Homozygous Father Research Articles

Search for additional variability in the herd of Holstein cows

Relevance. When breeding a large herd of the Holstein breed, the improvement of traits in the selection system is reflected, the integral basis (part of the basis) of which was belonging to the line. Against the background of a reduction in the number of lines, the selection of a limited number of sires related to each other into the herd, and the breeding of daughters from them by various types of rebounds, an increase in milk yield and mass fraction of protein (MPF) over three generations is reflected.Methods. The indices of the genetic value of sires in the population were evaluated according to the author's method, while taking into account the indicators of mothers and peers of the same herd. Comparison of the levels of variability of milk productivity traits in terms of standard deviations (±s) in breeding groups.Results. The relative increase in milk yield per generation for the highest lactations was 8.9–15.4% (P < 0.001), and MDL – 1.3–3.2% (P < 0.01). A comparison was made of groups of peer-daughters obtained by targeted selection of homo- and heterozygous fathers according to genetic markers of controlled EAB loci. Another aspect was the high standard deviation of milk yields in the group of daughters of homozygous fathers by 19% against the group of heterozygous, and among the crossed offspring – by 8.6% against intraline. It is assumed that this happened due to the introduction of new and rare alleles with sires into the herd.

Molecular Studies and an ex vivo Complement Assay on Endothelium Highlight the Genetic Complexity of Atypical Hemolytic Uremic Syndrome: The Case of a Pedigree With a Null CD46 Variant.

Atypical hemolytic uremic syndrome (aHUS) is an ultra-rare disease characterized by microangiopathic hemolysis, thrombocytopenia, and renal impairment and is associated with dysregulation of the alternative complement pathway on the microvascular endothelium. Outcomes have improved greatly with pharmacologic complement C5 blockade. Abnormalities in complement genes (CFH, CD46, CFI, CFB, C3, and THBD), CFH–CFHR genomic rearrangements, and anti-FH antibodies have been reported in 40–60% of cases. The penetrance of aHUS is incomplete in carriers of complement gene abnormalities; and multiple hits, including the CFH–H3 and CD46GGAAC risk haplotypes and the CFHR1*B risk allele, as well as environmental factors, contribute to disease development. Here, we investigated the determinants of penetrance of aHUS associated with CD46 genetic abnormalities. We studied 485 aHUS patients and found CD46 rare variants (RVs) in about 10%. The c.286+2T>G RV was the most prevalent (13/485) and was associated with <30% penetrance. We conducted an in-depth study of a large pedigree including a proband who is heterozygous for the c.286+2T>G RV who experienced a severe form of aHUS and developed end-stage renal failure. The father and paternal uncle with the same variant in homozygosity and six heterozygous relatives are unaffected. Flow cytometry analysis showed about 50% reduction of CD46 expression on blood mononuclear cells from the heterozygous proband and over 90% reduction in cells from the proband's unaffected homozygous father and aunt. Further genetic studies did not reveal RVs in known aHUS-associated genes or common genetic modifiers that segregated with the disease. Importantly, a specific ex vivo test showed excessive complement deposition on endothelial cells exposed to sera from the proband, and also from his mother and maternal uncle, who do not carry the c.286+2T>G RV, indicating that they share a circulating defect that results in complement dysregulation on the endothelium. These results highlight the complexity of the genetics of aHUS and indicate that CD46 deficiency may not be enough to induce aHUS. We hypothesize that the proband inherited from his mother a genetic abnormality in a complement circulating factor that has not been identified yet, which synergized with the CD46 RV in predisposing him to the aHUS phenotype.

Frequency of null alleles in paternity cases from Mexico for the locus Penta E

We report three peculiar motherless paternity cases received in our lab at consecutive months, which involved four father-child mismatches for Penta E, the autosomal STR loci included in the Powerplex Fusion system. In all these cases, based on the residual Paternity Index (PI), the kinship was confirmed between the father and three children (PI > 1.6 × 10+6). In the first case, the homozygous father 16/16 did not match with two homozygous children 5/5 for the Penta E locus. Unexpectedly, the next week we detected another unique mismatch for Penta E between one homozygous father 9/9 and the homozygous child 17/17. Finally, four months later, in another motherless case, the father was 14/14 and the child was 16/16 for Penta E. Consequently, these four paternity test mismatches were faced as null alleles, which are described as variants in the primer-binding region that can lead to allele dropout resulting as false homozygous. Interestingly, allele nulls for Penta E are not described in the STRBase (https://strbase.nist.gov/NullAlleles.htm). According with these findings and our previous lab’s records (García-Aceves et al. 2018), we estimated a preliminary null allele frequency for Penta E in Mexico (7/3300; 0.21%). Because this estimate comes from motherless paternity cases and only one Mexican paternity testing laboratory, eventually this frequency needs to be confirmed. In brief, this report provides helpful information for reliable interpretation of paternity cases with unique mismatches for Penta E in Mexico, promoting research or simple modification of the corresponding primers by the supplier.

