Mutation analysis of subjects with 46, XX sex reversal and 46, XY gonadal dysgenesis does not support the involvement of SOX3 in testis determination

Despite the identification of an increasing number of genes involved in sex determination and differentiation, no cause can be attributed to most cases of 46, XY gonadal dysgenesis, approximately 20% of 46, XX males and the majority of subjects with 46, XX true hermaphroditism. Perhaps the most interesting candidate for involvement in sexual development is SOX3, which belongs to the same family of proteins (SOX) as SRY and SOX9, both of which are involved in testis differentiation. As SOX3 is the most likely evolutionary precursor to SRY, it has been proposed that it has retained a role in testis differentiation. Therefore, we screened the coding region and the 5 and 3 flanking region of the SOX3 gene for mutations by means of single-stranded conformation polymorphism and heteroduplex analysis in eight subjects with 46, XX sex reversal (SRY negative) and 25 subjects with 46, XY gonadal dysgenesis. Although no mutations were identified, a nucleotide polymorphism (1056C/T) and a unique synonymous nucleotide change (1182A/C) were detected in a subject with 46, XY gonadal dysgenesis. The single nucleotide polymorphism had a heterozygosity rate of 5.1% (in a control population) and may prove useful for future X-inactivation studies. The absence of SOX3 mutations in these patients suggests that SOX3 is not a cause of abnormal male sexual development and might not be involved in testis differentiation.An erratum to this article can be found at     

SNPs associated with body weight and backfat thickness in two pig breeds identified by a genome-wide association study

Growth and fat deposition are important economic traits due to the influence on production in pigs. In this study, a dataset of 1200 pigs with 345,570 SNPs genotyped by sequencing (GBS) was used to conduct a GWAS with single-marker regression method to identify SNPs associated with body weight and backfat thickness (BFT) and to search for candidate genes in Landrace and Yorkshire pigs. A total of 27 and 13 significant SNPs were associated with body weight and BFT, respectively. In the region of 149.85–149.89 Mb on SSC6, the SNP (SSC6: 149876737) for body weight and the SNP (SSC6: 149876507) for BFT were in the same locus region (a gap of 230 bp). Two SNPs were located in the DOCK7 gene, which is a protein-coding gene that plays an important role in pigmentation. Two SNPs located on SSC8: 54567459 and SSC11: 33043081 were found to overlap weight and BFT; however, no candidate gene was found in these regions. In addition, based on other significant SNPs, two positional candidate genes, NSRP1 and CADPS, were proposed to influence weight. In conclusion, this is the first study report using GBS data to identify the significant SNPs for weight and BFT. A total of four particularly interesting SNPs and one potential candidate genes (DOCK7) were found for these traits in domestic pigs. This study improves our knowledge to better understand the complex genetic architecture of weight and BFT, but further validation studies of these candidate loci and genes are recommended in pigs     

Novel mutations in the SOX10 gene in the first two Chinese cases of type IV Waardenburg syndrome

Objective: We analyzed the clinical features and family-related gene mutations for the first two Chinese cases of type IV Waardenburg syndrome (WS4). Methods: Two families were analyzed in this study. The analysis included a medical history, clinical analysis, a hearing test and a physical examination. In addition, the EDNRB, EDN3 and SOX10 genes were sequenced in order to identify the pathogenic mutation responsible for the WS4 observed in these patients. Results: The two WS4 cases presented with high phenotypic variability. Two novel heterozygous mutations (c.254G>A and c.698-2A>T) in the SOX10 gene were detected. The mutations identified in the patients were not found in unaffected family members or in 200 unrelated control subjects. Conclusions: This is the first report of WS4 in Chinese patients. In addition, two novel mutations in SOX10 gene have been identified.     

