The fluid factor OVGP1 provides a significant oviductal microenvironment for the reproductive process in golden hamster†.

The mammalian oviductal lumen is a specialized chamber that provides an environment that strictly regulates fertilization and early embryogenesis, but the regulatory mechanisms to gametes and zygotes are unclear. We evaluated the oviductal regulation of early embryonic development using Ovgp1 (encoding an oviductal humoral factor, OVGP1)-knockout golden hamsters. The experimental results revealed the following: (1) female Ovgp1-knockout hamsters failed to produce litters; (2) in the oviducts of Ovgp1-knockout animals, fertilized eggs were sometimes identified, but their morphology showed abnormal features; (3) the number of implantations in the Ovgp1-knockout females was low; (4) even if implantations occurred, the embryos developed abnormally and eventually died; and (5) Ovgp1-knockout female ovaries transferred to wild-type females resulted in the production of Ovgp1-knockout egg-derived OVGP1-null litters, but the reverse experiment did not. These results suggest that OVGP1-mediated physiological events are crucial for reproductive process in vivo, from fertilization to early embryonic development. This animal model shows that the fate of the zygote is determined not only genetically, but also by the surrounding oviductal microenvironment.

Putting a finger on histidine methylation.

Specialized enzymes add methyl groups to the nitrogens of the amino acid histidine, altering the chemical properties of its imidazole ring and, in turn, the function of the modified (poly)peptide. In this issue of Genes & Development, Shimazu and colleagues (pp. 724-742) make the remarkable discovery that CARNMT1 acts as a dual-specificity histidine methyltransferase, modifying both the small-molecule dipeptide carnosine and a set of proteins, predominantly within RNA-binding C3H zinc finger (C3H ZF) motifs. As a result, CARNMT1 modulates the activity of its protein targets to affect RNA processing and metabolism, ultimately contributing an essential function during mammalian development.

Histidine N1-position-specific methyltransferase CARNMT1 targets C3H zinc finger proteins and modulates RNA metabolism.

Histidine (His) residues are methylated in various proteins, but their roles and regulation mechanisms remain unknown. Here, we show that carnosine N-methyltransferase 1 (CARNMT1), a known His methyltransferase of dipeptide carnosine (ßAla-His), is a major His N1-position-specific methyltransferase. We found that 52 His sites in 20 proteins underwent CARNMT1-mediated methylation. The consensus methylation site for CARNMT1 was identified as Cx(F/Y)xH, a C3H zinc finger (C3H ZF) motif. CARNMT1-deficient and catalytically inactive mutant mice showed embryonic lethality. Among the CARNMT1 target C3H ZF proteins, RNA degradation mediated by Roquin and tristetraprolin (TTP) was affected by CARNMT1 and its enzymatic activity. Furthermore, the recognition of the 3' splice site of the CARNMT1 target C3H ZF protein U2AF1 was perturbed, and pre-mRNA alternative splicing (AS) was affected by CARNMT1 deficiency. These findings indicate that CARNMT1-mediated protein His methylation, which is essential for embryogenesis, plays roles in diverse aspects of RNA metabolism by targeting C3H ZF-type RNA-binding proteins and modulating their functions, including pre-mRNA AS and mRNA degradation regulation.

High-fat diet reveals the impact of Sar1b defects on lipid and lipoprotein profile and cholesterol metabolism.

Biallelic pathogenic variants of the Sar1b gene cause chylomicron retention disease (CRD) whose central phenotype is the inability to secrete chylomicrons. Patients with CRD experience numerous clinical symptoms such as gastrointestinal, hepatic, neuromuscular, ophthalmic, and cardiological abnormalities. Recently, the production of mice expressing either a targeted deletion or mutation of Sar1b recapitulated biochemical and gastrointestinal defects associated with CRD. The present study was conducted to better understand little-known aspects of Sar1b mutations, including mouse embryonic development, lipid profile, and lipoprotein composition in response to high-fat diet, gut and liver cholesterol metabolism, sex-specific effects, and genotype-phenotype differences. Sar1b deletion and mutation produce a lethal phenotype in homozygous mice, which display intestinal lipid accumulation without any gross morphological abnormalities. On high-fat diet, mutant mice exhibit more marked abnormalities in body composition, adipose tissue and liver weight, plasma cholesterol, non-HDL cholesterol and polyunsaturated fatty acids than those on the regular Chow diet. Divergences were also noted in lipoprotein lipid composition, lipid ratios (serving as indices of particle size) and lipoprotein-apolipoprotein distribution. Sar1b defects significantly reduce gut cholesterol accumulation while altering key players in cholesterol metabolism. Noteworthy, variations were observed between males and females, and between Sar1b deletion and mutation phenotypes. Overall, mutant animal findings reveal the importance of Sar1b in several biochemical, metabolic and developmental processes.