Identification of TEX101-associated Proteins Through Proteomic Measurement of Human Spermatozoa Homozygous for the Missense Variant rs35033974*[S

TEX101 is a germ-cell-specific protein and a validated biomarker of male infertility. Mouse TEX101 was found essential for male fertility and was suggested to function as a cell surface chaperone involved in maturation of proteins required for sperm migration and sperm-oocyte interaction. However, the precise functional role of human TEX101 is not known and cannot be studied in vitro due to the lack of human germ cell lines. Here, we genotyped 386 men for a common missense variant rs35033974 of TEX101 and identified 52 heterozygous and 4 homozygous men. We then discovered by targeted proteomics that the variant allele rs35033974 was associated with the near-complete degradation (>97%) of the corresponding G99V TEX101 form and suggested that spermatozoa of homozygous men could serve as a knockdown model to study TEX101 function in humans. Differential proteomic profiling with label-free quantification measured 8,046 proteins in spermatozoa of eight men and identified eight cell-surface and nine secreted testis-specific proteins significantly down-regulated in four patients homozygous for rs35033974. Substantially reduced levels of testis-specific cell-surface proteins potentially involved in sperm migration and sperm-oocyte interaction (including LY6K and ADAM29) were confirmed by targeted proteomics and Western blotting assays. Because recent population-scale genomic data revealed homozygous fathers with biological children, rs35033974 is not a monogenic factor of male infertility in humans. However, median TEX101 levels in seminal plasma were found fivefold lower (p = 0.0005) in heterozygous than in wild-type men of European ancestry. We conclude that spermatozoa of rs35033974 homozygous men have substantially reduced levels of TEX101 and could be used as a model to elucidate the precise TEX101 function, which will advance biology of human reproduction.

Xist imprinting is promoted by the hemizygous (unpaired) state in the male germ line

The long noncoding X-inactivation-specific transcript (Xist gene) is responsible for mammalian X-chromosome dosage compensation between the sexes, the process by which one of the two X chromosomes is inactivated in the female soma. Xist is essential for both the random and imprinted forms of X-chromosome inactivation. In the imprinted form, Xist is paternally marked to be expressed in female embryos. To investigate the mechanism of Xist imprinting, we introduce Xist transgenes (Tg) into the male germ line. Although ectopic high-level Xist expression on autosomes can be compatible with viability, transgenic animals demonstrate reduced fitness, subfertility, defective meiotic pairing, and other germ-cell abnormalities. In the progeny, paternal-specific expression is recapitulated by the 200-kb Xist Tg. However, Xist imprinting occurs efficiently only when it is in an unpaired or unpartnered state during male meiosis. When transmitted from a hemizygous father (+/Tg), the Xist Tg demonstrates paternal-specific expression in the early embryo. When transmitted by a homozygous father (Tg/Tg), the Tg fails to show imprinted expression. Thus, Xist imprinting is directed by sequences within a 200-kb X-linked region, and the hemizygous (unpaired) state of the Xist region promotes its imprinting in the male germ line.

Novel findings in patients with primary hyperoxaluria type III and implications for advanced molecular testing strategies

Identification of mutations in the HOGA1 gene as the cause of autosomal recessive primary hyperoxaluria (PH) type III has revitalized research in the field of PH and related stone disease. In contrast to the well-characterized entities of PH type I and type II, the pathophysiology and prevalence of type III is largely unknown. In this study, we analyzed a large cohort of subjects previously tested negative for type I/II by complete HOGA1 sequencing. Seven distinct mutations, among them four novel, were found in 15 patients. In patients of non-consanguineous European descent the previously reported c.700+5G>T splice-site mutation was predominant and represents a potential founder mutation, while in consanguineous families private homozygous mutations were identified throughout the gene. Furthermore, we identified a family where a homozygous mutation in HOGA1 (p.P190L) segregated in two siblings with an additional AGXT mutation (p.D201E). The two girls exhibiting triallelic inheritance presented a more severe phenotype than their only mildly affected p.P190L homozygous father. In silico analysis of five mutations reveals that HOGA1 deficiency is causing type III, yet reduced HOGA1 expression or aberrant subcellular protein targeting is unlikely to be the responsible pathomechanism. Our results strongly suggest HOGA1 as a major cause of PH, indicate a greater genetic heterogeneity of hyperoxaluria, and point to a favorable outcome of type III in the context of PH despite incomplete or absent biochemical remission. Multiallelic inheritance could have implications for genetic testing strategies and might represent an unrecognized mechanism for phenotype variability in PH.

