Sanger Sequencing Of Exons Research Articles

Serological characteristics of ABO blood group and molecular genetic analysis of a Chinese pedigree with cisAB09 subtype

To explore the serological characteristics of ABO blood group and molecular genetic mechanism for a Chinese pedigree with cisAB09 subtype. A pedigree undergoing ABO blood group examination at the Department of Transfusion, Zhongshan Hospital Affiliated to Xiamen University on February 2, 2022 was selected as the study subjects. Serological assay was carried out to determine the ABO blood group of the proband and his family members. Activities of A and B glycosyltransferases in the plasma of the proband and his mother were measured with an enzymatic assay. Expression of A and B antigens on the red blood cells of the proband was analyzed by flow cytometry. Peripheral blood samples of the proband and his family members were collected. Following extraction of genomic DNA, exons 1 to 7 of the ABO gene and their flanking introns were sequenced, and Sanger sequencing of exon 7 was carried out for the proband, his elder daughter and mother. The results of serological assay suggested that the proband and his elder daughter and mother had an A2B phenotype, whilst his wife and younger daughter had an O phenotype. Measurement of plasma A and B glycosyltransferase activity suggested that the titers of B-glycosyltransferase activity were 32 and 256 for the proband and his mother, which were respectively below and above that of A1B phenotype-positive controls (128). Flow cytometry analysis showed that the expression of A antigen on the red blood cell surface of the proband has decreased, whilst the expression of B antigen was normal. Genetic sequencing confirmed that, in addition to an ABO*B.01 allele, the proband, his elder daughter and mother have harbored a c.796A>G variant in exon 7, which has resulted in substitution of the methionine at 266th position of the B-glycosyltransferase by valine and conformed to the characteristics of ABO*cisAB.09 allele. The genotypes of the proband and his elder daughter were determined as ABO*cisAB.09/ABO*O.01.01, his mother was ABO*cisAB.09/ABO*B.01, and his wife and younger daughter were ABO*O.01.01/ABO*O.01.01. The c.796A>G variant of the ABO*B.01 allele has resulted in an amino acid substitution p.Met266Val, which probably underlay the cisAB09 subtype. The ABO*cisA B.09 allele encodes a special glycosyltransferase which can synthesize normal level of B antigen and low level of A antigen on the red blood cells.

Molecular Diagnosis and Identification of Novel Pathogenic Variants in a Large Cohort of Italian Patients Affected by Polycystic Kidney Diseases.

Polycystic Kidney Diseases (PKDs) consist of a genetically and phenotypically heterogeneous group of inherited disorders characterized by numerous renal cysts. PKDs include autosomal dominant ADPKD, autosomal recessive ARPKD and atypical forms. Here, we analyzed 255 Italian patients using an NGS panel of 63 genes, plus Sanger sequencing of exon 1 of the PKD1 gene and MPLA (PKD1, PKD2 and PKHD1) analysis. Overall, 167 patients bore pathogenic/likely pathogenic variants in dominant genes, and 5 patients in recessive genes. Four patients were carriers of one pathogenic/likely pathogenic recessive variant. A total of 24 patients had a VUS variant in dominant genes, 8 patients in recessive genes and 15 patients were carriers of one VUS variant in recessive genes. Finally, in 32 patients we could not reveal any variant. Regarding the global diagnostic status, 69% of total patients bore pathogenic/likely pathogenic variants, 18.4% VUS variants and in 12.6% of patients we could not find any. PKD1 and PKD2 resulted to be the most mutated genes; additional genes were UMOD and GANAB. Among recessive genes, PKHD1 was the most mutated gene. An analysis of eGFR values showed that patients with truncating variants had a more severe phenotype. In conclusion, our study confirmed the high degree of genetic complexity at the basis of PKDs and highlighted the crucial role of molecular characterization in patients with suspicious clinical diagnosis. An accurate and early molecular diagnosis is essential to adopt the appropriate therapeutic protocol and represents a predictive factor for family members.

Somatic EPAS1 Variants in Pheochromocytoma and Paraganglioma in Patients With Sickle Cell Disease.

