Maternal Mutation Research Articles

Preimplantation genetic testing for Cockayne syndrome with a novel ERCC6 variant in a Chinese family.

Cockayne syndrome (CS) is a rare, multisystem, autosomal recessive disorder characterized by cachectic dwarfism, nervous system abnormalities, and premature aging. Mutations in the ERCC6 and ERCC8 genes are the predominant causes of Cockayne syndrome, with ERCC6 gene mutations present in approximately 75% of cases. Trio-based whole-exome sequencing (trio-WES) was employed to identify potential pathogenic variants associated with CS. Preimplantation genetic testing for monogenic disorders (PGT-M) was conducted to prevent the transmission of the pathogenic variant. Two compound heterozygous mutations were identified in ERCC6-c.1297G>T (p. Glu433*) and c.1607T>G (p. Leu536Trp)-with c.1297G>T representing a novel mutation. Four blastocysts resulting from intracytoplasmic sperm injection were subjected to biopsy. Genetic analyses revealed that E1 harbored maternal mutations in diploid embryos, E2 and E3 carried both paternal and maternal mutations in non-diploid embryos, and E4 did not carry paternal or maternal mutations in diploid embryos. Following the transfer of the E4 embryos, a single successful pregnancy was achieved. The successful application of PGT-M in this family offers a potential approach for addressing other monogenic diseases. The findings of this study broaden the variant spectrum of ERCC6 and will contribute to the molecular diagnosis and genetic counseling of CS. This case highlights the feasibility and effectiveness of PGT-M in preventing CS and provides valuable insights for similarly affected families.

LncRNAs in the Dlk1-Dio3 Domain Are Essential for Mid-Embryonic Heart Development.

The Dlk1-Dio3 domain is important for normal embryonic growth and development. The heart is the earliest developing and functioning organ of the embryo. In this study, we constructed a transcriptional termination model by inserting termination sequences and clarified that the lack of long non-coding RNA (lncRNA) expression in the Dlk1-Dio3 domain caused the death of maternal insertion mutant (MKI) and homozygous mutant (HOMO) mice starting from E13.5. Parental insertion mutants (PKI) can be born and grow normally. Macroscopically, dying MKI and HOMO embryos showed phenomena such as embryonic edema and reduced heart rate. Hematoxylin and eosin (H.E.) staining showed thinning of the myocardium in MKI and HOMO embryos. In situ hybridization (IHC) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) showed downregulation of lncGtl2, Rian, and Mirg expression in MKI and HOMO hearts. The results of single-cell RNA sequencing (scRNA-Seq) analysis indicated that the lack of lncRNA expression in the Dlk1-Dio3 domain led to reduced proliferation of epicardial cells and may be an important cause of cardiac dysplasia. In conclusion, this study demonstrates that Dlk1-Dio3 domain lncRNAs play an integral role in ventricular development.

Enhancing fetal outcomes in GCK-MODY pregnancies: a precision medicine approach via non-invasive prenatal GCK mutation detection.

Mutations in the GCK gene cause Maturity Onset Diabetes of the Young (GCK-MODY) by impairing glucose-sensing in pancreatic beta cells. During pregnancy, managing this type of diabetes varies based on fetal genotype. Fetuses carrying a GCK mutation can derive benefit from moderate maternal hyperglycemia, stimulating insulin secretion in fetal islets, whereas this may cause macrosomia in wild-type fetuses. Modulating maternal glycemia can thus be viewed as a form of personalized prenatal therapy, highly beneficial but not justifying the risk of invasive testing. We therefore developed a monogenic non-invasive prenatal diagnostic (NIPD-M) test to reliably detect the transmission of a known maternal GCK mutation to the fetus. A small amount of fetal circulating cell-free DNA is present in maternal plasma but cannot be distinguished from maternal cell-free DNA. Determining transmission of a maternal mutation to the fetus thus implies sequencing adjacent polymorphisms to determine the balance of maternal haplotypes, the transmitted haplotype being over-represented in maternal plasma. Here we present a series of such tests in which fetal genotype was successfully determined and show that it can be used to guide therapeutic decisions during pregnancy and improve the outcome for the offspring. We discuss several potential hurdles inherent to the technique, and strategies to overcome these. Our NIPD-M test allows reliable determination of the presence of a maternal GCK mutation in the fetus, thereby allowing personalized in utero therapy by modulating maternal glycemia, without incurring the risk of miscarriage inherent to invasive testing.