Osteonecrosis of the jaw and bisphosphonates in children and adolescents

Nutritional rickets most commonly results from vitamin D deficiency and is typically associated with inadequate sunlight. Thus, the existence of rickets in many tropical countries, where sunlight is abundant, is unexpected, and has suggested that genetic, hormonal, and other nutritional factors may cause rickets in susceptible children. We recently identified a missense mutation (Leu99Pro) in exon 2 of CYP2R1, the gene encoding the key hepatic vitamin D 25hydroxylase, in members of 2 of 10 Nigerian families in which more than 1 subject had rickets. One proband (T133), his affected brother (T133-1) and their father (T133-5) were homozygous (CYP−/−) for the L99P allele; his mother and two sisters were heterozygous (CYP−/+) for the L99P allele, and one of the sisters had a history of genu varum that resolved spontaneously as a toddler. The second proband (K170) and her affected sister were both heterozygous for the L99P mutation. Their mother was homozygous for the wild type allele while their clinically unaffected father was heterozygous. Biochemical analyses showed that homozygous brothers T133 and T133-1 were hypocalcemic with elevated alkaline phosphatase; their homozygous father T133-5 and all heterozygous subjects had normal serum calcium and alkaline phosphatase concentrations. Because in vitro analyses of CYP2R1 function had shown that the L99P enzyme retained modest residual activity, we administered 50,000 IU of ergocalciferol (D2) or cholecalciferol (D3) on separate occasions to subjects and determined serum levels of 25 (OH)D2 and 25(OH)D3 by isotope-dilution liquid chromatography-tandem mass spectrometry on days 0, 1, 3, and 7. Peak values were achieved on day 3 (see Table). We conclude that homozygous or heterozygous mutation of the CYP2R1 gene is a cause of rickets, and represents a new form of vitamin D dependent rickets. Remarkably, the phenotype appears to improve with age and treatment with calcium, suggesting the involvement of other genetic, hormonal or environmental factors that can compensate for loss of CYP2R1 vitamin D 25 hydroxylase activity. The role of variant alleles of CYP2R1 in the pathogenesis of vitamin D deficiency and/or metabolic bone disease worldwide remains to be determined.

Rhesus Box Variation: Multiple RHD Deletion Events and Implications for Testing RHD Heterozygosity.

The RHD gene encoding the Rhesus blood group antigen D is flanked by two highly homologous DNA segments of about 9,000 bp, the upstream and downstream Rhesus boxes. In haplotypes with an RHD deletion, the fusion of the two Rhesus boxes within the 1,463 bp identity region generates the single hybrid Rhesus box. Its detection has been applied to determine RHD zygosity. For instance, testing for RHD heterozygosity is of clinical interest, because all children of an RHD homozygous father will be D positive and at risk of hemolytic disease of the newborn, if the mother carries an anti-D. Aberrant Rhesus boxes can confound this application and appear to be frequent among Africans. A comprehensive, systematic analysis was lacking. We sequenced about 5,850 bp of the upstream and downstream Rhesus boxes in 18 samples that were representative for all four D clusters and of the hybrid Rhesus box in 4 samples that were mistyped in assays for the hybrid Rhesus box. The known differences between upstream and downstream Rhesus boxes were in part restricted to subsets of RHD alleles. We detected 46 additional polymorphism, which represented single nucleotide substitutions, short insertions or deletions. Gene conversions were found in the upstream Rhesus boxes of RHDψ, DAU-1 and DAU-3 and in the downstream Rhesus boxes of Ccdes, weak D type 4.1, type 4.2 and DAU-0. All 6 different gene conversions started or ended in or near the identity region, which may have been instrumental for generating these gene conversions. Recombinations between haplotypes were likely in several alleles like DIII type 4. Four non-standard hybrid Rhesus boxes were suggestive of multiple independent RHD deletion events.We found considerable variation of Rhesus box sequences, which explained the limitations for RHD heterozygosity testing that were previously observed in several laboratories. The general topology of the genomic Rhesus locus was however similar in all cases and consistent with our initial description. There was evidence for 5 probably independent RHD deletion events. Testing RHD heterozygosity in Africans is best based on quantitative PCR or amplification of the full length hybrid Rhesus box. Because in Europeans who harbored RHD alleles belonging to African D clusters the Rhesus box variation was also substantial, use of two complementary methods for hybrid Rhesus box detection may even be advisable in whites. We conclude that a long range PCR spanning the whole hybrid Rhesus box, while technically demanding, is not influenced by the kinds of aberrations observed in this study and may hence remain the standard for research purposes and quality assurance.