Somatic EPAS1 variants account for 5% to 8% of all pheochromocytoma and paragangliomas (PPGL) but are detected in over 90% of PPGL in patients with congenital cyanotic heart disease, where hypoxemia may select for EPAS1 gain-of-function variants. Sickle cell disease (SCD) is an inherited hemoglobinopathy associated with chronic hypoxia and there are isolated reports of PPGL in patients with SCD, but a genetic link between the conditions has yet to be established. To determine the phenotype and EPAS1 variant status of patients with PPGL and SCD. Records of 128 patients with PPGL under follow-up at our center from January 2017 to December 2022 were screened for SCD diagnosis. For identified patients, clinical data and biological specimens were obtained, including tumor, adjacent non-tumor tissue and peripheral blood. Sanger sequencing of exons 9 and 12 of EPAS1, followed by amplicon next-generation sequencing of identified variants was performed on all samples. Four patients with both PPGL and SCD were identified. Median age at PPGL diagnosis was 28 years. Three tumors were abdominal paragangliomas and 1 was a pheochromocytoma. No germline pathogenic variants in PPGL-susceptibility genes were identified in the cohort. Genetic testing of tumor tissue detected unique EPAS1 variants in all 4 patients. Variants were not detected in the germline, and 1 variant was detected in lymph node tissue of a patient with metastatic disease. We propose that somatic EPAS1 variants may be acquired through exposure to chronic hypoxia in SCD and drive PPGL development. Future work is needed to further characterize this association.

Association of methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms with vitamin B12 deficiency and adverse perinatal outcomes among pregnant women of rural South India - a cross sectional longitudinal study.

To determine the occurrence of MTHFR gene polymorphisms and to study their association with vitamin B12 deficiency and adverse perinatal outcomes among a cohort of pregnant women from Kaniyambadi block, Tamil Nadu. 120 consecutive pregnant women who were ≤20weeks of gestational age from the 82 villages of Kaniyambadi block were recruited. Genomic DNA was isolated from the peripheral blood. PCR amplification was done followed by Sangers sequencing. Maternal and neonatal outcomes were extracted. Data was entered and analysed. Our study found the occurrence of c.1298A>C variant in homozygous state in 14.2% and c.677C>T heterozygous state in 15%. Sanger sequencing of exon 7 identified another pathogenic variant c.1262G>T in heterozygous state in two of them. Both the mothers who harboured that variant had preterm delivery and one of them gave birth to a low-birth-weight neonate. In the entire cohort, 5% of the mothers had abortion, 4.2% of them had preterm delivery and 8.8% of the neonates had low birth weight. Presence of c.1298A>C or c.677C>T variants were associated with vitamin B12 deficiency [Pearson Chi squared value (χ2)=7.9 and 7.6 respectively; p=0.02]. Heterozygous pathogenic variant c.1262G>T was associated with both adverse maternal [χ2=11.5; p=0.001] and neonatal [χ2=18.3; p=0.009] outcomes. MTHFR gene polymorphisms could be associated with several adverse perinatal outcomes and vitamin B12 deficiency. Further larger studies are needed toprove the pathogenicity of c.1262G>T variant on pregnancy.

Clinicopathologic Evaluation of CTNNB1 Mutations in High-Intermediate Risk Endometrial Endometrioid Carcinoma.

CTNNB1 mutations convey increased risk of recurrence in low-risk endometrial endometrioid carcinoma (EEC). Results from previous high-intermediate risk (HIR) cohorts are mixed. The aims of this study were to correlate CTNNB1 mutational status with clinical outcomes and to evaluate the relationship between CTNNB1 mutations and the 4 prognostic subgroups defined by The Cancer Genome Atlas in HIR EEC. CTNNB1 mutational status was determined by Sanger sequencing of exon 3 of the CTNNB1 gene. Mismatch repair, POLE , p53, and L1 cell-adhesion molecule (L1CAM) status were also evaluated. Descriptive statistics and survival analyses were performed. Eighty-eight cases of HIR EEC were identified, of which 22 (25%) were CTNNB1 mutant ( CTNNB1 -mut) and 66 (75%) were wild-type ( CTNNB1 -WT). Median follow-up was 60 mo. Recurrence occurred in 13/88 (15%) patients. Recurrence rates were not significantly different between patients with CTNNB1- mut and CTNNB1- WT tumors (14% vs. 15%, P =0.86). Recurrence-free survival and overall survival were not significantly different (recurrence-free survival hazard ratio: 0.97, 95% confidence interval: 0.27-3.52, P =0.96; overall survival hazard ratio: 0.23, 95% confidence interval: 0.03-1.71, P =0.15). Mismatch repair deficiency was more prevalent in CTNNB1 -WT compared with CTNNB1 -mut tumors (46% vs. 14%, P =0.01); prevalence of POLE mutations and aberrant p53 were not significantly different. In contrast to patients with low-risk EEC, no differences in recurrence or survival were found in patients with HIR EEC with CTNNB1- mut compared with CTNNB1 -WT tumors.