Mammalian life depends on two distinct pathways of DNA damage tolerance.

DNA damage threatens genomic integrity and instigates stem cell failure. To bypass genotoxic lesions during replication, cells employ DNA damage tolerance (DDT), which is regulated via PCNA ubiquitination and REV1. DDT is conserved in all domains of life, yet its relevance in mammals remains unclear. Here, we show that inactivation of both PCNA-ubiquitination and REV1 results in embryonic and adult lethality, and the accumulation of DNA damage in hematopoietic stem and progenitor cells (HSPCs) that ultimately resulted in their depletion. Our results reveal the crucial relevance of DDT in the maintenance of stem cell compartments and mammalian life in unperturbed conditions.

Nigericin Abrogates Maternal and Embryonic Oxidative Stress in the Streptozotocin-Induced Diabetic Pregnant Rats.

Hyperglycemic exposure in diabetic pregnancy can lead to many developmental changes, such as delayed development, fetal malformations, and fetal/embryo death. These detrimental complications are collectively known as diabetic embryopathy or teratogenesis. The current study focuses to discover the therapeutic properties of the nigericin against the STZ-stimulated diabetic embryopathy via alleviation of maternal and embryonic oxidative stress. The male and female rats at a 1:1 ratio were permitted to mate overnight to establish the course of pregnancy. The pregnant rats were distributed into four groups control, diabetic pregnant (via administering 40 mg/kg of STZ), and diabetic + 10 and 20 mg/kg of nigericin-administered (via oral gavage from days 5 to 12) groups, respectively. The glucose level, urine output, diet intake, and body weight were determined carefully. The embryo and placenta weight and implantation rates were examined, and data were tabulated. The total protein and lipid profiles were assessed using respective kits. The oxidative stress markers and antioxidant enzymes were examined using respective assay kits. The 10 and 20 mg/kg of nigericin treatment decreased the glucose level and urine output and improved the diet intake and body weight gain in diabetic pregnant rats. The nigericin also decreased the total protein, cholesterol, triglycerides, and very-low-density lipoprotein (VLDL) and improved the high-density lipoprotein (HDL) in the serum of pregnant rats. The levels of malondialdehyde (MDA), reactive oxygen species (ROS), and protein carbonyls were decreased by the nigericin in both liver and embryos of the pregnant rats. The levels of glutathione (GSH), total thiols, and activities of catalase (CAT), glutathione reductase (GR), superoxide dismutase (SOD), glutathione peroxidase (GPX), and glutathione S-transferase (GST) were improved by the nigericin in the diabetic pregnant rats. Altogether, these results provide evidence that nigericin treatment remarkably attenuates the diabetes-stimulated embryopathy in rats. The nigericin effectively decreased embryo lethality, reduced glucose and dyslipidemia, and relieves oxidative stress via upregulating the antioxidant enzyme activities. Hence, it can be a talented therapeutic agent to treat diabetic pregnancy-associated complications.

The rat Downunder (Du) coat color mutation is associated with eye anomalies and embryonic lethality and maps to a 3.9-Mb region on chromosome 3.

Rodent coat color genes have been studied as a bioresource to understand developmental and cellular processes. The Downunder rat is a fancy variety with a marking on its belly that runs from the neck to the breech and appears to mirror the dorsal hooded marking. Here, we established a congenic strain carrying the Downunder (Du) gene in an F344 genetic background. In addition to the ventral marking, Du/+ rats exhibit anophthalmia or microphthalmia with incomplete penetrance. Du/Du embryos die in the early stages of organogenesis. Genetic linkage analysis mapped the Du gene to rat chromosome 3 and haplotype mapping with congenic rats localized the Du locus to a 3.9-Mb region. The Du locus includes two functional genes, glycosyltransferase-like domain-containing 1 (Gtdc1) and zinc finger E-box binding homeobox 2 (Zeb2). Although we found no functional variation within any of Zeb2's exons or intron-exon boundaries, Zeb2 mRNA levels were significantly lower in Du/+ rats compared with wild-type rats. It is known that melanocyte-specific Zeb2 deletion results in the congenital loss of hair pigmentation in mice. Taken together, our results indicate that the Du mutation exerts pleiotropic effects on hair pigmentation, eye morphology, and development. Moreover, the Zeb2 gene is a strong candidate for the Du mutation.