Mutational analysis of 23 autosomal short tandem repeats based on trio paternity testing in the Korean population

This study aimed to estimate A-STR mutation rates in 2,317 Korean parent–child trios by examining 20 Combined DNA Index System (CODIS) core loci (D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, vWA, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433, and D22S1045) and three non-CODIS loci (Penta E, Penta D, and SE33). Locus-specific mutation rate estimates varied from 0.00 to 8.63 × 10-3 per generation, with an average mutation rate of 1.62 × 10-3 (95 % CI, 1.39–1.88 × 10-3). We also combined data from previous studies to obtain comprehensive genetic values for the Korean population, and the average mutation rate was 1.59 × 10-3 (95 % CI, 1.38–1.82 × 10-3). Single-step mutations (95.69 %) and double-step mutations (3.35 %) were observed in the mutation pattern analysis, and cases expected to have multi-step mutations (0.96 %) were also observed. Large-sized alleles exhibited more loss mutations than gain mutations, and paternal mutations (62.68 %) were more frequently observed than maternal mutations (19.62 %). The calculated values and features of the 23 A-STRs explored in this study are expected to play a crucial role in establishing criteria for forensic genetic interpretation.

Thrombophilia and newborn

Introduction: The term thrombophilia (TF) describes disorders associated with an increased predisposition of developing venous thromboembolism. Thrombotic accidents during pregnancy are now widely recognized as a cause of morbidity and mortality. Objectives: To compare maternal and term newborn (NB) characteristics between healthy and thrombophilic pregnancies. Methods: The study included 31 full-term newborns whose mothers were diagnosed with TF before the current pregnancy and treatment was carried out through it. In the control group were included 37 full-term NBs. NB indicators which were used are gender, gestation age, birth weight, Apgar score, morbidity during the early neonatal period, hospital stay, treatment during the early neonatal period. The indicators for the mothers are the type of TF, mechanism of childbirth, sequence of pregnancy and sequence of childbirth. Results: In the observed groups of patients, we found that mothers with TF have an average higher age and have a higher number of unsuccessful pregnancies. Тhey more often give birth by operation. The higher the number of maternal TF mutations, the more unsuccessful pregnancies. The NB of mothers with TF have a lower Apgar score, develop RDS more often during the early neonatal period and need a larger volume of therapeutic manipulations and have a significantly longer hospital stay. Conclusions: TF in pregnant women, even when adequate treatment is administered during pregnancy, is associated with increased morbidity in their full-term NBs during the early neonatal period. This requires additional therapeutic measures in full-term newborns and is associated with a prolonged hospital stay.

Analysis of data and common mutations encountered during routine parentage testing in Zimbabwe

We analyzed parentage data collected over a ten-year period in a Zimbabwean DNA testing laboratory. Parentage case types, prevalence, exclusion data, mutations rates and observed genotyping irregularities were analyzed. We report analysis results from 1303 cases. DNA extraction and STR typing was conducted using standard commercial kits. Paternity was the most requested test (87.37%) followed by the indirect biological kinship tests (7.01%). Duo paternity (motherless) was the most common paternity test for both regular and court cases. We observed 367 paternity exclusions from 1135 cases, giving an overall paternity exclusion rate of 32.33%. Maternity had the lowest exclusion rate (8.33%), with criminal cases having the highest paternity (61.11%) and maternity (33.33%) exclusion rates. The number of mismatched STR loci ranged from 2–12 for duo cases and 4–18 for the trio cases. FGA, D2S1338, D18S51 and D2S441 were the most informative markers for exclusion. We detected 30 mutations out of 837 cases with an estimated paternal and maternal mutation rate of 0.0021 and 0.0011 respectively. Triallelic patterns were only observed at the TPOX locus with allele 10 and 11 being the extra alleles transmitted. Our report provides forensic parameters which can improve parentage and forensic analysis in Zimbabwe.