Genetic characteristic and clinical features of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL)

Introduction. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is one of the most common genetic causes of small-vessel cerebral diseases.Objective. The aim of our study was to examine the frequency and severity of phenotypic spectrum in patients with CADASIL, including the study of the prevalence of the NOTCH3 gene mutations in patients with suspected CADASIL.Material and methods. Sanger sequencing of exons 2–7, 11 of NOTCH3 gene was conducted in 314 patients with suspected CADASIL (confirmed by anamnesis and magnetic resonance imaging (MRI)). Clinical and MRI data were collected and analyzed for 14 patients with CADASIL.Results. NOTCH3 gene aberrations in exons 2–7, 11 were detected in 34 of 314 examined patients, that is 11% of all cases. The most frequent aberrations are localized in exon 4 (70.4%), exon 3 and exon 6 (8.8%) of the NOTCH3 gene. A detailed analysis of clinical and instrumental data was conducted in 14 cases of confirmed CADASIL with pathogenic mutations.Conclusion. The age of manifestation of CADASIL in the Russian population varies significantly. Patients without a previous history of TIA/stroke may have an atypical course of the disease, including cerebellar ataxia and epilepsy. MRI pattern of the CADASIL patients of the studied cohort showed no severe damage of external capsules and temporal lobes. Spinal cord lesion are not to be excluded as a CADASIL symptom.

Pseudoachondroplasia: Phenotype and genotype in 11 Indian patients.

Pseudoachondroplasia (PSACH) is an autosomal dominant disorder characterized by rhizomelic short-limbed skeletal dysplasia. The primary clinical and radiographic features include disproportionate dwarfism, joint laxity and hyperextensibility, exaggerated lumbar lordosis, and late ossification of the epiphyses. Identification of disease-causing variants in heterozygous state in COMP establishes the molecular diagnosis of PSACH. We examined 11 families with clinical features suggestive of PSACH. In nine families, we used Sanger sequencing of exons 8-19 of COMP (NM_000095.2) and in two families exome sequencing was used for confirming the diagnosis. We identified 10 de novo variants, including five known variants (c.925G>A, c.976G>A, c.1201G>T, c.1417_1419del, and c.1511G>A) and five variants (c.874T>C, c.1201G>C, c.1309G>A, c.1416_1421delCGACAA, and c.1445A>T) which are not reported outside Indian ethnicity. We hereby report the largest series of individuals with molecular diagnosis of PSACH from India and reiterate the well-known genotype-phenotype corelation in PSACH.

Clinicopathologic evaluation of CTNNB1 gene mutations in high-intermediate risk endometrial cancer

<h3>Objectives:</h3> <i>CTNNB1</i> mutations convey increased risk of recurrence in low risk endometrioid endometrial cancers. However, sub-analyses of high-intermediate risk (HIR) endometrioid endometrial cancers in other cohorts have not demonstrated a clearly increased recurrence risk and have often been equivocal. The objective of this study is to perform a clinicopathologic evaluation of patients with HIR endometrioid endometrial cancer in relation to <i>CTNNB1</i> mutational status. <h3>Methods:</h3> A single-institution chart review of patients diagnosed with endometrioid endometrial cancer between 2006 and 2018 was undertaken. Patients were classified as HIR according to GOG249 criteria. Mutational status was determined by Sanger sequencing of exon 3 of the <i>CTNNB1</i> gene in primary tumor specimens. Immunohistochemical (IHC) analysis of B-catenin was performed to evaluate for the presence of nuclear staining. Factors associated with recurrence were assessed by logistic regression; recurrence-free (RFS) and overall survival (OS) were assessed by Kaplan-Meier survival analysis. <h3>Results:</h3> 93 cases of HIR endometrioid endometrial cancer were identified, of which 23 (24.7%) were <i>CTNNB1</i> mutant and 70 (75.3%) were wild type. Median follow-up was 59.7 months. Recurrence occurred in 14/93 (15.1%) patients. Recurrence rates were not significantly different between patients with <i>CTNNB1</i> mutant and wild type tumors (13.0% vs. 15.9%, p=0.76). In both univariable and multivariable analyses, RFS and OS were not significantly different (RFS HR 0.88, p=0.85, 95% CI 0.24 - 3.17; OS HR 0.20, p=0.12, 95% CI 0.03 - 1.54). Predicted oncogenic mutations comprised 87% of all identified mutations and were present in 100% of recurrent tumors and 85% of non-recurrent tumors (p=0.47). Nuclear B-catenin expression by IHC demonstrated a sensitivity of 87% and a specificity of 83% for <i>CTNNB1</i> mutations. <h3>Conclusions:</h3> <i>CTNNB1</i> mutations have a prevalence of 25% in HIR endometrioid endometrial cancer. In this single institution retrospective study, there were no differences in RFS or OS in patients with <i>CTNNB1</i> mutant compared to wild-type tumors.