High-Throughput Discovery of Mutations in Tef Semi-Dwarfing Genes by Next-Generation Sequencing Analysis

Tef (Eragrostis tef) is a major cereal crop in Ethiopia. Lodging is the primary constraint to increasing productivity in this allotetraploid species, accounting for losses of ∼15–45% in yield each year. As a first step toward identifying semi-dwarf varieties that might have improved lodging resistance, an ∼6× fosmid library was constructed and used to identify both homeologues of the dw3 semi-dwarfing gene of Sorghum bicolor. An EMS mutagenized population, consisting of ∼21,210 tef plants, was planted and leaf materials were collected into 23 superpools. Two dwarfing candidate genes, homeologues of dw3 of sorghum and rht1 of wheat, were sequenced directly from each superpool with 454 technology, and 120 candidate mutations were identified. Out of 10 candidates tested, six independent mutations were validated by Sanger sequencing, including two predicted detrimental mutations in both dw3 homeologues with a potential to improve lodging resistance in tef through further breeding. This study demonstrates that high-throughput sequencing can identify potentially valuable mutations in under-studied plant species like tef and has provided mutant lines that can now be combined and tested in breeding programs for improved lodging resistance.     

Phenotypic similarities in pigs with SOX10c.321dupC and SOX10c.325A>T mutations implied the correlation of SOX10 haploinsufficiency with Waardenburg syndrome

SOX10 is a causative gene of Waardenburg syndrome (WS) that is a rare genetic disorder characterized by hearing loss and pigment disturbance. More than 100 mutations of SOX10 have been found in patients with Type 2 WS (WS2), Type 4 WS (WS4), and more complex syndromes. However, no mutation hotspot has been detected in SOX10, and most cases are sporadic, making it difficult to establish a correlation between the high phenotypic and genetic variability. In this study, a duplication of the 321th cytosine (c.321dupC) was introduced into SOX10 in pigs, which induced premature termination of the translation of SOX10 (p.K108QfsX45). The premature stop codon in Exon 3 triggered the degradation of mutant mRNA through nonsense-mediated mRNA decay. However, SOX10^c.321dupC induced a highly similar phenotype of WS2 with heterogeneous inner ear malformation compared with its adjacent missense mutation SOX10^c.325A>T. In addition, a site-saturation mutation analysis of the SOX10 N-terminal nuclear localization signal (n-NLS), where these two mutations located, revealed the correlation between SOX10 haploinsufficiency and WS by an in vitro reporter assay. The analysis combining the in vitro assay with clinical cases may provide a clue to clinical diagnoses.     

Identification and functional analysis of a novel mutation in the SOX10 gene associated with Waardenburg syndrome type IV

Waardenburg syndrome type IV (WS4) is a rare genetic disorder, characterized by auditory–pigmentary abnormalities and Hirschsprung disease. Mutations of the EDNRB gene, EDN3 gene, or SOX10 gene are responsible for WS4. In the present study, we reported a case of a Chinese patient with clinical features of WS4. In addition, the three genes mentioned above were sequenced in order to identify whether mutations are responsible for the case. We revealed a novel nonsense mutation, c.1063C>T (p.Q355*), in the last coding exon of SOX10. The same mutation was not found in three unaffected family members or 100 unrelated controls. Then, the function and mechanism of the mutation were investigated in vitro. We found both wild-type (WT) and mutant SOX10 p.Q355* were detected at the expected size and their expression levels are equivalent. The mutant protein also localized in the nucleus and retained the DNA-binding activity as WT counterpart; however, it lost its transactivation capability on the MITF promoter and acted as a dominant-negative repressor impairing function of the WT SOX10.     

Oncogene Panel Sequencing Analysis Identifies Candidate Actionable Genes in Advanced Well-Differentiated Gastroenteropancreatic Neuroendocrine Tumors