Outcomes of BRCA pre-implantation genetic testing according to the parental mutation origin: a cohort study

BackgroundThe process of gamete formation and early embryonic development involves rapid DNA replication, chromosome segregation and cell division. These processes may be affected by mutations in the BRCA1/2 genes. The aim of this study was to evaluate BRCA mutation inheritance and its effect on early embryonic development according to the parental origin of the mutation. The study question was approached by analyzing in vitro fertilization cycles (IVF) that included pre-implantation testing (PGT-M) for a BRCA gene mutation.MethodsThis retrospective cohort study compared cycles of pre-implantation genetic testing for mutations (PGT-M) between male and female patients diagnosed with BRCA 1/2 mutations (cases), to a control group of two other mutations with dominant inheritance (myotonic dystrophy (MD) and polycystic kidney disease (PKD)). Results were compared according to mutation type and through a generalized linear model analysis.ResultsThe cohort included 88 PGT-M cycles (47 BRCA and 41 non-BRCA) among 50 patients. Maternal and paternal ages at oocyte retrieval were comparable between groups. When tested per cycle, FSH dose, maximum estradiol level, oocytes retrieved, number of zygotes, and number of embryos available for biopsy and affected embryos, were not significantly different among mutation types. All together 444 embryos were biopsied: the rate of affected embryos was comparable between groups. Among BRCA patients, the proportion of affected embryos was similar between maternal and paternal mutation origin (p = 0.24). In a generalized linear model analysis, the relative oocyte yield in maternal BRCA patients was significantly lower (0.7, as related to the non BRCA group)(p < 0.001). Zygote formation and blastulation were not affected by the BRCA gene among paternal cases (P = 0.176 and P = 0.293 respectively), nor by paternal versus maternal BRCA carriage (P = 0.904 and P = 0.149, respectively).ConclusionsBRCA PGT-M cycles performed similarly compared to non-BRCA cycles. Inheritance rate and cycle parameters were not affected by the parental origin of the mutation.

Spontaneous miscarriage driven by maternal genetic mutation at position of PAI-1-844G/A: shed light on a race-specific genetic polymorphism

ObjectiveAssociation between a genetic polymorphism and disease, either positively or negatively, within a population may not necessarily predict association in other race-ethnic populations. The aim of this study was to genotype well recognized thrombophilia associated polymorphisms as common risk factors for miscarriage and investigate their benefit to use as risk factors in southwest region of Iran females (Khuzestan) in the Arabs ethnic minority group with spontaneous miscarriage. We developed a Reverse Dot Blot Assay for the genotyping of four polymorphisms.ResultsThere were significant differences in the genotype distribution and allelic frequencies of the MTHFR 1298 A > C, MTHFR 677 C > T, Factor V Leiden 1691 G > A, PAI-1-844G > A polymorphisms between the case and control groups. The MTHFR 1298 A > C, MTHFR 677 C > T and Factor V Leiden 1691 G > A polymorphisms were significantly associated with spontaneous miscarriage risk. Unlike some other race-ethnic populations, PAI-1-844G > A polymorphism was associated with risk of developing unplanned miscarriage in Iranian Arabs ethnic minority group females.

FRI597 Impact Of Theophylline On BMI In Pseudohypoparathyroidism Type 1a - Results Of An Open Label Extension Trial

Abstract Disclosure: A.H. Shoemaker: None. J. Tamaroff: None. H. Jueppner: None. Background: Pseudohypoparathyroidism type 1a (PHP1A) is a rare genetic disorder characterized by early onset obesity, short stature and multi hormone resistance. The underlying causes are maternally inherited, heterozygous inactivating mutations in GNAS, which encodes the α-subunit of stimulatory G protein (Gsα). Due to tissue specific silencing of paternal Gsα expression, maternal GNAS mutations lead to impaired Gsα-protein coupled receptor (GPCR) function and multihormone resistance. The GPCR signaling cascade begins with increased cAMP which is rapidly degraded by phosphodiesterase (PDE). We hypothesized that paternal Gsα expression is reduced, but not absent, and therefore the nonselective PDE inhibitor theophylline may potentiate the cAMP/PKA-dependent cellular events down-stream of GPCRs. We hypothesized that theophylline would reduce BMI in PHP1A patients while being safe and well tolerated. Methods: Children and adults with PHP1A who completed the randomized, placebo controlled, double-blind clinical trials (RCT) NCT03029429 and NCT04551170 were eligible for enrollment in an open-label extension (OLE) study of theophylline treatment (NCT04240821). As the RCT is ongoing and treatment assignment is blinded, we compared baseline enrollment data (prior to any exposure to theophylline or placebo) with the last study visit in the OLE study. The RCTs are 1 year long and then patients in the OLE study are followed for up to 2 additional years. The primary outcome is change in BMI, expressed as % of the 95th %ile (BMI95) to account for differences in gender and age. Results: To date, 23 patients have enrolled in, and 16 patients have completed the RCTs. Two patients completed RCTs before the OLE was available. One patient was ineligible due to study drug noncompliance. One patient was Canadian and unable to participate due to COVID-19 travel restrictions. The remaining 12 completers enrolled in the OLE. One withdrew prior to OLE visit 2 due to difficulty with travel and is not included in the analysis. Patients were 17.2 ± 12.9 years old (range 9-55 years) and 10 of 11 were female. All participants were white, one patient was Hispanic. Patients were followed for an average of 30 months. Baseline BMI95 was 133.5 ± 19.9% and at the last OLE visit, BMI95 had decreased to 124.7 ± 18.6% (p= 0.06 by paired t-test). A decrease in BMI95 was seen in 73% of patients and 36% had a decrease in BMI95 &amp;gt;20%. Review of the 3 patients without improvement in BMI95 showed one had an increasing BMI95 during the RCT, followed by decreasing BMI95 during the OLE, and another patient had persistently sub-therapeutic theophylline levels. The most common adverse event attributed to the drug was transient nausea/vomiting. There were no OLE withdrawals due to adverse events. Conclusion: Most patients with PHP1A in a long term, OLE study of theophylline demonstrated an improvement in BMI95. Theophylline was safe and well tolerated. Presentation: Friday, June 16, 2023