A family with Milroy disease caused by the FLT4/VEGFR3 gene variant c.2774\xa0T\u2009>\u2009A

BackgroundMilroy disease (MD) is a rare, autosomal-dominant disorder. Variants in the Fms-related tyrosine kinase 4 (FLT4/VEGFR3) gene cause the symptoms of this disease. In this report, we investigated the variant in a large Chinese family with MD.MethodsWe conducted Sanger sequencing of exons 17–26 of FLT4/VEGFR3 (NM_182925.4). We assessed its pathogenicity based on the ACMG criteria and predicted it with an in silico program.ResultsA heterozygous substitution (NM_182925.4 (FLT4/VEGFR3):c.2774 T>A, p. (Val925Glu)) was detected in all patients with MD but not in any healthy controls. The variant was evaluated as pathogenic according to the ACMG criteria and was predicted to be pathogenic using an in silico program.ConclusionsIn this report, we described a large family with MD caused by a missense variant in FLT4/VEGFR3 (NM_182925.4 (FLT4/VEGFR3_v001):c.2774 T>A, p. (Val925Glu)). There are phenotypic heterogeneities among family members, and further research should be conducted to explore the possible reasons.

Abstract PO014: Clinicopathologic evaluation of CTNNB1 gene mutations in high-intermediate risk endometrial cancer

Abstract Background: CTNNB1 mutations convey increased risk of recurrence in low risk endometrioid endometrial cancers. However, sub-analyses of high-intermediate risk (HIR) endometrioid endometrial cancers in other cohorts have not demonstrated a clearly increased recurrence risk and have often been equivocal. The objective of this study is to perform a clinicopathologic evaluation of patients with HIR endometrioid endometrial cancer in relation to CTNNB1 mutational status. Methods: A single-institution chart review of patients diagnosed with endometrioid endometrial cancer between 2006 and 2018 was undertaken. Patients were classified as HIR according to GOG249 criteria. Mutational status was determined by Sanger sequencing of exon 3 of the CTNNB1 gene in primary tumor specimens. Immunohistochemical (IHC) analysis of B-catenin was performed to evaluate for the presence of nuclear staining. Factors associated with recurrence were assessed by logistic regression; recurrence-free (RFS) and overall survival (OS) were assessed by Kaplan-Meier survival analysis. Results: 93 cases of HIR endometrioid endometrial cancer were identified, of which 23 (24.7%) were CTNNB1 mutant and 70 (75.3%) were wild type. Median follow-up was 54.7 months. Recurrence occurred in 14/93 (15.1%) patients. Recurrence rates were not significantly different between patients with CTNNB1 mutant and wild type tumors (13.0% vs. 15.9%, p=0.76). In both univariable and multivariable analyses, RFS and OS were not significantly different (RFS HR 0.88, p=0.85, 95% CI 0.24 – 3.17; OS HR 0.20, p=0.12, 95% CI 0.03 – 1.54). Predicted oncogenic mutations comprised 87% of all identified mutations and were present in 100% of recurrent tumors and 85% of non-recurrent tumors (p=0.47). Nuclear B-catenin expression by IHC demonstrated a sensitivity of 87% and a specificity of 83% for CTNNB1 mutations. Conclusions: CTNNB1 mutations have a prevalence of 25% in HIR endometrioid endometrial cancer. In this single institution retrospective study, there were no differences in RFS or OS in patients with CTNNB1 mutant compared to wild-type tumors. Citation Format: Jennifer G. Haag, Rebecca J. Wolsky, Marisa R. Moroney, Jamie Sheren, Jeanelle Sheeder, Benjamin G. Bitler, Bradley R. Corr. Clinicopathologic evaluation of CTNNB1 gene mutations in high-intermediate risk endometrial cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO014.

Screening of West Siberian patients with primary congenital glaucoma for CYP1B1 gene mutations.

