Novel compound heterozygous mutations in OCA2 gene were identified in a Chinese family with oculocutaneous albinism.

BACKGROUND: Oculocutaneous albinism (OCA) is a group of rare autosomal recessive disorders characterized by clinical genetic heterogeneity. OCA type II (OMIM: 203200) is the most common subtype among African and African Americans, primarily caused by pathogenic variants in the OCA2 (HGNC ID: 8101) gene. In this study, we presented a Chinese family with OCA and reported two novel variants in the OCA2 gene. METHODS: Whole-exome sequencing (WES) was performed to identify pathogenic variants in the proband. The candidate variants were subsequently validated using Sanger sequencing and QPCR assay. Additionally, bioinformatics analyses were employed to predict the deleteriousness and conservation of the identified mutations. RESULTS: In the 16-year-old male proband, two novel compound heterozygous OCA2 variants, NM_000275.3: c.1640T>G (NP_000266.2: p.L547R) and an exons 10-19 deletion variant, were identified. Meanwhile, a reported heterozygous variant c.1441G>A/p.A481T (NM_000275.3, NP_000266.2) in the OCA2 gene was also found in the proband. Sanger sequencing confirmed that the two variants c.1441G>A/p.A481T and c.1640T>G/p.L547R were inherited from his father. Moreover, qPCR assay revealed that the exons 10-19 deletion was inherited from the mother, his sister also carried this variant. Fortunately, the variant was not detected in the amniotic fluid of the proband's sister. Multiple online bioinformatics tools predicted the variant c.1640T>G to be damaging, leading to the replacement of a highly conserved leucine with an arginine. The gross exon 10-19 deletion in the OCA2 gene resulted in a truncated, non-functional protein losing the 3-9 transmembrane α-helices domains. According to the American College of Medical Genetics and Genomics classification, these three variants in the OCA2 gene were evaluated as likely pathogenic. CONCLUSION: This study has identified two novel compound variants in the OCA2 gene and a previously reported variant in a Chinese family with OCA. By expanding the mutation spectrum of the OCA2 gene, our findings contribute to a better understanding of the genetic basis of OCA.

Identification of novel and de novo GABRB1 mutation in Chinese patient with developmental and epileptic encephalopathy 45.

Developmental and epileptic encephalopathy 45 (DEE45) is an autosomal dominant disease caused by variation in the gamma-aminobutyric acid type A receptor subunit beta 1 (GABRB1) gene. Affected individuals have severely impaired intellectual development, hypotonia, and other persistent neurological deficits. However, DEE45 is rare; only four infants with DEE45 have been reported worldwide and no case has been reported in China. Confirming a diagnosis of DEE45 is of great significance for guiding further treatment, assessing patient prognosis, and genetic counseling. The clinical characteristics of DEE45 and the medical history of DEE45 patients requires supplementation and clarification. Here, we present the clinical and genetic findings of a 7-year-old girl with DEE45 carrying a novel de novo GABRB1 mutation (c.858_859delinsTT, p.286_287delinsIleSer) identified by whole exome sequencing (WES). The mutation is phylogenetic conserved in the second helix of the ß1-subunit's transmembrane region. Western blot and RT-qPCR both indicated significant increase in the expression levels of GABRB1 mutant when compared with wild. The proband has epileptic encephalopathy and experienced refractory epilepsy onset at age 2 months and showed developmental delay at age 8 months. Electroencephalography (EEG) displayed hypsarrhythmia. Magnetic resonance imaging (MRI) showed no significant abnormalities in the internal structure of the patient's brain, which is displayed in two previously reported cases. The patient's symptoms of hypotonia, ataxia, profound mental retardation, and dysmetria became evident with development. In summary, we report the genetic and clinical characteristics of the first Chinese patient with DEE45 and explores the relationship between mutation and clinical symptoms.

A novel loss-of-function variant in PNLDC1 inducing oligo-astheno-teratozoospermia and male infertility.

Male infertility is a major reproductive disorder, which is clinically characterized by highly heterogeneous phenotypes of abnormal sperm count or quality. To date, five male patients with biallelic loss-of-function (LOF) variants of PARN-like ribonuclease domain-containing exonuclease 1 ( PNLDC1 ) have been reported to experience infertility with nonobstructive azoospermia. The aim of this study was to identify the genetic cause of male infertility with oligo-astheno-teratozoospermia (OAT) in a patient from a Chinese Han family. Whole-exome and Sanger sequencing analyses identified a homozygous LOF variant (NM_173516.2, c.142C>T, p.Gln48Ter) in PNLDC1 . Hematoxylin and eosin staining revealed that the spermatozoa of the patient with OAT had an irregular head phenotype, including microcephaly, head tapering, and globozoospermia. Consistently, peanut agglutinin staining of the spermatozoa revealed a complete or partial loss of the acrosome. Furthermore, the disomy rate of chromosomes in the patient's spermatozoa was significantly increased compared with that of a fertile control sample. We reported an LOF variant of the PNLDC1 gene responsible for OAT.

