Chowdhry M. Prakash B. Karna P. Retrospective analysis of forward and reverse ABO typing discrepancies among patients and blood donors in a tertiary care hospital. Transfus Med. 2019; 29: 103-109 Crossref PubMed Scopus (10) Google Scholar ]. Weak ABO subgroups are categorized as type II (Weak/missing antigen) or type IV (due to presence of Anti–A1) ABO discrepancy [ [8] Jain A. Gupta A. Malhotra S. Marwaha N. Sharma R.R. An ABO blood grouping discrepancy: Probable B (A) phenotype. Transfus Apher Sci. 2017; 56: 459-460 Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar ]. Mistyped A and B weak blood group as O and B(A) as cisAB and AxB during routine serologic tests can have serious consequences, such as hemolytic adverse transfusion reactions [ [9] Jiao L.X. Zhang J.Y. Chen L. Yu X.L. Han Y. Yang F. et al. Gene identification of rare B (A) blood group. Transfus Apher Sci. 2017; 56: 855-857 Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar ].

The PINK1 p.Asn521Thr Variant Is Associated with Earlier Disease Onset in GRN/C9orf72 Frontotemporal Lobar Degeneration.

Genetic frontotemporal lobar degeneration (FTLD) is characterized by heterogeneous phenotypic expression, with a disease onset highly variable even in patients carrying the same mutation. Herein we investigated if variants in lysosomal genes modulate the age of onset both in FTLD due to GRN null mutations and C9orf72 expansion. In a total of 127 subjects (n = 74 GRN mutations and n = 53 C9orf72 expansion carriers), we performed targeted sequencing of the top 98 genes belonging to the lysosomal pathway, selected based on their high expression in multiple brain regions. We described an earlier disease onset in GRN/C9orf72 pedigrees in subjects carrying the p.Asn521Thr variant (rs1043424) in PTEN-induced kinase 1 (PINK1), a gene that is already known to be involved in neurodegenerative diseases. We found that: (i) the PINK1 rs1043424 C allele is significantly associated with the age of onset; (ii) every risk C allele increases hazard by 2.11%; (iii) the estimated median age of onset in homozygous risk allele carriers is 10-12 years earlier than heterozygous/wild type homozygous subjects. A replication study in GRN/C9orf72 negative FTLD patients confirmed that the rs1043424 C allele was associated with earlier disease onset (-5.5 years in CC versus A carriers). Understanding the potential mechanisms behind the observed modulating effect of the PINK1 gene in FTLD might prove critical for identifying biomarkers and/or designing drugs to modify the age of onset, especially in GRN/C9orf72-driven disease.

Analyses of the autism-associated neuroligin-3 R451C mutation in human neurons reveal a gain-of-function synaptic mechanism.

Mutations in many synaptic genes are associated with autism spectrum disorders (ASD), suggesting that synaptic dysfunction is a key driver of ASD pathogenesis. Among these mutations, the R451C substitution in the NLGN3 gene that encodes the postsynaptic adhesion molecule Neuroligin-3 is noteworthy because it was the first specific mutation linked to ASDs. In mice, the corresponding Nlgn3 R451C-knockin mutation recapitulates social interaction deficits of ASD patients and produces synaptic abnormalities, but the impact of the NLGN3 R451C mutation on human neurons has not been investigated. Here, we generated human knockin neurons with the NLGN3 R451C and NLGN3 null mutations. Strikingly, analyses of NLGN3 R451C-mutant neurons revealed that the R451C mutation decreased NLGN3 protein levels but enhanced the strength of excitatory synapses without affecting inhibitory synapses; meanwhile NLGN3 knockout neurons showed reduction in excitatory synaptic strengths. Moreover, overexpression of NLGN3 R451C recapitulated the synaptic enhancement in human neurons. Notably, the augmentation of excitatory transmission was confirmed in vivo with human neurons transplanted into mouse forebrain. Using single-cell RNA-seq experiments with co-cultured excitatory and inhibitory NLGN3 R451C-mutant neurons, we identified differentially expressed genes in relatively mature human neurons corresponding to synaptic gene expression networks. Moreover, gene ontology and enrichment analyses revealed convergent gene networks associated with ASDs and other mental disorders. Our findings suggest that the NLGN3 R451C mutation induces a gain-of-function enhancement in excitatory synaptic transmission that may contribute to the pathophysiology of ASD.