Primary congenital glaucoma (PСG) is a visual organ pathology that leads to progressive blindness and poor vision in children. Its main cause is an anomaly of the anterior chamber angle. Most cases of PСG are sporadic, but familial cases with an autosomal recessive (predominantly) and autosomal dominant (rare) type of inheritance have been described. Congenital glaucoma is a rare condition (1 case per 10,000–20,000 newborns), but its prevalence is substantially higher (up to 1 case per 250 newborns) in countries where consanguineous marriages are common. Mutations in the CYP1B1 gene, which encodes cytochrome P450 1B1, are the most common cause of autosomal recessive primary congenital glaucoma. This enzyme is known to be involved in retinoic acid metabolism and is necessary for normal eye development. The aim of this work was to assess the polymorphism of the CYP1B1 gene among West Siberian patients with primary congenital glaucoma. Direct automatic Sanger sequencing of exons and adjacent splicing sites of the CYP1B1 gene was carried out in 28 people with the PCG phenotype from a West Siberian region. As a result, in the sample of the white population we examined, pathogenic variants previously described in other ethnic groups were revealed: E387K (rs55989760), R444* (rs377049098), R444Q (rs72549376), and P437L (rs56175199), as well as novel single-nucleotide deletion p.F114Lfs*38 in the CYP1B1 gene. The latter can cause a frame shift, changed amino acid composition, and a formation of truncated in the protein. None of the detected mutations were found in the control sample of ophthalmologically examined individuals without PCG (100 people). Variants R444* (rs377049098) and R444Q (rs72549376) were not found in the general population sample either (576 randomly selected West Siberia residents). All the detected mutations caused the development of the autosomal recessive form of primary congenital glaucoma. The most severe clinical phenotype was observed in carriers of mutations in codon 444 of the gene. Consequently, in children with signs of increased intraocular pressure, molecular genetic analysis of the CYP1B1 gene is advisable for early diagnosis and timely initiation of PCG therapy.

KIT Mutation Incidence and Pattern of Melanoma in Central Europe

Data on the KIT mutation rate in melanoma in the central European region is missing. Accordingly, in a cohort of 79 BRAF/NRAS double wild type cutaneous melanoma and 17 mucosal melanoma KIT mutation was assessed by Sanger sequencing of exons 9,11,13,17 and 18. In this cutaneous melanoma cohort KIT mutation frequency was found to be 34/79 (43.04%) with a significantly higher rate in acrolentiginous melanoma (ALM) as compared to UV-induced common variants (20/34, 58.8% versus 14/45, 31.1%, p = 0.014). In the double wild type mucosal melanoma cohort the KIT mutation frequency was found to be comparable (41.2%). The actual frequency of KIT mutation in the original 227 patient cutaneous melanoma cohort was 34/227, 14.9%. Exon 11 was the most frequent mutation site (44.7%) followed by exon 9 (21.1%) equally characterizing UV-induced common histotypes and ALM tumors. In mucosal melanoma exon 9 was the most frequently involved exon followed by exon 13 and 17. KIT mutation hotspots were identified in exon 9 (c482/491/492), in exon 11 (c559,c572, c570), in exon 13 (c642), in exon 17 (c822) and in exon 18 (c853). The relatively high KIT mutation rate in cutaneous melanoma in this central-European cohort justifies regular testing of this molecular target in this entity, not only in mucosal variants.

Implication of BRAF V600E and CTNNB1 gene mutations in the pathological classification of craniopharyngioma

Objective: To investigate the clinicopathological significance of BRAF V600E and CTNNB1 gene mutations in adamantinomatous craniopharyngiomas (ACP) and papillary craniopharyngiomas (PCP). Methods: The retrospective study included a total of 67 craniopharyngiomas diagnosed from October 2009 to August 2018 at Xuanwu Hospital, Capital Medical University. The immunohistochemical staining for β-catenin and BRAF V600E expression, Sanger sequencing of exon 3 of CTNNB1, BRAF mutation analysis by scorpions amplification refractory mutation system (ARMS) fluorescence quantitative PCR were performed. Univariate survival analysis was used to correlate with tumor recurrence. Results: Of the 67 patients, 53 were ACPs and 14 were PCPs. Four patients underwent multiple operations and one of them presented with malignant transformation into squamous cell carcinoma. Histologically, ACPs were characterized by whorl-like cell clusters, peripheral palisaded layer, stellate reticulum, finger-shaped protrusions, ghost cells and wet keratinous substances. While PCPs usually consisted of mature squamous epithelium associated with fibrovascular stroma resulting in papillary appearance. The nuclear immunopositivity for β-catenin was observed in 73.6% (39/53) of ACPs, and it was absent in PCPs (0/14). The nuclear translocation of β-catenin usually presented at whorl-like structures or around ghost cells. Of all the cases, mutations analysis in exon 3 of β-catenin gene CTNNB1 were successful in 46 cases and 42.1% (16/38) of ACP showed CTNNB1 gene mutation, while none of the PCPs harbored CTNNB1 gene mutation (0/8). The cytoplasmic immunopositivity for BRAF V600E mutant protein was found in all PCPs (14/14) and negative in all ACPs (0/53). ARMS-PCR results showed that BRAF V600E mutations were observed in 13/14 of PCPs but not seen in ACPs (0/53). Follow-up data were available in 35 patients with duration of 2 to 120 months. Ten patients experienced recurrences after the first surgery. Upon univariate survival analysis, only subtotal excision was found to be associated with increased recurrence (P=0.032), while pathological type, postoperative radiotherapy and CTNNB1 gene mutation were not (P>0.05). Conclusions: There is significant difference in the expression of BRAF V600E and CTNNB1 genes between ACP and PCP, and their immunohistochemical and molecular detection therefore can be used in the diagnosis and differential diagnoses of craniopharyngiomas.