MOS mutation causes female infertility with large polar body oocytes.

The Moloney sarcoma oncogene (MOS) encodes a protein serine/threonine kinase and MOS is expressed at high levels in oocytes undergoing meiotic maturation. The MOS/MAPK pathway is normally required for the maintenance of microtubules and chromatin in a metaphasic state during the meiotic divisions. To determine the pathogenic genes in a female infertile patient due to large polar body oocytes, whole-exome sequencing was performed on the patient and available family members. We identified a novel homozygous missense mutation c.591T > G in MOS. Bioinformatics analysis showed that the mutation is harmful. These findings suggest that MOS mutation results in oocytes with a large polar body and poor embryonic development in patients. The MOS variant may regulate oocyte asymmetric division by MAPK/WAVE2/Arp2/3/actin signaling pathway. This will help to understand the comprehensive role of MOS in early human reproductive process and provide genetic markers for future genetic counseling for more individualized treatments.

A novel mutation in ryanodine receptor 2 (RYR2) genes at c.12670G>T associated with focal epilepsy in a 3-year-old child.

Background: Ryanodine receptor 2 (RYR2) encodes a component of a calcium channel. RYR2 variants were well-reported to be associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), but rarely reported in epilepsy cases. Here, we present a novel heterozygous mutation of RYR2 in a child with focal epilepsy. Methods: At the age of 2 years and 7 months, the patient experienced seizures, such as eye closure, tooth clenching, clonic jerking and hemifacial spasm, as well as abnormal electroencephalogram (EEG). Then, he was analyzed by whole-exome sequencing (WES). The mutations of both the proband and his parents were further confirmed by Sanger sequencing. The pathogenicity of the variant was further assessed by population-based variant frequency screening, evolutionary conservation comparison, and American Association for Medical Genetics and Genomics (ACMG) scoring. Results: WES sequencing revealed a novel heterozygous truncating mutation [c.12670G > T, p.(Glu4224*), NM_001035.3] in RYR2 gene of the proband. Sanger sequencing confirmed that this mutation was inherited from his mother. This novel variant was predicted to be damaging by different bioinformatics methods. Cardiac investigation showed that the proband had no structural abnormalities, but sinus tachycardia. Conclusion: We proposed that RYR2 is a potential candidate gene for focal epilepsy, and epilepsy patients carried with RYR2 variants should be given more attention, even if they do not show cardiac abnormalities.

Identifying Circulating Tumor DNA Mutations Associated with Neoadjuvant Chemotherapy Efficacy in Local Advanced Breast Cancer.

Circulating tumor DNA (ctDNA) correlates with tumor burden and provides early detection of treatment response and tumor genetic alterations in breast cancer. Neoadjuvant chemotherapy (NACT) has become standard therapy for local advanced breast cancer (LABC). The aim of our study was to investigate plasma ctDNA as a prognostic marker for outcome in patients with LABC treated with NACT. A total of 56 patients with LABC were involved in this study. ctDNA mutations were investigated by using a 100 gene panel-target capture next-generation sequencing. The patients then received standard NACT therapy: adriamycin and cyclophosphamide and paclitaxel (AC-T) or AC-TH (AC-T+ Trastuzumab) regimen. The efficacy of NACT was evaluated by Miller-Payne grading system. A predictive and weight model was used to screen ctDNA point mutation biomarkers for NACT. The ctDNA mutational profile of LABC patients was identified. For nonsynonymous mutations, the top 5 mutated genes were MTHFR (51/56, 91.1%), XPC (50/56, 89.3%), ABCB1 (48/51, 94.1%), BRCA2 (38/56, 67.9%), and XRCC1 (38/56, 67.9%). In addition, the mutation frequencies of PIK3CA and TP53 were 32.1% (18/56) and 26.8% (15/56), respectively. The predictive model indicated that XRCC1 44055726 (TG>-) mutation (25/56, 44.6%) was significantly associated with Miller-Payne 4-5 and Miller-Payne 3-5 responses. While mTOR 11249132(G>C) mutation (23/56, 41.1%) was associated with Miller-Payne 1-4 or Miller-Payne 1-3 responses. Furthermore, XRCC1 44055726 (TG>-) accompanied by mTOR wild type predicted a good NACT efficacy in all response classification systems. The ROC curves to discriminate good neoadjuvant chemotherapy efficiency (Miller-Payne 4-5) and poor efficiency (Miller-Payne 1-3) were created, and AUC value was 0.77. Our results suggested that ctDNA mutation of XRCC1 44055726 (TG>-) might be a positive biomarker for NACT therapy in LABC, while mTOR 11249132(G>C) mutation was potentially associated with NACT resistance.