OR14-03 The Thyroid Hormone Receptor Beta Genotype Of The Fetus Influences The Maternal Thyroid Function During Pregnancy

Abstract Disclosure: F. Salas Lucia: None. X. Liao: None. H. Jiang: None. A.M. Dumitrescu: None. S. Refetoff: None. J. Anselmo: None. When women with resistance to thyroid hormone beta (RTHβ) are pregnant, their high serum thyroid hormone (TH) levels are perceived as normal by affected fetuses (AfFe) carrying the same THRB mutation as their mothers. On the contrary, normal fetuses (NlFe) that do not carry the maternal THRB mutation are exposed to supraphysiologic TH levels and have unfavorable outcomes. This shows the impact maternal TH levels have on the fetus. However, whether the fetus might influence the maternal thyroid function remains unknown. To explore this possibility, we studied the maternal thyroid function tests (TFT) TSH, FT3, and FT4 throughout the pregnancies from 8 women harboring the R243Q THRB mutation (AfMo) during a total of 10 pregnancies: 6 carrying AfFe and 4 NlFe fetuses. Cord blood TFT from 3 of the NlFe and 3 AfFe at birth were also obtained. In addition, 8 pregnancies from 7 normal mothers (NlMo) carrying 4 AfFe who inherited the THRB mutation from the father and 4 NlFe were also evaluated for TFT. Cord blood from these pregnancies was not available. Results were compared to those in pregnancies of normal WT women. In all RTHβpregnancies, the maternal TSH values were within the range of pregnancies in WT women (0.35-4.94 mU/L) with an hCG-mediated nadir at the end of the first trimester. Maternal FT4 and FT3 values in RTHβpregnancies were above the upper limit of normal (ULN) of 100, peaking during weeks 5-16 (150±20% and 175±15% ULN, respectively) and progressively decreasing afterward. We found differences in maternal TFTdepending on the THRB genotype of the fetus. From weeks 25-32, the AfMo carrying an AfFe exhibited lower FT4 than when carrying a NlFe (96±2 vs. 108±5.3% ULN; p&amp;lt;0.05). Similarly, from weeks 25-40, the AfMo carrying an AfFe exhibited lower FT3 values than when carrying a NlFe ULN (110±11 vs. 134±13% ULN; p&amp;lt;0.05). While gestational age at delivery was similar in all RTHβ pregnancies, the NlFe had a lower birth body weight than the AfFe (2.3±0.1 vs. 3.1±0.2 kg; p&amp;lt;0.01). Cord blood TSH in NlFe was undetectable (&amp;lt;0.1 mU/L) but was 9.51 mU/L (2.2-10; range in normal neonates) in AfFe, which indicates that the thyroid function in NlFe could be restricted during pregnancy, but not in AfFe. Cord blood FT4 levels were similar in NlFe and AfFe (98.4±16.4 vs. 89.5±9.4% ULN). However, FT3 levels were significantly higher in NlFe than in AfFe (67.7±3.0 vs. 42.0±9.6% ULN; p&amp;lt;0.05) and represented ∼68, and ∼42% of the serum maternal FT3 level at birth, respectively. NlMo carrying either AfFe or NlFe showed similar TSH and FT3 levels during pregnancies. However, from weeks 33 to 40, FT4 levels were lower when carrying an AfFe than when carrying a NlFe (51.8±2.8 vs. 62.5±3.2% ULN; p&amp;lt;0.001). Our findings indicate that maternal thyroid function are influenced by THRB genotype of the fetus. The quantitation of placental and fetal TH transporters, deiodinases, and TH metabolites could provide insights into the mechanisms involved. Presentation: Friday, June 16, 2023