The GATA factor ELT-3 specifies endoderm in Caenorhabditis angaria in an ancestral gene network.

Endoderm specification in Caenorhabditis elegans occurs through a network in which maternally provided SKN-1/Nrf, with additional input from POP-1/TCF, activates the GATA factor cascade MED-1,2→END-1,3→ELT-2,7. Orthologues of the MED, END and ELT-7 factors are found only among nematodes closely related to C. elegans, raising the question of how gut is specified in their absence in more distant species in the genus. We find that the C. angaria, C. portoensis and C. monodelphis orthologues of the GATA factor gene elt-3 are expressed in the early E lineage, just before their elt-2 orthologues. In C. angaria, Can-pop-1(RNAi), Can-elt-3(RNAi) and a Can-elt-3 null mutation result in a penetrant 'gutless' phenotype. Can-pop-1 is necessary for Can-elt-3 activation, showing that it acts upstream. Forced early E lineage expression of Can-elt-3 in C. elegans can direct the expression of a Can-elt-2 transgene and rescue an elt-7 end-1 end-3; elt-2 quadruple mutant strain to viability. Our results demonstrate an ancestral mechanism for gut specification and differentiation in Caenorhabditis involving a simpler POP-1→ELT-3→ELT-2 gene network.

Wide spectrum of neuronal and network phenotypes in human stem cell-derived excitatory neurons with Rett syndrome-associated MECP2 mutations

Rett syndrome (RTT) is a severe neurodevelopmental disorder primarily caused by heterozygous loss-of-function mutations in the X-linked gene MECP2 that is a global transcriptional regulator. Mutations in the methyl-CpG binding domain (MBD) of MECP2 disrupt its interaction with methylated DNA. Here, we investigate the effect of a novel MECP2 L124W missense mutation in the MBD of an atypical RTT patient with preserved speech in comparison to severe MECP2 null mutations. L124W protein had a limited ability to disrupt heterochromatic chromocenters due to decreased binding dynamics. We isolated two pairs of isogenic WT and L124W induced pluripotent stem cells. L124W induced excitatory neurons expressed stable protein, exhibited increased input resistance and decreased voltage-gated Na+ and K+ currents, and their neuronal dysmorphology was limited to decreased dendritic complexity. Three isogenic pairs of MECP2 null neurons had the expected more extreme morphological and electrophysiological phenotypes. We examined development and maturation of L124W and MECP2 null excitatory neural network activity using micro-electrode arrays. Relative to isogenic controls, L124W neurons had an increase in synchronous network burst frequency, in contrast to MECP2 null neurons that suffered a significant decrease in synchronous network burst frequency and a transient extension of network burst duration. A biologically motivated computational neural network model shows the observed changes in network dynamics are explained by changes in intrinsic Na+ and K+ currents in individual neurons. Our multilevel results demonstrate that RTT excitatory neurons show a wide spectrum of morphological, electrophysiological and circuitry phenotypes that are dependent on the severity of the MECP2 mutation.

Glucose-6-phosphate dehydrogenase exerts antistress effects independently of its enzymatic activity

G6PD (glucose-6-phosphate dehydrogenase) is the rate-limiting enzyme in the oxidative pentose phosphate pathway that can generate cytosolic NADPH for biosynthesis and oxidative defense. Since cytosolic NADPH can be compensatively produced by other sources, the enzymatic activity deficiency alleles of G6PD are well tolerated in somatic cells but the effect of null mutations is unclear. Herein, we show that G6PD KO sensitizes cells to the stresses induced by hydrogen peroxide, superoxide, hypoxia, and the inhibition of the electron transport chain. This effect can be completely reversed by the expressions of natural mutants associated with G6PD deficiency, even without dehydrogenase activity, exactly like the WT G6PD. Furthermore, we demonstrate that G6PD can physically interact with AMPK (AMPK-activated protein kinase) to facilitate its activity and directly bind to NAMPT (nicotinamide phosphoribosyltransferase) to promote its activity and maintain the NAD(P)H/NAD(P)+ homeostasis. These functions are necessary to the antistress ability of cells but independent of the dehydrogenase activity of G6PD. In addition, the WT G6PD and naturally inactive mutant also can similarly regulate the metabolism of glucose, glutamine, fatty acid synthesis, and GSH and interact with the involved enzymes. Therefore, our findings reveal the previously unidentified functions of G6PD that can act as the important physiological neutralizer of stresses independently of its enzymatic activity.

