Identification of Point Mutation in Exon 3 of Leptin Gene in Munjal Sheep

Background: Leptin is a varied hormone which plays vital role in body development by regulating the balance between food intake and energy expenditure by signaling to the brain. Leptin has diverse effect on controlling appetite, energy metabolism, growth, reproduction, body composition and immunity. The present study was aimed to screen candidate point mutation (g.332G greater than A) in the targeted genomic region of leptin gene in Munjal sheep. Methods: A total of 50 Munjal sheep were selected and genomic DNA was isolated in Automated Maxell RSC DNA/ RNA purification system by using Maxwell RSC whole blood DNA kit. Reported set of primers was used to amplify 463bp fragment encompassing targeted region (exon 3) of leptin gene. PCR-RFLP was performed to genotype targeted point mutation in our resource population. PCR products were digested by Cail 1 restriction enzyme to genotype g.332G greater than A (at 332th nucleotide of exon 3 leptin gene) non-synonymous mutation (Arg to Gln). Result: All studied samples resolved into monomorphic banding pattern, revealed only AA (463bp single band bp) genotype. The absence of candidate mutation in our resource population might be due to small sample size.

A Trimethylguanosine Synthase1-like (TGS1) homologue is implicated in vernalisation and flowering time control

Key message A plant-specificTrimethylguanosine Synthase1-likehomologue was identified as a candidate gene for theeflmutation in narrow-leafed lupin, which alters phenology by reducing vernalisation requirement. Abstract The vernalisation pathway is a key component of flowering time control in plants from temperate regions but is not well understood in the legume family. Here we examined vernalisation control in the temperate grain legume species, narrow-leafed lupin (Lupinus angustifolius L.), and discovered a candidate gene for an ethylene imine mutation (efl). The efl mutation changes phenology from late to mid-season flowering and additionally causes transformation from obligate to facultative vernalisation requirement. The efl locus was mapped to pseudochromosome NLL-10 in a recombinant inbred line (RIL) mapping population developed by accelerated single seed descent. Candidate genes were identified in the reference genome, and a diverse panel of narrow-leafed lupins was screened to validate mutations specific to accessions with efl. A non-synonymous SNP mutation within an S-adenosyl-L-methionine-dependent methyltransferase protein domain of a Trimethylguanosine Synthase1-like (TGS1) orthologue was identified as the candidate mutation giving rise to efl. This mutation caused substitution of an amino acid within an established motif at a position that is otherwise highly conserved in several plant families and was perfectly correlated with the efl phenotype in F2 and F6 genetic population and a panel of diverse accessions, including the original efl mutant. Expression of the TGS1 homologue did not differ between wild-type and efl genotypes, supporting altered functional activity of the gene product. This is the first time a TGS1 orthologue has been associated with vernalisation response and flowering time control in any plant species.

A Novel Homozygous Missense Mutation of WEE2 Causes Female Infertility Characterized by Fertilization Failure

Purpose: To investigate the genetic cause of infertility in a female patient due to repeated fertilization failure.Methods: Whole exome sequencing was performed to obtain the candidate mutation. Sanger sequencing was used to identify the mutation of the proband and other family members. SIFT, Polyphen-2, and Mutation Taster were used to predict the pathogenicity of mutations. The online software Arpeggio and the mCSM online service were used to analyze the effect of the mutation on protein structure and stability. Results: We identified a novel homozygous missense mutation c.T1199A:p.L400Q (Leu400Gln) in WEE2 gene in a female proband with infertility caused by fertilization failure. Conclusions: We discovered a novel homozygous missense mutation c.T1199A:p.L400Q (Leu400Gln) of the WEE2 gene in an infertile female whose oocytes had undergone complete fertilization failure, either after ICSI or RICSI. Our findings extend the mutant spectrum of WEE2 , a genetic cause for fertilization failure, and provide a theoretical basis for clinical diagnosis of the pathogenic causes of infertility.