SINE-VNTR-Alu retrotransposon insertion as a novel mutational event underlying Glanzmann thrombasthenia

BackgroundGlanzmann thrombasthenia (GT) is an autosomal recessive platelet aggregation disorder caused by mutations in ITGA2B or ITGB3. ObjectivesWe aimed to assess the phenotype and investigate the genetic etiology of a GT pedigree. MethodsA patient with bleeding manifestations and mild mental retardation was enrolled. Complete blood count, coagulation, and platelet aggregation tests were performed. Causal mutations were identified via whole exome and genome sequencing and subsequently confirmed through polymerase chain reaction and Sanger sequencing. The transcription of ITGB3 was characterized using RNA sequencing and reverse transcription polymerase chain reaction. The αⅡb and β3 biosynthesis was investigated via whole blood flow cytometry and in vitro studies. ResultsGT was diagnosed in a patient with defective platelet aggregation. Novel compound heterozygous ITGB3 variants were identified, with a maternal nonsense mutation (c.2222G>A, p.Trp741∗) and a paternal SINE-VNTR-Alu (SVA) retrotransposon insertion. The 5' truncated SVA element was inserted in a sense orientation in intron 11 of ITGB3, resulting in aberrant splicing of ITGB3 and significantly reducing β3 protein content. Meanwhile, both the expression and transportation of β3 were damaged by the ITGB3 c.2222G>A. Almost no αⅡb and β3 expressions were detected on the patient's platelets surface. ConclusionNovel compound heterozygous ITGB3 mutations were identified in the GT pedigree, resulting in defects of αⅡbβ3 biosynthesis. This is the first report of SVA retrotransposon insertion in the genetic pathogenesis of GT. Our study highlights the importance of combining multiple high-throughput sequencing technologies for the molecular diagnosis of genetic disorders.

Noninvasive prenatal diagnosis of Mendelian disorders for consanguineous couples by relative genotype dosage.

Noninvasive prenatal diagnosis relies on the presence in maternal blood of circulating cell-free fetal DNA released by apoptotic trophoblast cells. Widely used for aneuploidy screening, it can also be applied to monogenic diseases (NIPD-M) in case of known parental mutations. Due to the confounding effect of maternal DNA, detection of maternal or biparental mutations requires relative haplotype dosage (RHDO), a method relying on the presence of SNPs that are heterozygous in one parent and homozygous in the other. Unavoidably, there is a risk of test failure by lack of such informative SNPs, an event particularly likely for consanguineous couples who often share common haplotypes in regions of identity-by-descent. Here we present a novel approach, relative genotype dosage (RGDO) that bypasses this predicament by directly assessing fetal genotype with SNPs that are heterozygous in both parents (frequent in regions of identity-by-descent). We show that RGDO is as sensitive as RHDO and that it performs well over a large range of fetal fractions and DNA amounts, thereby opening NIPD-M to most consanguineous couples. We also report examples of couples, consanguineous or not, where combining RGDO and RHDO allowed a diagnosis that would not have been possible with only one approach.

Attenuated glucose uptake promotes catabolic metabolism through activated AMPK signaling and impaired insulin signaling in zebrafish.