Identification of two rare NPRL3 variants in two Chinese families with familial focal epilepsy with variable foci 3: NGS analysis with literature review.

Background: The GAP Activity Towards Rags 1 (GATOR1) complex, which includes DEPDC5, NPRL2, and NPRL3, plays a key role in epilepsy. It has been reported that focal epilepsy is associated with mutations in the NPRL3 gene in some cases. We report two rare mutations in the NPRL3 gene in two unrelated Chinese families with focal epilepsy in this study. Methods: The proband and her brother in family E1 first experienced seizures at 1.5 and 6 years of age, respectively. Despite resection of epileptogenic foci, she still suffered recurrent seizures. The first seizure of a 20-year-old male proband in family E2 occurred when he was 2 years old. To identify pathogenic variants in these families, whole-exome sequencing (WES) was performed on genomic DNA from peripheral blood. Results: In family E1, the trio-WES analysis of the proband and her brother without apparent structural brain abnormalities identified a heterozygous variant in the NPRL3 gene (c.954C>A, p.Y318*, NM_001077350.3). In family E2, the proband carried a heterozygous NPRL3 mutation (c.1545-1G>C, NM_001077350.3). Surprisingly, the mothers of the two probands each carried the variants, but neither had an attack. Bioinformatics analysis predicted that the mutation (c.954C>A) was in the highly conserved amino acid residues of NPRL3, which affected the α-helix of NPRL3 protein, leading to a truncated protein. The splice variant (c.1545-1G>C) resulted in the loss of the last exon of the NPRL3 gene. Conclusion: The results of this study provide a foundation for diagnosing NPRL3-related epilepsy by enriching their genotypes and phenotypes and help us identify the genetic etiologies of epilepsy in these two families.

Novel associations of SNPs MYLIP rs3757354 and ABCA1 2230806 gene with early-onset-preeclampsia: A case-control candidate genetic study.

OBJECTIVE: To investigate the association between MYLIP rs3757354 and ABCA1 2230806 single nucleotide polymorphisms in women with preeclampsia in China. STUDY DESIGN: The case-control study involved 205 patients with preeclampsia and 145 controls. All women with preeclampsia were divided into two groups: 78 patients with early-onset preeclampsia and 127 with late-onset preeclampsia. MAIN OUTCOME MEASURE: MYLIP rs3757354 and ABCA1 rs2230806 SNPs were analyzed through multiplex PCR for targeted next-generation sequencing technology. A secondary outcome was lipid profile changes and liver function in women with PE. RESULTS: Maternal age (OR: 1.073, 95% CI = 1.006-1.145), BMI (OR: 1.118, 95% CI = 1.040-1.201), TG/HDL-C (OR: 1.536, 95% CI = 1.080-2.183), and TT genotype of SNP rs3757354 (OR: 3.238, 95% CI = 1.313-7.990) were associated with EOPE risk. Our study found that patients with TT genotype of ABCA1 rs2230806 had more severe hepatic dysfunction and higher HDL levels in the EOPE group compared with CC/CT genotype. There was no association between rs2230806 and the risk of PE. CONCLUSION: The polymorphisms of rs3757354 are associated with the risk of EOPE in Chinese pregnant women. The TT genotype in ABCA1 rs2230806 is a strong predictive risk for elevated aminotransferase levels in pregnant women with EOPE.

Novel association between FOXO3 rs2232365 polymorphism and late-onset preeclampsia: a case-control candidate genetic study.

BACKGROUND: Both genetic susceptibility and dysregulated lipid metabolism are important susceptibilities to preeclampsia. In the study, we devote to investigate the associations of FOXO3 and TLR7 genetic polymorphisms with preeclampsia in a Chinese population. METHODS: This case-control study involved 335 Han Chinese pregnant women, including 177 pregnant women with preeclampsia and 158 healthy controls. The preeclampsia group was further sub-grouped into early-onset preeclampsia (EOPE, n = 70)and late-onset preeclampsia (LOPE, n = 107. Three single nucleotide polymorphisms (SNPs), including FOXO3 (rs2232365, rs3761548), and TLR7 rs3853839 were genotyped by multiplex PCR for targeted next-generation sequencing. The χ2 test and multiple interaction effect analyses were performed to determine the association of three SNPs with serum lipid levels and thyroid function in women with preeclampsia. RESULTS: The genotype (CC vs. TT + CT) distribution of rs2232365 revealed a significant association with LOPE (P = 0.004, odds ratio = 3.525 (0.95 CI: 1.498-8.164)). No significant difference was found in the genotype and allele frequencies of rs3761548 and rs3853839 between controls and cases (P > 0.05). Moreover, the genotype CT/TT of rs2232365 was significantly correlated with increased TG/HDL levels in the LOPE group (p = 0.014). CONCLUSIONS: The polymorphisms of rs2232365 are associated with the risk of LOPE and may modulate TG/HDL levels in pregnant women with LOPE.