SUN-198 GENETIC CHARACTERISTICS OF KOREAN AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE BY TARGETED EXOME SEQUENCING

Autosomal dominant polycystic kidney disease (ADPKD) is one of the main causes of end-stage renal disease (ESRD). Since it is caused by mutations either in PKD1 or PKD2, genetic information is utmost important in understanding pathogenesis of ADPKD. Therefore, this study aimed to demonstrate genetic characteristics of ADPKD and its effect on renal function in 749 Korean ADPKD subjects from 524 unrelated families. Genetic analysis of PKD 1 and PKD2 were performed using targeted exome sequencing. The SureSelectXT Custom probe set and the Agilent SureSelect™ target enrichment protocol (version B.3, June 2015) was used and sequencing was performed using the HiSeq™ 2500 platform (Illumina, San Diego, CA, USA). For cases with inadequate results, Sanger sequencing for exon 1 of PKD1 and multiple ligation probe assay were done. For determining pathogenicity, familial segregation analysis was performed. A total of 476 variants were detected among 524 families. Among them, nonsense variants consist of 26.3%, frameshift 30.9%, typical splicing 5%, large deletion 2.7%, missense 32.6%, and small in-frame indel 2.5%. The overall mutation detection rate was 80.7% (423/524 families). Among them, novel mutation was 70.7%. The prevalence of PKD1 and PKD2 was 84% and 16%, respectively. Mutations were divided into PKD1 protein truncating (PKD1-PT), PKD1 non-truncating (PKD1-NT), and PKD2 genotypes according to their effect on protein function. PKD1-PT genotype was associated with younger age at 1st visit (40.2±12.9 vs. 43.3±12.6 and 45.9±13.0 years, p<0.001), larger height-adjusted total kidney volume (HtTKV) (median 898 vs. 609 and 629 mL/m, p<0.001), lower estimated glomerular filtration rate (eGFR) (63.5±42.2 vs. 66.6±37.8 and 73.1±33.8 mL/min/1.73m2, p=0.052) and higher proportion of Mayo imaging classification 1C-1E (76.3 vs. 58.9 and 47.9%, p<0.001) compared to PKD1-NT and PKD2 genotypes. The PKD1 genotype showed earlier onset of ESRD compared to PKD2 genotype (52.6 ± 9.3 vs. 60.5 ± 10.7 years, P=0.003). In frailty model adjusted for age, gender, and familial clustering effect, PKD2 genotype had 0.2 times lower risk for reaching ESRD than PKD1-PT genotype (p=0.037). Our results demonstrated higher prevalence of novel mutations (70.7%) in Korean ADPKD cohort. Subjects with PKD1-PT genotype had early onset of hypertension, larger htTKV, lower eGFR and higher events of ESRD compared to other genotypes. Genotyping can contribute to selecting rapid progressors for new emerging therapeutic interventions.

Detection of single nucleotide polymorphisms at major prolificacy genes in the Mehraban sheep and association with litter size

Abstract The present study aimed to investigate the presence of polymorphisms at four known genes controlling ovine prolificacy i.e. BMP15, GDF9, BMPR1B and B4GALNT2 in a sample of 115 Iranian Mehraban ewes and their association with litter size (LS) and lambs’ birth weight (BW) traits. Using Sanger sequencing of exons and polymorphism specific genotyping, ten SNPs (Single Nucleotide Polymorphisms) were observed in only two genes, GDF9 and BMPR1B. Seven SNPs were found in the GDF9 gene on the chromosome 5. Among them, six were already described in the coding sequence, and a new one (g.41840985C&gt;T) was found in the 3’UTR. In the BMPR1B gene on the chromosome 6, three novel SNPs were detected in the exon 7 (g.29382184G&gt;A; g.29382337G&gt;A and g.29382340G&gt;A). Allelic frequencies were established for six SNPs among the ten identified and they were in Hardy-Weinberg equilibrium. A significant association was found between the novel SNPs found in the exon 7 of BMPR1B and LS. Present results indicate the potential role of the BMPR1B locus in controlling prolificacy of Mehraban sheep and provide genetic markers for further exploitation in selection to improve reproductive efficiency.