Glucose metabolism in fish remains a controversial area of research as many fish species are traditionally considered glucose-intolerant. Although energy homeostasis remodeling has been observed in fish with inhibited fatty acid β-oxidation (FAO), the effects and mechanism of the remodeling caused by blocked glucose uptake remain poorly understood. In this study, we blocked glucose uptake by knocking out glut2 in zebrafish. Intriguingly, the complete lethality, found in Glut2-null mice, was not observed in glut2-/- zebrafish. Approxiamately 30% of glut2-/- fish survived to adulthood and could reproduce. The maternal zygotic mutant glut2 (MZglut2) fish exhibited growth retardation, decreased blood and tissue glucose levels, and low locomotion activity. The decreased pancreatic β-cell numbers and insulin expression, as well as liver insulin receptor a (insra), fatty acid synthesis (chrebp, srebf1, fasn, fads2, and scd), triglyceride synthesis (dgat1a), and muscle mechanistic target of rapamycin kinase (mtor) of MZglut2 zebrafish, suggest impaired insulin-dependent anabolic metabolism. Upregulated expression of lipolysis (atgl and lpl) and FAO genes (cpt1aa and cpt1ab) in the liver and proteolysis genes (bckdk, glud1b, and murf1a) in muscle were observed in the MZglut2 zebrafish, as well as elevated levels of P-AMPK proteins in both the liver and muscle, indicating enhanced catabolic metabolism associated with AMPK signaling. In addition, decreased amino acids and elevated carnitines of the MZglut2 zebrafish supported the decreased protein and lipid content of the whole fish. In summary, we found that blocked glucose uptake impaired insulin signaling-mediated anabolism via β-cell loss, while AMPK signaling-mediated catabolism was enhanced. These findings reveal the mechanism of energy homeostasis remodeling caused by blocked glucose uptake, which may be a potential strategy for adapting to low glucose levels.

Genetics analysis of Belarusian pediatric patients with factor VII deficiency

IntroductionCoagulation factor VII (FVII) deficiency (OMIM 227500) is the most common of the rare hereditary coagulation disorders characterized by autosomal recessive inheritance with variable penetrance. The estimated incidence of coagulation factor VII deficiency is 1:500,000. Clinical heterogeneity is a special feature of this coagulation disorder, the severity of which ranges from severe to mild or even asymptomatic forms. Materials and methodsIn the study were included three unrelated pediatric patients (3 female) with confirmed factor VII deficiency and members of their families (2 male and 2 female) Genetic analysis was carried out by next generation sequencing using Nextera XT, examining all exon and the exon/intron boundaries of F7 gene using a genetic analyzer MiSeq (Illumina, USA). All clinically significant observations were confirmed by Sanger sequencing. ResultsWe identified the pathogenic variant in all pediatric patients with factor VII deficiency in F7 gene. Four unique variants were identified: two missense mutation (c.1061C>T, p.Ala354Val; c.283G>A, p.Glu95Lys), one frameshift variant (c.1391delC, p.Pro464His fsTer32), one point mutation of proximal promoter region (c.1–32A>C). Genetic analysis indicated that patient 1 had heterozygous compound of maternal missense mutation c.1061 C > T and frameshift variant c.1391delC and paternal point mutation of proximal promoter region c.1–32A>C. Patient 2 was identified homozygous of two mutation c.1061C > T and c.1391delC, while her both parents were heterozygous of this mutations. In patient 3 was detected heterozygous compound of missense mutation c.1061C>Tand missense mutation c.283G > A. DNA samples of parents of patient 3 was unavailable for genetic analysis. All these variants have been previously reported in the EAHAD F7 databases, except for the missense variant c.283G > A, p. Glu95Lys, which is classified as variant of uncertain significance. ConclusionWe have performed genetic analysis for the identification of pathogenic variants in coagulation factor VII deficiency patients for the first time in Belarus. Genetic analysis of coagulation factor VII deficiency patients is useful to confirm diagnosis, to determine the carrier status and genetic counseling.

Noninvasive Prenatal Diagnosis of Beta-Thalassemia Disease by Using Digital PCR Analysis of Cell-Free Fetal DNA in Maternal Plasma