MiR-145 is upregulated in the retarded preimplantation embryos and modulates cholesterol levels in mice preimplantation embryos through targeting Abca1.

BACKGROUND: Preimplantation embryonic lethality is a driver of female infertility. Certain microRNAs (miRNAs) have previously been demonstrated to play important roles in the regulation of embryogenesis. METHODS: Normally developing blastocysts and arrested embryos were collected from patients undergoing intracytoplasmic sperm injection (ICSI), and the expression of specific miRNAs therein was evaluated by qPCR. The overexpression of target molecule miR-145 in early mice embryos was achieved via oocyte microinjection, enabling the subsequent monitoring of how such overexpression impacted embryonic development. Bioinformatics approaches were utilized to identify putative miR-145 target mRNAs, and luciferase reporter assessments were implemented to confirm the ability of miR-145 to regulate Abca1 in HEK293T cells. The functional relationship between miR-145 and Abca1 in the mice's embryonic development was then confirmed through rescue assays. RESULTS: Abnormally increased miR-145 expression was observed in patients' arrested embryos, and the exogenous overexpression of this miRNA significantly suppressed mural blastocyst formation. Mechanistically, miR-145 was found to bind to the 3'-untranslated area of the Abca1 mRNA in HK293T cells, thus suppressing its expression and increasing embryonic cholesterol levels. In line with the importance of these cholesterol levels to embryogenesis, the upregulation of Abca1 was sufficient to rescue the observed change in cholesterol levels and the associated retardation of mice embryonic development that occurred in response to the overexpression of miR-145. CONCLUSION: The regulatory dynamics of the miR-145/Abca1 axis play an important role in shaping normal embryonic development.

Mfsd2b and Spns2 are essential for maintenance of blood vessels during development and in anaphylactic shock.

Sphingosine-1-phosphate (S1P) is a potent lipid mediator that is secreted by several cell types. We recently showed that Mfsd2b is an S1P transporter from hematopoietic cells that contributes approximately 50% plasma S1P. Here we report the characterization of compound deletion of Mfsd2b and Spns2, another S1P transporter active primarily in endothelial cells. Global deletion of Mfsd2b and Spns2 (global double knockout [gDKO]) results in embryonic lethality beyond embryonic day 14.5 (E14.5), with severe hemorrhage accompanied by defects of tight junction proteins, indicating that Mfsd2b and Spns2 provide S1P for signaling, which is essential for blood vessel integrity. Compound postnatal deletion of Mfsd2b and Spns2 using Mx1Cre (ctDKO-Mx1Cre) results in maximal 80% reduction of plasma S1P. ctDKO-Mx1Cre mice exhibit severe susceptibility to anaphylaxis, indicating that S1P from Mfsd2b and Spns2 is indispensable for vascular homeostasis. Our results show that S1P export from Mfsd2b and Spns2 is essential for developing and mature vasculature.

Roan coat color in livestock.

Since domestication, a wide variety of phenotypes including coat color variation has developed in livestock. This variation is mostly based on selective breeding. During the beginning of selective breeding, potential negative consequences did not become immediately evident due to low frequencies of homozygous animals and have been occasionally neglected. However, numerous studies of coat color genetics have been carried out over more than a century and, meanwhile, pleiotropic effects for several coat color genes, including disorders of even lethal impact, were described. Similar coat color phenotypes can often be found across species, caused either by conserved genes or by different genes. Even in the same species, more than one gene could cause the same or similar coat color phenotype. The roan coat color in livestock species is characterized by a mixture of white and colored hair in cattle, pig, sheep, goat, alpaca, and horse. So far, the genetic background of this phenotype is not fully understood, but KIT and its ligand KITLG (MGF) are major candidate genes in livestock species. For some of these species, pleiotropic effects such as subfertility in homozygous roan cattle or homozygous embryonic lethality in certain horse breeds have been described. This review aims to point out the similarities and differences of the roan phenotype across the following livestock species: cattle, pig, sheep, goat, alpaca, and horse; and provides the current state of knowledge on genetic background and pleiotropic effects.