Neonatal-onset autoinflammation and immunodeficiency caused by heterozygous missense mutation of the proteasome subunit β-type 9

ABSTRACTBACKGROUNDDefective proteasome activities due to genetic mutations lead to an autoinflammatory disease, termed as proteasome-associated autoinflammatory syndromes (PRAAS). In PRAAS relapsing inflammations and progressive wasting are common, but immunodeficiency has not been reported.METHODSWe studied two unrelated Japanese infants with PRAAS-like manifestations. We have also generated and analyzed the mice carrying the candidate mutation found in the patients.RESULTSBoth patients showed neonatal-onset skin rash, myositis and basal ganglia calcification, similar to PRAAS patients. Meanwhile, they manifested distinct phenotypes, including pulmonary hypertension and immunodeficiency without lipoatrophy. We identified a novel de novo heterozygous missense mutation, G156D, in a proteasome subunit gene, PSMB9, encoding β1i, in the two patients. Maturation and activity of the immunoproteasome were impaired, but ubiquitin accumulation was hardly detected not only in patient-derived cells and samples but also in Psmb9G156D/+ mice. As an immunodeficient phenotype, one patient showed decrease of B cells and increase of monocytes, while the other patient showed decrease of CD8 T cells. The proteasome defects and immunodeficient phenotypes were recapitulated in Psmb9G156D/+ mice.CONCLUSIONSThe PSMB9 G156D is a unique mutation in proteasome subunits in causing defects by its heterozygosity, affecting two β rings interaction and leading to immunodeficiency. The mutant mice are the first mice model for analyzing proteasome dysfunctions in PRAAS. We here propose the term, proteasome-associated autoinflammation and immunodeficiency disease (PRAID), as an umbrella name for our cases, PRAAS with immunodeficiency, as well as PRAAS described so far.

Evidence that autosomal recessive spastic cerebral palsy-1 (CPSQ1) is caused by a missense variant in HPDL

A subset of individuals diagnosed with cerebral palsy will have an underlying genetic diagnosis. Previously, a missense variant in GAD1 was described as a candidate mutation in a single family diagnosed with autosomal recessive spastic cerebral palsy-1 (CPSQ1; OMIM 603513). Following the ascertainment of a further branch of the CPSQ1 kindred, we found that the previously reported GAD1 variant did not segregate with the neurological disease phenotype in the recently ascertained branch of the kindred. Following genetic linkage studies to map autozygous regions and whole-exome sequencing, a missense variant (c.527 T > C; p. Leu176Pro, rs773333490) in the HPDL gene was detected and found to segregate with disease status in both branches of the kindred. HPDL encodes a 371-amino acid protein (4-Hydroxyphenylpyruvate Dioxygenase Like) that localizes to mitochondria but whose function is uncertain. Recently, biallelic loss of function variants and missense substitution-causing variants in HPDL were reported to cause a childhood onset progressive spastic movement disorder with a variable presentation. These findings suggest that HPDL-related neurological disease may mimic spastic cerebral palsy and that GAD1 should not be included in diagnostic gene panels for inherited cerebral palsy.

Accurate prediction of somatic variants using deep learning model.

e13659 Background: Efficient and accurate identification of somatic variant is important for understanding the formation, progression, and treatment of cancer. It is necessary to conduct manual review by Integrative Genomic Viewer (IGV) in traditional variant calling process. However, the traditional manual is heavy workload when evaluating tumor with a high variant burden. In this study, a new convolutional neural network (CNN) method was created to train models for somatic mutation identification, which was suitable for Panel sequencing platform with different tumor purities. Methods: A total of 1000 tumor samples from next generation sequencing (NGS)-based genetic testing by a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Through variant calling program, like GATK, the candidate mutation locations were identified and standardized by manual confirmation. For each candidate mutation location, reads of both tumor and control tissue were extracted. A 2-dimensional feature matrix M of size (2k+1) * 32 in each candidate base was created. The rows of 2k+1 represented the length of candidate region, and the 32 columns included the reads coverage frequency, mapping quality messages, and genome local scores of different tumor and control tissues. CNN model, which includes nine convolutional layers structured by Temporal Convolutional Networks (TCN) but with a different structure to adapt to the proposed input matrix, was used for training. The training data set including manually validated sequence data was used as benchmark test, and optimized by Stochastic Gradient Descent (SGD) optimizer with a learning rate of 0.01 was used for training. Results: The validation data set included 15 mixed samples which were composed of different proportions of known cell lines and real mixed blood samples. The pooled DNA contained 2,359 somatic variants, with expected variant allele frequencies ranged from 3% to 97% in each pool. The overall sensitivity and positive predictive value (PPV) of single nucleotide variants (SNVs) were 99.3% and 99.8%, respectively. Conclusions: A novel and sensitive computational tool for somatic variation detection in DNA Panel sequencing was developed. Our result showed that the deep learning CNN model could call variant in Panel sequencing data.