Introduction: Prenatal diagnosis of thalassemia disease was usually based on invasive technique. Noninvasive diagnosis using cell-free fetal DNA (cff-DNA) was described with various laboratory techniques. The aim of this study was to identify the performance of dPCR for analyzing cff-DNA in maternal plasma to diagnose fetal beta-thalassemia diseases. Methods: Thirty-five couples at risk of fetal beta-thalassemia disease caused by four common mutations of HBB were enrolled at 12–18 weeks. The dPCR assay was designed to detect and quantify paternally inherited beta-thalassemia allele (PIB) and maternally inherited beta-thalassemia allele (MIB) from cff-DNA in maternal plasma. Results: Of 29 couples with different paternal/maternal mutations, all cases who inherited paternal mutation had detectable PIB-M. The MIB-mutant/wild-type (MIB-M/MIB-N) ratio in the mothers whose fetuses did not inherit maternal mutation was 0.87 ± 0.07 which was significantly lower than that of the mothers whose fetuses inherited maternal mutation, 1.01 ± 0.05. The sensitivity and specificity of MIB-M/MIB-N ratio >0.95 in predicting fetus inheriting maternal mutation were 100 and 92.3%, respectively. In four couples with same paternal/maternal mutation, IB-M/IB-N ratio of >0.95 correctly predicted the presence of an inheritance of at least one beta-thalassemia allele. In two couples with paternal Hb E/beta-thalassemia, the presence of PIB-M and the MIB-M/MIB-N ratio of >0.95 correctly predicted the presence of paternal/maternal mutations, respectively. Conclusions: The method of analyzing cff-DNA in maternal plasma by dPCR is efficient for prenatal diagnosis of beta-thalassemia.

Craniofacial and cardiac defects in chd7 zebrafish mutants mimic CHARGE syndrome.

Congenital heart defects occur in almost 80% of patients with CHARGE syndrome, a sporadically occurring disease causing craniofacial and other abnormalities due to mutations in the CHD7 gene. Animal models have been generated to mimic CHARGE syndrome; however, heart defects are not extensively described in zebrafish disease models of CHARGE using morpholino injections or genetic mutants. Here, we describe the co-occurrence of craniofacial abnormalities and heart defects in zebrafish chd7 mutants. These mutant phenotypes are enhanced in the maternal zygotic mutant background. In the chd7 mutant fish, we found shortened craniofacial cartilages and extra cartilage formation. Furthermore, the length of the ventral aorta is altered in chd7 mutants. Many CHARGE patients have aortic arch anomalies. It should be noted that the aberrant branching of the first branchial arch artery is observed for the first time in chd7 fish mutants. To understand the cellular mechanism of CHARGE syndrome, neural crest cells (NCCs), that contribute to craniofacial and cardiovascular tissues, are examined using sox10:Cre lineage tracing. In contrast to its function in cranial NCCs, we found that the cardiac NCC-derived mural cells along the ventral aorta and aortic arch arteries are not affected in chd7 mutant fish. The chd7 fish mutants we generated recapitulate some of the craniofacial and cardiovascular phenotypes found in CHARGE patients and can be used to further determine the roles of CHD7.

Genetic aetiology of Down syndrome birth: novel variants of maternal DNMT3B and RFC1 genes increase risk of meiosis II nondisjunction in the oocyte.

The aim of the present work was to explore the intriguing association of maternal folate regulator gene polymorphisms and mutations with the incidence of chromosome 21 nondisjunction and Down syndrome birth. We tested polymorphisms/mutations of DNMT3B and RFC1 genes for their association with meiotic errors in oocyte among the 1215 Down syndrome child-bearing women and 900 controls. We observed that 23 out of 31 variants of DNMT3B and RFC1 exhibited an association with meiosis II nondisjunction in maternal age-independent manner. Additionally, we have reported 17 novel mutations and 1 novel polymorphic variant that are unique to the Indian Bengali speaking cohort and increased odds in favour of meiosis II nondisjunction. We hypothesize that the risk variants and mutations of DNMT3B and RFC1 genes may cause reduction in two or more recombination events and also cause peri-centromeric single exchange that increases the risk of nondisjunction at any age of women. In silico analyses predicted the probable damages of the transcripts or proteins from the respective genes owing to the said polymorphisms. These findings from the largest population sample tested ever revealed that mutations/polymorphisms of the genes DNMT3B and RFC1 impair recombination that leads to chromosome 21 nondisjunction in the oocyte at meiosis II stage and bring us a significant step closer towards understanding the aetiology of chromosome 21 nondisjunction and birth of a child with Down syndrome to women at any age.