Objective: To report the rate of candidate actionable somatic mutations in patients with locally advanced and metastatic gastro-enteropancreatic (GEP) neuroendocrine tumors (NET) and of other genetic alterations that may be associated with tumorigenesis.Methods: A phase II mutation targeted therapy trial was conducted in patients with advanced well-differentiated G1/G2 GEP-NET. Mutations found in the mTOR pathway-associated genes led to treatment with the mTOR inhibitor everolimus, and were defined as actionable. Tumor deoxyribonucleic acid (DNA) from GEP-NET were sequenced and compared with germline DNA, using the OncoVAR-NET assay, designed for hybrid capture sequencing of 500 tumor suppressor genes and oncogenes. Somatic variants were called and copy-number (CN) variant analysis was performed.Results: Thirty patients (14 small-intestine, 8 pancreatic, 3 unknown primary NET, and 5 of other primary sites) harbored 37 lesions (4 patients had DNA of multiple lesions sequenced). Only 2 patients with sporadic NET (n = 26) had an actionable mutation leading to treatment with everolimus. Driver somatic mutations were detected in 18 of 30 patients (21/37 lesions sequenced). In the remaining samples without a driver mutation, CN alterations were found in 11/16 tumors (10/12 patients), including CN loss of chromosome (Chr) 18 (P<.05), CN gain of Chr 5, and loss of Chr 13. CN losses in Chr 18 were more common in patients without driver mutations detected. Pronounced genetic heterogeneity was detected in patients with multiple lesions sequenced.Conclusion: Genome-wide DNA sequencing may identify candidate actionable genes and lead to the identification of novel target genes for advanced well-differentiated GEP-NET.Abbreviations: Chr = chromosome; CN = copy number; DNA = deoxyribonucleic acid; FDA = Food and Drug Administration; GEP = gastro-enteropancreatic; MEN-1 = multiple endocrine neoplasia syndrome type 1; mTOR = mammalian target of rapamycin; NET = neuroendocrine tumor; PFS = progression-free survival; PNET = pancreatic neuroendocrine tumors; SINET = small-intestine neuroendocrine tumor     

Mutations in SOX9, the Gene Responsible for Campomelic Dysplasia and Autosomal Sex Reversal

Campomelic dysplasia (CD) is a skeletal malformation syndrome frequently accompanied by 46,XY sex reversal. A mutation-screening strategy using SSCP was employed to identify mutations in SOX9, the chromosome 17q24 gene responsible for CD and autosomal sex reversal in man. We have screened seven CD patients with no cytologically detectable chromosomal aberrations and two CD patients with chromosome 17 rearrangements for mutations in the entire open reading frame of SOX9. Five different mutations have been identified in six CD patients: two missense mutations in the SOX9 putative DNA binding domain (high mobility group, or HMG, box); three frameshift mutations and a splice-acceptor mutation. An identical frameshift mutation is found in two unrelated 46,XY patients, one exhibiting a male phenotype and the other displaying a female phenotype (XY sex reversal). All mutations found affect a single allele, which is consistent with a dominant mode of inheritance. No mutations were found in the SOX9 open reading frame of two patients with chromosome 17q rearrangements, suggesting that the translocations affect SOX9 expression. These findings are consistent with the hypothesis that CD results from haploinsufficiency of SOX9.     

Mutations in the sarcosine dehydrogenase gene in patients with sarcosinemia

Sarcosinemia is an autosomal recessive metabolic trait manifested by relatively high concentrations of sarcosine in blood and urine. Sarcosine is a key intermediate in 1-carbon metabolism and under normal circumstances is converted to glycine by the enzyme sarcosine dehydrogenase. We encountered six families from two different descents (French and Arab), each with at least one individual with elevated levels of sarcosine in blood and urine. Using the “candidate gene approach” we sequenced the gene encoding sarcosine dehydrogenase (SARDH), which plays an important role in the conversion of sarcosine to glycine, and found four different mutations (P287L, V71F, R723X, R514X) in three patients. In an additional patient, we found a uniparental disomy in the region of SARDH gene. In two other patients, we did not find any mutations in this gene. We have shown for the first time that mutations in the SARDH gene are associated with sarcosinemia. In addition, our results indicate that other genes are most probably involved in the pathogenesis of this condition.     