The Hypothesis of the Prolonged Cell Cycle in Turner Syndrome.

Turner syndrome (TS) is a chromosomal disorder that is caused by a missing or structurally abnormal second sex chromosome. Subjects with TS are at an increased risk of developing intrauterine growth retardation, low birth weight, short stature, congenital heart diseases, infertility, obesity, dyslipidemia, hypertension, insulin resistance, type 2 diabetes mellitus, metabolic syndrome, and cardiovascular diseases (stroke and myocardial infarction). The underlying pathogenetic mechanism of TS is unknown. The assumption that X chromosome-linked gene haploinsufficiency is associated with the TS phenotype is questioned since such genes have not been identified. Thus, other pathogenic mechanisms have been suggested to explain this phenotype. Morphogenesis encompasses a series of events that includes cell division, the production of migratory precursors and their progeny, differentiation, programmed cell death, and integration into organs and systems. The precise control of the growth and differentiation of cells is essential for normal development. The cell cycle frequency and the number of proliferating cells are essential in cell growth. 45,X cells have a failure to proliferate at a normal rate, leading to a decreased cell number in a given tissue during organogenesis. A convergence of data indicates an association between a prolonged cell cycle and the phenotypical features in Turner syndrome. This review aims to examine old and new findings concerning the relationship between a prolonged cell cycle and TS phenotype. These studies reveal a diversity of phenotypic features in TS that could be explained by reduced cell proliferation. The implications of this hypothesis for our understanding of the TS phenotype and its pathogenesis are discussed. It is not surprising that 45,X monosomy leads to cellular growth pathway dysregulation with profound deleterious effects on both embryonic and later stages of development. The prolonged cell cycle could represent the beginning of the pathogenesis of TS, leading to a series of phenotypic consequences in embryonic/fetal, neonatal, pediatric, adolescence, and adulthood life.

Targeted disruption of Gdi2 causes early embryonic lethality.

INTRODUCTION: GDI2 regulates the GDP/GTP exchange reaction of Rab proteins by inhibiting the dissociation of GDP and the subsequent binding of GTP, dysregulation of GDI2 has been reported in many different cancers. Recently, we found that GDI2 bound to the ITIM domain of Siglec-G under normal homeostasis, whereas Rab1a was recruited to the ITIM domain during bacterial infection. Therefore, GDI2 and Rab1a may regulate the immune response through interaction with the ITIM domain during bacterial infection. However, the regulation of the inflammatory response by GDI2 in vivo and its regulatory mechanism remain unknown. METHODS: We generated a Gdi2 null mutant mouse with a trapped Gdi2 gene and examined the expression by X-gal and immunohistochemistry staining. TUNEL staining was used to determine the apoptosis cells. RESULTS: Here we show that Gdi2 is essential for embryonic development. One functional Gdi2 allele is sufficient for murine embryo development, but complete loss of Gdi2 leads to embryonic lethality. Developmental retardation of Gdi2-/- mice is apparent at E10.5 to E14.5, with no viable Gdi2-/- embryos detected after E14.5. Histological analysis revealed extensive cell death and cell loss in Gdi2-/- embryos. Apoptosis was confirmed by staining with cleaved caspase-3, suggesting that Gdi2 maintain homeostasis by regulating the apoptosis of the cells. There was no significant difference in cytokine production and survival between wild-type and Gdi2+/- mice after LPS challenge. DISCUSSION: These findings suggest that one Gdi2 allele is sufficient to maintain function. However, the detailed molecular mechanism underlying Gdi2 in regulating the embryonic development needs further identification.

Mice lacking DCAF2 in placenta die at the gastrulation stage.