Compound but non-linked heterozygous p.W14X and p.L279\xa0V LPL gene mutations in a Chinese patient with long-term severe hypertriglyceridemia and recurrent acute pancreatitis

BackgroundVariants in the lipoprotein lipase (LPL), apolipoprotein C-II (APOC2), apolipoprotein A-V (APOA5), GPIHBP1 and LMF1 genes may cause severe hypertriglyceridemia (HTG), which is now the second-leading aetiology of acute pancreatitis in China.MethodsThe patient and his family were assessed for gene variants by Sanger sequencing of exons and exon-intron junctions of the LPL, GPIHBP1, APOA5, APOC2, and LMF1 genes. Post-heparin blood was collected for LPL mass and activity detection.ResultsThe patient had suffered from long-term severe hypertriglyceridemia and recurrent abdominal pain for over 30 years, since age 26, and 3 bouts of acute pancreatitis. Two heterozygous LPL single-nucleotide polymorphisms (SNPs) were compound but dislinked: a single-nucleotide substitution (c.42G > A) resulting in the substitution of tryptophan with a stop codon (p.W14X) in one allele, and a single-nucleotide substitution (c.835C > G) resulting in a leucine-to-valine substitution (p.L279 V) in another allele. Only one SNP, p.L279 V, was detected in his son. Post-heparin LPL activity and mass were also lower in the patient.ConclusionTwo heterozygous LPL SNPs, W14X and L279 V, were newly found to be compound but dislinked, which may cause long-term severe hypertriglyceridemia and recurrent acute pancreatitis.

Micropapillary urothelial carcinoma: evaluation of HER2 status and immunohistochemical characterization of the molecular subtype

Comprehensive molecular analyses of urothelial bladder cancer (UBC) have defined distinct subtypes with potential therapeutic implications. In this study, we focused on micropapillary urothelial carcinoma (MPUC), an aggressive, histomorphologically defined rare variant. Apart from genetic alterations shared with conventional UBC, alterations of the HER2 gene have been reported in higher frequencies. However, only small cohorts of MPUCs have been analyzed, and the real impact is still unclear. We collected a cohort of 94 MPUCs and immunohistochemically tested HER2, basal (CD44, CK5, EGFR, p63) and luminal (CD24, FOXA1, GATA3, CK20) markers to allocate MPUC to a molecular subtype. Additionally, HER2 amplification status was assigned by chromogenic in situ hybridization. Sanger sequencing of exon 4 and 8 was used to test for HER2 mutations. Kruskal-Wallis test was calculated to compare marker distribution between proportions of the MPUC component. HER2 2+/3+ staining scores were identified in 39.6% of 91 analyzed MPUCs and were not differentially distributed among the proportion of the MPUC component (P = .89). Additionally, CISH analysis revealed 30% of HER2-amplified tumors independently of the MPUC fraction. In 6/90 evaluable MPUCs, a p.S310F HER2 mutation was detected. Overexpression of luminal markers was observed in the majority of MPUC. Our investigations of the largest cohort of analyzed MPUC demonstrate that HER2 overexpression and amplifications are common genetic alterations and identification of overexpressed luminal markers allows subclassification to the luminal subtype. These findings highlight the need of histomorphological recognition of MPUC and analysis of HER2 status and the luminal molecular subtype for potential targeted therapeutic strategies.

Hepatic angiomyolipoma: mutation analysis and immunohistochemical pitfalls in diagnosis.