Low-density lipoprotein receptor genotypes modify the sera metabolome of patients with homozygous familial hypercholesterolemia

Homozygous familial hypercholesterolemia (HoFH) is an extremely rare metabolism disorder usually caused by low-density lipoprotein receptor (LDLR) mutations. LDLR genotype is commonly known to determine blood concentrations of LDL cholesterol. However, effects of LDLR genotype on holistic metabolome remain unclear. Herein, we present metabolomic, genetic, and clinical datasets from a large multi-center panel of 142 patients with LDLR-mutated HoFH. Wefound that true homozygotes and compound heterozygotes showed few differences in clinical and metabolomic phenotypes. Compared with defective/defective mutation carriers, patients carrying one or two null mutation showed profound alterations in clinical laboratory lipids and serum cholesterol esters, lysophosphocholines, bile acids, and amino acids. Importantly, these altered metabolites are implicated in multiple biochemical reactions and associated with LDL cholesterol. This study extends the first map of different LDLR genotypes influencing the metabolome and suggests that the small-molecule metabolites serve as potential targets to mitigate the deleterious impact of LDLR mutations on HoFH.

Null allele can bring to interpretative problems in a deficitary paternity case

An STR null allele is an allele at a microsatellite locus that fails to amplify. A possible cause is poor primer annealing due to nucleotide sequence divergence in the flanking primers. In this study, a woman (ZAM) wanted to know whether a man (PGAF) was the father of her child (ZGC). During the court settlement, PGAF died. PGAF’s parents refused to undergo DNA investigation and denied the access to biological fragments from their dead son. Although, DNA specimens were obtained from buccal swabs of ZAM, ZGC and PGAF’s paternal sister (PTFS). Initially, only autosomal profiles were studied, and kinship assignment was inconclusive. Following our requests, PGAF’s parents (PRGF and LLGM) led us to obtain their DNA specimens. Only with the PTFS genetic profile, we were not able to demonstrate a kinship assignment. PTFS showed a homozygosis at D8S1179 locus. Then, merely comparing PTFS, LLGM and ZGC autosomal genetic profiles it was possible to underline that they were three different homozygous at D8S1179 locus. Hence, comparing the peak heights in different loci and according to literature, they had to carry a null allele at this locus. Parental studies were completed by Y haplotype analysis.

Genomics of sexual cell fate transdifferentiation in the mouse gonad.

Sex determination in mammals hinges on a cell fate decision in the fetal bipotential gonad between formation of male Sertoli cells or female granulosa cells. While this decision normally is permanent, loss of key cell fate regulators such as the transcription factors Dmrt1 and Foxl2 can cause postnatal transdifferentiation from Sertoli to granulosa-like (Dmrt1) or vice versa (Foxl2). Here, we examine the mechanism of male-to-female transdifferentiation in mice carrying either a null mutation of Dmrt1 or a point mutation, R111G, that alters the DNA-binding motif and causes human XY gonadal dysgenesis and sex reversal. We first define genes misexpressed during transdifferentiation and then show that female transcriptional regulators driving transdifferentiation in the mutant XY gonad (ESR2, LRH1, FOXL2) bind chromatin sites related to those normally bound in the XX ovary. We next define gene expression changes and abnormal chromatin compartments at the onset of transdifferentiation that may help destabilize cell fate and initiate the transdifferentiation process. We model the R111G mutation in mice and show that it causes dominant gonadal dysgenesis, analogous to its human phenotype but less severe. We show that R111G partially feminizes the testicular transcriptome and causes dominant disruption of DMRT1 binding specificity in vivo. These data help illuminate how transdifferentiation occurs when sexual cell fate maintenance is disrupted and identify chromatin sites and transcripts that may play key roles in the transdifferentiation process.