Mutation analysis of SUOX in isolated sulfite oxidase deficiency with ectopia lentis as the presenting feature: insights into genotype–phenotype correlation

BackgroundIsolated sulfite oxidase deficiency (ISOD) caused by sulfite oxidase gene (SUOX) mutations is a rare neurometabolic disease associated with ectopia lentis (EL). However, few genotype–phenotype correlations have been established yet.MethodsPotentially pathogenic SUOX mutations were screened from a Chinese cohort of congenital EL using panel-based next-generation sequencing and analyzed with multiple bioinformatics tools. The genotype–phenotype correlations were evaluated via a systematic review of SUOX mutations within our data and from the literature.ResultsA novel paternal missense mutation, c.205G > C (p.A69P), and a recurrent maternal nonsense mutation, c.1200 C > G (p.Y400*), of SUOX were identified in a 4-year-old boy from 312 probands. The biochemical assays manifested elevated urine sulfite and S-sulfocysteine accompanied by decreased homocysteine in the blood. The patient had bilateral EL and normal fundus, yet minimal neurological involvement and normal brain structure. Molecular modeling simulation revealed the p.A69P mutant had an unstable structure but an unchanged affinity for sulfite, while the truncated p.Y400* mutant showed decreased binding capacity. Genotype–phenotype analysis demonstrated patients with biallelic missense mutations had milder symptoms (P = 0.023), later age of onset (P < 0.001), and a higher incidence of regression (P = 0.017) than other genotypes. No correlations were found regarding EL and other neurological symptoms.ConclusionThe data from this study not only enrich the known mutation spectrum of SUOX but also suggest that missense mutations are associated with mild and atypical symptoms.

How frequently are Autosomal and X-STRs multistep mutations perceived as single-step?

Short tandem repeats (STRs) incur in length mutations that involve the loss or gain of repeats. STR mutation rates are usually estimated considering the rates of observed Mendelian incompatibilities in one generation familial configurations. When considering multistep mutations, for the autosomal and X-chromosomal modes of genetic transmission, underestimations are inevitable when using this approach (MIA), due to the occurrence of mutational events deceptively perceived as involving fewer steps. The rate of this occurrence depends on the mode of genetic transmission considered, the parental origin of the mutation, the type of familial configuration considered, and the genotypic background of the progenitor(s). MIA biases were weighted and compared for the diploid and haplodiploid modes of transmission, using familial genotypic configurations (parent(s)-child duos and trios) generated resorting to Python™ and real population databases from Norway, Somalia, and Spain for 10 Aut-STRs and Argentina, Eastern Asia, and Northern Europe for 12 X-STRs. One two- or one three-step mutation was simulated in each of the 1,000,000 familial configurations. The frequency with which mutations could be interpreted as involving fewer steps, when the most parsimonious reasoning is employed, was computed. Results showed that the magnitude and type of biases depend on the type of familial data and the genetic mode of transmission considered, being higher in duos than in trios, both in autosomes and the X chromosome. Indeed, whether X- or Aut-STRs are analyzed, trios generally provide better estimates and should be favored over duos. The pooling of the two types of data is not advised. The greater the number of steps involved in the mutation, the worst the estimates obtained. In X-chromosomal analyzes, trios with a paternal mutation presented the best estimates and mother-daughter duos the worst; mother-son duos showed similar estimates to trios when a maternal mutation was considered.

The Concordance of Paternal and Maternal Retinoblastoma-1 Gene Mutation Pattern with Clinical Manifestation and Disease Staging in Patients Suffering from Retinoblastoma

Background: Retinoblastoma (RB) is a common neoplastic disease in children, leading to high mortality if not diagnosed and treated timely. Mutations in both versions of the Retinoblastoma1 (RB1) gene are responsible for this disease. A wide range of mutations has been reported throughout the RB1 gene. Objectives: The present study aimed to assess the concordance of paternal and maternal RB-1 gene mutation with clinical manifestation and disease staging in patients suffering from RB. Methods: This cross-sectional study was performed on 23 patients with unilateral or bilateral RB. Paternal and maternal peripheral blood samples were extracted for genome analysis. Information related to clinical manifestations and disease staging was collected from the patients’ hospital records. Multiplex-ligation dependent probe amplification (MLPA) method or Sanger sequencing method was employed to assess the gene mutation and its genomic pattern. Results: No significant association was revealed between the presence of both maternal and paternal RB1 gene mutations and the disease staging, while the study could show a significant relationship between the presence and heterozygous pattern of RB1 gene mutation and the presence of disease-related clinical manifestations that bilateral involvement was strongly associated with the presence of a heterozygous pattern of gene mutation compared to unilateral involvement (P = 0.001). Conclusions: This study showed a significant correlation between the presence of RB1 gene mutation and bilateral involvement in RB, but the association between the disease staging and gene mutation remains insignificant.