UV-damaged DNA-binding protein 1 (DDB1) and cullin 4-associated factor 2 (DCAF2, also known as DTL or CDT2) is an evolutionarily highly conserved substrate recognition factor in the cullin 4 RING E3 ubiquitin ligase (CRL4) complex. This complex degrades multiple DNA replication and cell cycle-associated proteins to maintain genome stability. To clarify the function of DCAF2 in vivo, we used Cre recombinase driven by the Elf5 promoter to generate knockout mouse model that was specifically deleted Dcaf2 in the trophoblast lineage (Elf5-Cre; Dcaf2fl/fl, Dcaf2 cKO). Here, we show that mice with the genotype Elf5-Cre; Dcaf2fl/+ are normal and fertile. However, after mating of Elf5-Cre; Dcaf2fl/+ mice with Dcaf2fl/fl, no Dcaf2 cKO pups were born. Timed pregnancy studies have shown that Dcaf2 cKO mice developed abnormally on embryonic day 5.5 and died at gastrulation stage. It is worth noting that the extraembryonic ectoderm of Dcaf2 cKO mice is severely reduced or missing and leading to embryonic death. We also proved that stronger DNA damage accumulated in the trophoblastic cells of Dcaf2 cKO mice at E8.5. In addition, higher expression of Caspase-3 was found in the embryonic and trophoblastic cells of these cKO mice. In general, our research shows that the placental DCAF2 is crucial to the formation of gastrula.

Loxl2 and Loxl3 Paralogues Play Redundant Roles during Mouse Development.

Lysyl oxidase-like 2 (LOXL2) and 3 (LOXL3) are members of the lysyl oxidase family of enzymes involved in the maturation of the extracellular matrix. Both enzymes share a highly conserved catalytic domain, but it is unclear whether they perform redundant functions in vivo. In this study, we show that mice lacking Loxl3 exhibit perinatal lethality and abnormal skeletal development. Additionally, analysis of the genotype of embryos carrying double knockout of Loxl2 and Loxl3 genes suggests that both enzymes have overlapping functions during mouse development. Furthermore, we also show that ubiquitous expression of Loxl2 suppresses the lethality associated with Loxl3 knockout mice.

Crlz-1 Homozygous Null Knockout Mouse Embryos Are Lethally Stopped in Their Early Development.

Although the conditional gene knockout (KO) is a better choice for observing its phenotype in a specific cell, tissue, and/or organ, the simple null gene KO could nevertheless be attempted initially to scan its overall phenotypes at the level of the whole-body system, especially for a new gene such as Crlz-1. Therefore, with a hope to glean phenotypic clues for Crlz-1 at the whole-body system, we attempted to generate its null KO mice. Contrary to our original desire, Crlz-1 homozygous null KO mice were not born. However, in the chasing of their homozygous KO embryos, they were found to be lethally impaired from early development, remaining in a state of small globular mass without ever leading to a body shape, indicating the critical role of Crlz-1 as a Wnt target gene for the proliferation and/or differentiation of cells during early mouse embryonic development.

Genetic resiliency associated with dominant lethal TPM1 mutation causing atrial septal defect with high heritability.

Analysis of large-scale human genomic data has yielded unexplained mutations known to cause severe disease in healthy individuals. Here, we report the unexpected recovery of a rare dominant lethal mutation in TPM1, a sarcomeric actin-binding protein, in eight individuals with large atrial septal defect (ASD) in a five-generation pedigree. Mice with Tpm1 mutation exhibit early embryonic lethality with disrupted myofibril assembly and no heartbeat. However, patient-induced pluripotent-stem-cell-derived cardiomyocytes show normal beating with mild myofilament defect, indicating disease suppression. A variant in TLN2, another myofilament actin-binding protein, is identified as a candidate suppressor. Mouse CRISPR knock-in (KI) of both the TLN2 and TPM1 variants rescues heart beating, with near-term fetuses exhibiting large ASD. Thus, the role of TPM1 in ASD pathogenesis unfolds with suppression of its embryonic lethality by protective TLN2 variant. These findings provide evidence that genetic resiliency can arise with genetic suppression of a deleterious mutation.

Targeted disruption of Rab1a causes early embryonic lethality.

Guanosine nucleotide diphosphate (GDP) dissociation inhibitor 2 (GDI2) regulates the GDP/guanosine triphosphate (GTP) exchange reaction of Rab proteins by inhibiting the dissociation of GDP and the subsequent binding of GTP. The present study aimed to determine the function of Rab1a in vivo, and thus generated mice with a trapped Rab1a gene. It was demonstrated that Rab1a is essential for embryonic development. It was also found that one functional Rab1a allele was sufficient for development in a heterozygous murine embryo, whereas a double mutant led to embryonic lethality. The dissection of uteri on embryonic day (E)10.5­14.5 yielded no homozygous embryos, indicating that homozygotes die between E10.5 to E11.5. The gene trap construct contains a ß­galactosidase/neomycin reporter gene, allowing for heterozygotes to be stained for ß­galactosidase to determine the tissue­specific expression of Rab1a. Rab1a was found to be highly expressed in the small intestine of both adult mice and embryos, although its expression levels were low in the brains of embryos. Moreover, there was no significant change in cytokine production and survival in wild­type and heterozygous Rab1a+/­ mice following a challenge with lipopolysaccharide. On the whole, the present study demonstrates that the disruption of the Rab1a gene causes embryonic lethality and homozygotes die between E10.5 and E11.5, suggesting that Rab1a is essential for the early development of mouse embryos.