Protective Effects of Arginine on Saccharomyces cerevisiae Against Ethanol Stress.

Yeast cells are challenged by various environmental stresses in the process of industrial fermentation. As the currently main organism for bio-ethanol production, Saccharomyces cerevisiae suffers from ethanol stress. Some amino acids have been reported to be related to yeast tolerance to stresses. Here the relationship between arginine and yeast response to ethanol stress was investigated. Marked inhibitions of ethanol on cell growth, expression of genes involved in arginine biosynthesis and intracellular accumulation of arginine were observed. Furthermore, extracellular addition of arginine can abate the ethanol damage largely. To further confirm the protective effects of arginine on yeast cells, yeast strains with different levels of arginine content were constructed by overexpression of ARG4 involved in arginine biosynthesis or CAR1 encoding arginase. Intracellular arginine was increased by 18.9% or 13.1% respectively by overexpression of ARG4 or disruption of CAR1, which enhanced yeast tolerance to ethanol stress. Moreover, a 41.1% decrease of intracellular arginine was observed in CAR1 overexpressing strain, which made yeast cells keenly sensitive to ethanol. Further investigations indicated that arginine protected yeast cells from ethanol damage by maintaining the integrity of cell wall and cytoplasma membrane, stabilizing the morphology and function of organellae due to low ROS generation.

[Improvement of acetic acid tolerance and fermentation performance of industrial Saccharomyces cerevisiae by overexpression of flocculent gene FLO1 and FLO1c].

Flocculent gene FLO1 and its truncated form FLO1c with complete deletion of repeat unit C were expressed in a non-flocculent industrial strain Saccharomyces cerevisiae CE6 to generate recombinant flocculent strains 6-AF1 and 6-AF1c respectively. Both strains of 6-AF1 and 6-AF1c displayed strong flocculation and better cell growth than the control strain CE6-V carrying the empty vector under acetic acid stress. Moreover, the flocculent strains converted glucose to ethanol at much higher rates than the control strain CE6-V under acetic acid stress. In the presence of 0.6% (V/V) acetic acid, the average ethanol production rates of 6-AF1 and 6-AF1c were 1.56 and 1.62 times of that of strain CE6-V, while the ethanol production rates of 6-AF1 and 6-AF1c were 1.21 and 1.78 times of that of strain CE6-V under 1.0% acetic acid stress. Results in this study indicate that acetic acid tolerance and fermentation performance of industrial S. cerevisiae under acetic acid stress can be improved largely by flocculation endowed by expression of flocculent genes, especially FLO1c.

[Effect of tandem repeats adjacent to 3'-terminal of FLO1 on the flocculation function of Saccharomyces cerevisiae].

OBJECTIVE: Many tandem repeats exist in FLO1 gene of Saccharomyces cerevisiae, which might have great regulatory effect on the conformation and function of flocculation protein (flocculin). In this study, we analyzed the effect of 3'-terminal tandem repeats B, C and D complete deletion on the function of flocculin. METHODS: We constructed the derived gene FLO1 bcd with complete deletion of tandem repeats B, C and D of FLO1 by fusion PCR. We then constructed plasmid pYCF1 bcd by insertion of FLO1 bcd into YCp50, and transformed such plasmid, pYCF1 and YCp50 into S. cerevisiae YS58 separately to generate recombinant strains YSF1 bcd, YSF1 and YSP50. We compared the flocculation ability and characteristics of these strains. RESULT: Compared to YSF1, YSF1 bcd displayed only a slight reduction (4%) in flocculation ability in optimal flocculation buffer (50 mmol/L NaAC, pH 4.5). Moreover, the dependence of flocculation on Ca2+, sensitivity to metal ions and ethanol, and the specificity to different sugars showed no obvious difference between strains YSF1 and YSF1 bcd. However, strain YSF1 bcd displayed much higher flocculation levels than strain YSF1 under conditions with extreme pH, high temperature, or high concentration of mannose. CONCLUSION: Combined deletion of tandem repeats B, C and D adjacent to the 3'-terminal of FLO1 increases the conformation stability of flocculin in response to changes of pH, temperature or concentration of mannose in environment, but does not influence the other characteristics of flocculation.