Identification of rare thalassemia variants using third-generation sequencing.

Routine PCR, Sanger sequencing, and specially designed GAP-PCR are often used in the genetic analysis of thalassemia, but all these methods have limitations. In this study, we evaluated a new third-generation sequencing-based approach termed comprehensive analysis of thalassemia alleles (CATSA) in subjects with no variants identified by routine PCR, Sanger sequencing, and specially designed GAP-PCR. Hemoglobin testing and routine PCR tests for 23 common variants were performed for 3,033 subjects. Then, Sanger sequencing and specially designed GAP-PCR were performed for a subject with no variants identified by routine PCR, no iron deficiency, and positive hemoglobin testing. Finally, the new CATSA method was conducted for the subjects with no variants identified by Sanger sequencing and specially designed GAP-PCR. In the 49 subjects tested by CATSA, eight subjects had variants identified. Sanger sequencing and independent PCR confirmed the CATSA result. In addition, it is the first time that Hb Lepore was identified in Hunan Province. In total, traditional methods identified variants in 759 of the 3,033 subjects, while CATSA identified additional variants in eight subjects. CATSA showed great advantages compared to the other genetic testing methods.

Novel Mutations in the GTPBP3 Gene for Mitochondrial Disease and Characteristics of Related Phenotypic Spectrum: The First Three Cases From China.

Combined Oxidative Phosphorylation Deficiency 23 (COXPD23) caused by mutations in GTPBP3 gene is a rare mitochondrial disease, and this disorder identified from the Chinese population has not been described thus far. Here, we report a case series of three patients with COXPD23 caused by GTPBP3 mutations, from a severe to a mild phenotype. The main clinical features of these patients include lactic acidosis, myocardial damage, and neurologic symptoms. Whole genome sequencing and targeted panels of candidate human mitochondrial genome revealed that patient 1 was a compound heterozygote with novel mutations c.413C > T (p. A138V) and c.509_510del (p. E170Gfs∗42) in GTPBP3. Patient 2 was a compound heterozygote with novel mutations c.544G > T (p. G182X) and c.785A > C (p.Q262P), while patient 3 was a compound heterozygote with a previously reported mutation c.424G > A (p.E142K) and novel mutation c.785A > C (p.Q262P). In conclusion, we first describe three Chinese individuals with COXPD23, and discuss the genotype-phenotype correlations of GTPBP3 mutations. Our findings provide novel information in the diagnosis and genetic counseling of patients with mitochondrial disease.

Prenatal case of Simpson-Golabi-Behmel syndrome with a de novo 370Kb-sized microdeletion of Xq26.2 compassing partial GPC3 gene and review.

BACKGROUND: Simpson-Golabi-Behmel syndrome type 1 (SGBS1) is a rare X-linked recessive disorder characterized by pre- and postnatal overgrowth and a broad spectrum of anomalies including craniofacial dysmorphism, heart defects, renal, and genital anomalies. Due to the ultrasound findings are not pathognomonic for this syndrome, most clinical diagnosis of SGBS1 are made postnatally. METHODS: A pregnant woman with abnormal prenatal sonographic findings was advised to perform molecular diagnosis. Single nucleotide polymorphism array (SNP array) was performed in the fetus, and the result was validated with multiplex ligation-dependent probe amplification (MLPA) and real-time quantitative PCR (qPCR). RESULTS: The prenatal sonographic presented with increased nuchal translucency at 13 gestational weeks, and later at 21 weeks with cleft lip and palate, heart defect, increased amniotic fluid index and over growth. A de novo 370Kb-deletion covering the 5'-UTR and exon 1 of GPC3 gene was detected in the fetus by SNP array, which was subsequently confirmed by MLPA and qPCR. CONCLUSION: The de novo 370Kb hemizygous deletion of 5'-UTR and exon 1 of GPC3 results in the SGBS1 of this Chinese family. Combination of ultrasound and genetics tests helped us effectively to diagnose the prenatal cases of SGBS1. Our findings also enlarge the spectrum of mutations in GPC3 gene.

Clinical significance of S100B protein in pregnant woman with early- onset severe preeclampsia.