Hepatic angiomyolipoma (AML) often shows epithelioid morphology with inconspicuous fat. Epithelioid component can mimic hepatocellular adenoma (HCA) or carcinoma (HCC). The aims of this study were to examine the expression of commonly used markers for HCA or HCC in hepatic AML and highlight pitfalls in diagnosis. Resected hepatic AMLs (n = 16) were reviewed; reticulin stain, immunohistochemistry for glutamine synthetase (GS), β-catenin and liver fatty acid binding protein (LFABP) were performed along with Sanger sequencing of exon 3 of CTNNB1 and next-generation sequencing (NGS). Predominant epithelioid component (≥50%) was seen in 80% of cases. Foamy macrophage was present in 33% of cases. High-risk histological features were often present in tumours with benign outcome: marked atypia (19%), mitoses (20%) and necrosis (33%). GS staining (≥10% of tumour) was seen in epithelioid components in 13 (87%) cases, and was diffuse (>50% of tumour) in six (40%) cases. LFABP staining or nuclear β-catenin staining was not seen in any case. Sanger sequencing and NGS did not reveal CTNNB1 mutation in any tested case. NGS demonstrated TSC2 mutations in all five cases tested. The predominance of epithelioid component resembling HCA or HCC is common in hepatic AML. Absence of LFABP and presence of fat can be mistaken for HNF1α-inactivated HCA. Diffuse GS staining can be mistaken for β-catenin-activated HCA or HCC. Diffuse GS expression is not related to CTNNB1 mutation. All tested cases showed TSC2 mutation, supporting this as the driving genetic event for hepatic AML.

Clinical and molecular genetic study of Cockayne syndrome accompanied with nephrotic syndrome in a family

Objective To analyze the clinical and genetics characteristics in twin sisters with Cockayne syndrome. Methods The identical twin sisters visited the Affiliated Children′s Hospital of Capital Institute of Pediatrics in December 2016.The clinical presentations, course of treatment, blood biochemistry, metabolic screening and whole exon sanger sequencing were analyzed. Results These two patients were referred at 4 years and 5 months of age for growth failure and developmental delay.The younger sister manifested short stature (only 97 cm), low weight (14.0 kg) and little head circumference (43 cm), and the elder sister manifested short stature (only 98 cm), low weight (15.5 kg) and little head circumference (43 cm). They were born with out adverse event, and then they kept the head up at 8 months of age.They could sit at 10 months of age, but they had not acquired independent walking ability up till now.They spoke their first words at 2 year of age, and made little progress after that.They had a variety of abnormal clinical features including cognitive deficits, microcephaly, thin pointy nose, sunken eyes, small chin, photosensitive rash, hearing impairment, volitional tremor and hypermyotonia.They had been diagnosed as nephrotic syndrome at 4.5 years old, with little response to prednisone.The renal biopsy revealed minimal change nephropathy.Cerebrum and cerebellum atrophy was detected by magnetic resonance image scanning.Two heterozygous ERCC8 mutations in both patients, c.394_398delTTACA and large fragment deletion, were identified in the patient.The c. 394_398delTTACA mutation originated from his father.The exon 4 deletion from his mother caused the defection of the protein. Conclusions Cockayne syndrome is a rare autosomal recessive disease.It is not only characterized by developmental delay, microcephaly, sunken eyes, photosensitive rash and auditory abnormalities, but also can be involved in nephrotic syndrome.Cockayne syndrome can be caused by compound heterozygous mutation, including c. 394_398delTTACA and a large fragment deletion of exon 4 in ERCC8. Key words: Cockayne syndrome; Nephrotic syndrome; ERCC8 gene; Microcephaly

A disease-causing novel missense mutation in the ST14 gene underlies autosomal recessive ichthyosis with hypotrichosis syndrome in a consanguineous family.

Autosomal recessive ichthyosis with hypotrichosis (ARIH; MIM 602400) syndrome is characterized by diffused congenital ichthyosis and generalized non-scarring hypotrichosis. The underlying genetic cause of ARIH syndrome has been associated with sequence variants of the gene ST14, encoding type II transmembrane serine protease matriptase, which maps to chromosome 11q24.3. The current report aimed to investigate the clinical features and genetic cause of ARIH syndrome in a large consanguineous family of Pakistani origin. The technique of homozygosity mapping with highly polymorphic microsatellite markers was employed to establish linkage within the family. Sanger sequencing of exons and intron-exon boundaries of ST14 was performed to identify the potential pathogenic sequence variants, followed by structural analysis of the mutated protein. Linkage was established to chromosome 11q24.3, comprising the gene ST14. Sequence analysis led to the identification of a novel homozygous missense variant (c.1315G>A, p.Gly439Ser) in the ST14 gene that co-segregated with the disease phenotype in all affected members. Homology modelling and molecular docking analysis of ST14 with wild-type TMEFF1 protein was performed which revealed that glycine at position 439 is crucial for maintaining normal structural confirmation and interaction with the EGF domain of TMEFF1 protein. Taken together, the data strongly advocate this ST14 variant as the underlying genetic cause of ARIH syndrome in this first reported affected family from Pakistan. Moreover, the present study adds to the spectrum of mutations in the ST14 gene, implicating them in the pathogenesis of ARIH syndrome.