The functions and mechanisms of sequence differences of DGAT1 gene on milk fat synthesis between dairy cow and buffalo

In this research communication we describe the DGAT1 sequence and promoter region in dairy cows and buffalo and compare the activities of DGAT1 between the two species in order to increase knowledge of the cause of milk fat variation. pGL-3 basic vectors were used to construct the reporter gene. Based on the predicted promoter region, 4 truncated plasmid vectors were constructed in cow-DGAT1 and 3 plasmid vectors in buffalo-DGAT1. Each reporter plasmid was transfected into the bovine mammary epithelial cell (BMEC), 293T cell, and CHO cells to analyze the activity using Dual-Luciferase Reporter Assay System. The results show that the region between −93 to −556 bp was essential for cow promoter activity while −84 to −590 bp was essential for buffalo promoter activity revealing these regions contain core promoter. The buffalo has higher promoter activity than cow yet it was not statistically significant. Comparison of candidate mutation K232A between cow and buffalo population revealed the presence of both the allelic population in dairy cows (lysine and alanine) however, only K (lysine) allelic amino acid was found in buffalo population. The absence of the alanine allelic population from buffalo explains the higher fat content of buffalo milk.

A Novel COMP Mutation in a Chinese Family with Multiple Epiphyseal Dysplasia

Background: Multiple epiphyseal dysplasia (MED) is a skeletal disorder characterized by delayed and irregular ossification of the epiphyses and early-onset osteoarthritis. At least 66% of the reported autosomal dominant MED (AD-MED) cases are caused by COMP mutations. Methods: We recruited a four-generation Chinese family with early-onset hip osteoarthritis, flatfoot, brachydactyly, and mild short stature. The assessment of family history, detailed physical examinations, and radiographic evaluations were performed on the proband and other family members, followed by the performance of whole-exome sequencing (WES). The pathogenicity of the candidate mutation was also analyzed. Results: We recruited an AD-MED family with 10 affected members and 17 unaffected members. The main radiographic findings were symmetrical changes in the dysplastic acetabulum and femoral heads, irregular contours of the epiphyses, a shortened femoral neck, and flatfoot. Lower bone density was also observed in the ankle joints, wrist joints, and knees, as well as irregular vertebral end plates. In the proband, we identified the missense mutation c.1153G>T (p. Asp385Tyr), located in exon 11 of the COMP gene. This mutation was assessed as “pathogenic” because of its low allele frequency and its high likelihood of co-segregation with disease in the reported family. Sanger sequencing identified the novel heterozygous mutation c.1153G>T (p. Asp385Tyr) in exon 11 of COMP in all affected individuals in the family. Conclusions: Our result underlined a key role of the Asp385 amino acid in the protein function of COMP, and confirmed the pathogenicity of the COMP (c.1153G>T; p. Asp385Tyr) mutation in AD-MED disease. We have therefore expanded the known mutational spectrum of COMP and revealed new phenotypic information for AD-MED.

A Missense Mutation of the HSPB7 Gene Associated with Heat Tolerance in Chinese Indicine Cattle

The small heat shock proteins (HSPB) are expressed in response to heat stress, and the heat shock protein family B (small) member 7 (HSPB7) gene has been reported to play an important role in heat tolerance pathways. Only a missense mutation (NC_037329.1: g.136054902 C > G: p.Ala69Gly) was identified in the HSPB7 gene in indicine cattle, which might be a candidate mutation associated with the heat tolerance. Here, we explore the allele frequency of this mutation in 774 individuals belonging to 32 Chinese indigenous cattle breeds using polymerase chain reaction (PCR) and DNA sequencing methods. The distribution of alleles of NC_037329.1: g.136054902 C > G displays significant geographical difference across native Chinese cattle breeds that the allele C was dominant in northern cattle groups, while allele G was dominant in southern indicine cattle groups. Additionally, the association analysis indicated that the G allele was significantly associated with mean annual temperature (T), relative humidity (RH), and temperature humidity index (THI) (p < 0.01), suggesting that cattle carrying allele G were distributed in regions with higher T, RH, and THI. Our results demonstrate that the mutation of the HSPB7 gene in Chinese indicine cattle might be a candidate gene associated with the heat tolerance.