Genomic analysis identifies candidate pathogenic variants in 9 of 18 patients with unexplained West syndrome

West syndrome, which is narrowly defined as infantile spasms that occur in clusters and hypsarrhythmia on EEG, is the most common early-onset epileptic encephalopathy (EOEE). Patients with West syndrome may have clear etiologies, including perinatal events, infections, gross chromosomal abnormalities, or cases followed by other EOEEs. However, the genetic etiology of most cases of West syndrome remains unexplained. DNA from 18 patients with unexplained West syndrome was subjected to microarray-based comparative genomic hybridization (array CGH), followed by trio-based whole-exome sequencing in 14 unsolved families. We identified candidate pathogenic variants in 50 % of the patients (n = 9/18). The array CGH revealed candidate pathogenic copy number variations in four cases (22 %, 4/18), including an Xq28 duplication, a 16p11.2 deletion, a 16p13.1 deletion and a 19p13.2 deletion disrupting CACNA1A. Whole-exome sequencing identified candidate mutations in known epilepsy genes in five cases (36 %, 5/14). Three candidate de novo mutations were identified in three cases, with two mutations occurring in two new candidate genes (NR2F1 and CACNA2D1) (21 %, 3/14). Hemizygous candidate mutations in ALG13 and BRWD3 were identified in the other two cases (14 %, 2/14). Evaluating a panel of 67 known EOEE genes failed to identify significant mutations. Despite the heterogeneity of unexplained West syndrome, the combination of array CGH and whole-exome sequencing is an effective means of evaluating the genetic background in unexplained West syndrome. We provide additional evidence for NR2F1 as a causative gene and for CACNA2D1 and BRWD3 as candidate genes for West syndrome.     

A case of intersexuality in pigs associated with a de novo paracentric inversion 9 (p1.2; p2.2)

In several mammalian species, genetic defects can be responsible for the interruption of and/or the deviation from the sequential steps of normal gonadal differentiation, leading to a sex-reversal syndrome. In pigs, female-to-male sex-reversal conditions are particularly frequent, but their aetiologies remain unclear. Chromosomal abnormalities that co-occur with sex-reversal disorders can be useful in the identification of loci containing responsible or susceptibility genes. This report describes a female-to-male SRY-negative intersex pig with a de novo paracentric inversion of the short arm of one chromosome 9 (p1.2; p2.2). We have fine mapped the proximal chromosomal breakpoint of this rearrangement because it corresponded to a region potentially involved in the pig intersexuality. Fluorescent in situ hybridization (FISH) experiments carried out with Bacterial Artificial Chromosome (BAC) clones located within the critical region defined by genetic linkage analysis and ordered on the porcine RH map allowed us to locate the proximal breakpoint between markers SW2571 and SW539. Further investigations are currently in progress to find new markers inside this interval, in order to determine the BAC in which the break occurred.     

Single step genome-wide association studies based on genotyping by sequence data reveals novel loci for the litter traits of domestic pigs

In this study, data genotyping by sequence (GBS) was used to perform single step GWAS (ssGWAS) to identify SNPs associated with the litter traits in domestic pigs and search for candidate genes in the region of significant SNPs. After quality control, 167,355 high-quality SNPs from 532 pigs were obtained. Phenotypic traits on 2112 gilt litters from 532 pigs were recorded including total number born (TNB), number born alive (NBA), and litter weight born alive (LWB). A single-step genomic BLUP approach (ssGBLUP) was used to implement the genome-wide association analysis at a 5% genome-wide significance level. A total of 8, 23 and 20 significant SNPs were associated with TNB, NBA, and LWB, respectively, and these significant SNPs accounted for 62.78%, 79.75%, and 58.79% of genetic variance. Furthermore, 1 (SSC14: 16314857), 4 (SSC1: 81986236, SSC1: 66599775, SSC1: 161999013, and SSC1: 267883107), and 5 (SSC9: 29030061, SSC2: 32368561, SSC5: 110375350, SSC13: 45619882 and SSC13: 45647829) significant SNPs for TNB, NBA, and LWB were inferred to be novel loci. At SSC1, the AIM1 and FOXO3 genes were found to be associated with NBA; these genes increase ovarian reproductive capacity and follicle number and decrease gonadotropin levels. The genes SLC36A4 and INTU are involved in cell growth, cytogenesis and development were found to be associated with LWB. These significant SNPs can be used as an indication for regions in the Sus scrofa genome for variability in litter traits, but further studies are expected to confirm causative mutations.