A class I histone deacetylase HDA-2 is essential for embryonic development and size regulation of fertilized eggs in Caenorhabditis elegans.

BACKGROUND: Caenorhabditis elegans encodes three class I histone deacetylases (HDACs), HDA-1, HDA-2, and HDA-3. Although HDA-1 is known to be involved in embryogenesis, the regulatory roles of HDA-2 and HDA-3 in embryonic development remain unexplored. OBJECTIVE: To elucidate the functional roles of the three class I HDACs in C. elegans embryonic development. METHODS: The roles of Class I HDACs, HDA-1, HDA-2, and HDA-3 in Caenorhabditis elegans during embryogenesis were investigated through the analysis of embryonic lethality via gene knockdown or deletion mutants. Additionally, the size of these knockdown and mutant eggs was observed using a differential interference contrast microscope. Finally, expression pattern and tissue-specific role of hda-2 and transcriptome of the hda-2 mutant were analyzed. RESULTS: Here, we report that HDA-1 and HDA-2, but not HDA-3, play essential roles in Caenorhabditis elegans embryonic development. Our observations of the fertilized egg size variance demonstrated that HDA-2 is involved in regulating the size of fertilized eggs. Combined analysis of expression patterns and sheath cell-specific rescue experiments indicated that the transgenerational role of HDA-2 is involved in the viability of embryonic development and fertilized egg size regulation. Furthermore, transcriptome analysis of hda-2 mutant embryos implies that HDA-2 is involved in epigenetic regulation of embryonic biological processes by downregulating and upregulating the gene expression. CONCLUSION: Our finding suggests that HDA-2 regulates the embryonic development in Caenorhabditis elegans by controling a specific subset of genes, and this function might be mediated by transgenerational epigenetic effect.

Lipid signatures reflect the function of the murine primary placentation†.

The placenta regulates maternal-fetal communication, and its defect leads to significant pregnancy complications. The maternal and embryonic circulations are primitively connected in early placentation, but the function of the placenta during this developmentally essential period is relatively unknown. We thus performed a comparative proteomic analysis of the placenta before and after primary placentation and found that the metabolism and transport of lipids were characteristically activated in this period. The placental fatty acid (FA) carriers in specific placental compartments were upregulated according to gestational age, and metabolomic analysis also showed that the placental transport of FAs increased in a time-dependent manner. Further analysis of two mutant mice models with embryonic lethality revealed that lipid-related signatures could reflect the functional state of the placenta. Our findings highlight the importance of the nutrient transport function of the primary placenta in the early gestational period and the role of lipids in embryonic development. SUMMARY SENTENCE: The placenta is activated characteristically in terms of lipid transport during primary placentation, and the lipid-related signatures closely reflect the functional state of the placenta.

Integrated analysis of whole genome and transcriptome sequencing reveals a frameshift mutation associated with recessive embryonic lethality in Holstein cattle.

Embryo loss is an important factor affecting fertility in dairy production. HH2 was identified as a haplotype on chromosome 1 associated with embryonic lethality in Holstein cattle. In the current study, both short- and long-read WGS was performed on four carriers and four non-carriers of HH2 to screen for variants in concordance with HH2 haplotype status. Sequence variation analysis revealed five putative functional variants of protein-coding genes, including a frameshift mutation (g.107172616delT) in intraflagellar transport protein 80 (IFT80) gene. Transcriptome analysis of whole blood indicated that no gene exhibited significantly differential expression or allele-specific expression between carriers and non-carriers in the candidate region. This evidence points to g.107172616delT as the highest priority causative mutation for HH2. Protein prediction reveals that the frameshift mutation results in a premature stop codon to reduce the peptide chain from 760 to 383 amino acids and greatly alters the structure and function of IFT80 protein. Our results demonstrate that the use of a combination of multiple high-throughput sequencing technologies is an efficient strategy to screen for the candidate causative mutations responsible for Mendelian traits, including genetic disorders.