Prenatal RHD gene determination and dosage analysis by PCR: clinical evaluation.

Use of the polymerase chain reaction (PCR) for detection of the RHD gene can measure the RHD gene status for unborn babies at risk for hemolytic disease of the newborn (HDN). The occurrence of D gene variants has led to errors in prenatal typing. Previous reports have highlighted the danger of assigning a positive fetus as negative, resulting in intrauterine fetal deaths. To evaluate the effectiveness of a testing strategy whereby PCR was not only performed to determine the presence/absence of the RHD gene, but also used to assess the D gene copy number (zero, one or two RHD genes) in family studies for at risk pregnancies. Samples comprising maternal (57) and paternal (42) peripheral blood samples, amniotic fluid (64), and matching cord blood (64) were collected. Rhesus (Rh) serotyping was performed on all blood samples. For RHD genotyping, DNA was extracted from all samples except for 28 cord samples, where only serotyping was performed (total 199 DNA genotyping). RHD gene PCR amplified exon 4 and exon 7 regions of the RHD gene. The dosage of RHD gene was determined by comparing the intensity of the RHD gene to that of the RHCE gene. A total of 197/199 samples showed concordance between exon 4 and exon 7 PCR results. Two discrepant results occurred in one family: the father carried one normal D gene and one D gene variant where PCR was tested to be positive using exon 4 but negative using exon 7. One of a pair of dizygotic twins inherited this abnormal D gene and was mildly affected by HDN. This was correctly identified antenatally and the pregnancy successfully managed. The concordance rate between serotypes and genotypes for 135 blood samples was 100%. Amongst the family groups, 8/14 heterozygous fathers transmitted the D gene and 26/26 homozygous fathers transmitted the D gene to the babies. The concordance rate between RHD genotypes from amniotic fluid and Rh D serotypes from cord blood was also 100%. The present study demonstrates the effectiveness of using PCR in a clinical setting. It verifies the importance of testing more than one region of the gene, and also the need for a testing strategy where both maternal and paternal testing for RHD gene dosages are performed.

Alloimmune thrombocytopenia in the fetus and newborn.

Alloimmune thrombocytopenia is an interesting and challenging disease. Identification in the fetus and newborn by screening remains to be clarified. The primary clinical criterion for neonatal diagnosis appears to be a neonatal platelet count of <50 x 10(9)/L. Treatment of the neonate can be accomplished with intravenous immunoglobulin (IVIG) +/- steroids or with matched platelet transfusion. Cranial ultrasonography is important. Testing can be performed on the parents and requires a highly experienced laboratory. If an affected fetus is identified, based on a previous affected neonate and a homozygous father, antenatal management is needed. Studies have been completed that inform the still controversial decision. IVIG remains the basis of therapy but appears to require a higher dose (2 g/kg/week) and/or the addition of 1 mg/kg of prednisone in the highest risk cases, those with antenatal intracranial hemorrhage.

Fibrinogen Kurashiki (γ G268E) Fibrin Network Structure

Abstract Upon thrombin-catalyzed proteolytic conversion of fibrinogen to fibrin, fibrinopeptide A release exposes an N-terminal polymerization site (‘EA’) in the central E domain that interacts with a constitutive complementary site in each outer D domain (‘Da’) to drive the molecular assembly process by end-to-middle domain non-covalent ‘Da:EA’ interactions. End-to-end aligned doublestranded fibrils are formed in coordination with another recently described constitutive self-association site (termed ‘D:D’)that is situated at the outer end of each D domain. The D:D site contributes to end-to-end molecular alignment of fibrin molecules within each fibril strand. Concomitant branching and lateral fibril associations to form thick fibers, lead to formation of the mature fibrin clot. Fibrinogen ‘Kurashiki’ is a unique congenital dysfibrinogenemia that was identified in a homozygous father and his heterozygous son. The abnormality involves a γ chain amino acid mutation (γ G268E) and is characterized by delayed and abnormal fibrin polymerization.