OBJECTIVES: Preeclampsia is one of the most feared complications of pregnancy, which can progress rapidly to serious complications such as death of both mother and fetus. To present, the leading cause of preeclampsia is still debated. The purpose of this article was to explore the clinical significance of S100B protein, a kind of Ca2+ -sensor protein, in the early-onset severe preeclampsia. MATERIAL AND METHODS: Nine pregnant women with early-onset severe preeclampsia (the study group) and 13 healthy pregnant women (the control group) were included in this study. The level of S100B in the amniotic fluid, maternal blood, and umbilical cord blood were detected by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance imaging (SPRi) methods. Diagnostic values of S100B for early-onset severe preeclampsia were assessed by Receiver Operating Characteristic (ROC) curve analysis. RESULTS: The levels of S100B in maternal blood and amniotic fluid in the study group were higher than those in the control group (p < 0.05). ROC curve analysis showed that S100B detected by SPRi method (SPRi-S100B) showed a cut-off level of 181 ng/mL with sensitivity of 100%, a specificity of 84.6%, and a Youden index of 0.846 in the maternal blood, which had better clinical significance and diagnostic value (at than that detected by ELISA (ELISA-S100B). CONCLUSIONS: The levels of S100B detected by SPRi in maternal blood can indicate early-onset severe preeclampsia and perinatal brain injury.

Clinical and genetic analysis of classical Ehlers-Danlos syndrome patient caused by synonymous mutation in COL5A2.

BACKGROUND: Classical Ehlers-Danlos syndrome (cEDS) is a heterogeneous connective tissue disorder that mainly results from the germline mutation of COL5A1 and COL5A2. The majority of the COL5A2 mutations reported to date represent structural mutations, including missense or in-frame exon-skipping splice mutations. The only reported synonymous mutation was expected to affect on splicing of exon 29 by prediction programs which should be further confirmed. METHODS: Whole exome sequencing was performed to identify the genetic variants of a Chinese boy who was characterized by skin hyperextensibility, abnormal scarring, hypermobile joints and scoliosis. Sanger sequencing was used to validate the variants in his parents. Reverse transcription polymerase chain reaction (RT-PCR) was performed to analyze the functional effects of the variant. RESULTS: A de novo heterozygous synonymous variant (NM_000393.5:c.1977 G>A) of COL5A2 gene was identified in the patient. The results of RT-PCR revealed that the synonymous variant led to skipping of exon 29 in the RNA transcript. CONCLUSIONS: Our study supplies further supporting evidence that the synonymous COL5A2 mutation c.1977 G>A can cause skipping of exon 29 in the RNA transcript, thus resulting in the production of mutant α2(V)-chains and clinical phenotype of cEDS. This result highlights the need to include splicing-altering synonymous mutations into the screening for cEDS.

Integrated CNV-seq, karyotyping and SNP-array analyses for effective prenatal diagnosis of chromosomal mosaicism.

BACKGROUND: Emerging studies suggest that low-coverage massively parallel copy number variation sequencing (CNV-seq) more sensitive than chromosomal microarray analysis (CMA) for detecting low-level mosaicism. However, a retrospective back-to-back comparison evaluating accuracy, efficacy, and incremental yield of CNV-seq compared with CMA is warranted. METHODS: A total of 72 mosaicism cases identified by karyotyping or CMA were recruited to the study. There were 67 mosaic samples co-analysed by CMA and CNV-seq, comprising 40 with sex chromosome aneuploidy, 22 with autosomal aneuploidy and 5 with large cryptic genomic rearrangements. RESULTS: Of the 67 positive mosaic cases, the levels of mosaicism defined by CNV-seq ranged from 6 to 92% compared to the ratio from 3 to 90% by karyotyping and 20% to 72% by CMA. CNV-seq not only identified all 43 chromosomal aneuploidies or large cryptic genomic rearrangements detected by CMA, but also provided a 34.88% (15/43) increased yield compared with CMA. The improved yield of mosaicism detection by CNV-seq was largely due to the ability to detect low level mosaicism below 20%. CONCLUSION: In the context of prenatal diagnosis, CNV-seq identified additional and clinically significant mosaicism with enhanced resolution and increased sensitivity. This study provides strong evidence for applying CNV-seq as an alternative to CMA for detection of aneuploidy and mosaic variants.

Whole genome sequencing reveals translocation breakpoints disrupting TP63 gene underlying split hand/foot malformation in a Chinese family.

BACKGROUND: Split hand/foot malformation (SHFM) is a congenital limb developmental disorder, which impairs the fine activities of hand/foot in the affected individuals seriously. SHFM is commonly inherited as an autosomal dominant disease with incomplete penetrance. Chromosomal aberrations such as copy number variations and translocations have been linked to SHFM. This study aimed to identify the genetic cause for three patients with bilateral hand and foot malformation in a Chinese family. METHODS: Karyotyping, single-nucleotide polymorphism (SNP) array, whole exome sequencing, whole genome sequencing, and Sanger sequencing were applied to identify the pathogenic variant. RESULTS: Karyotyping revealed that the three patients had balanced reciprocal translocation, 46, XX, t(3;15) (q29;q22). SNP array identified no pathogenic copy number variation in the proband. Trio-WES (fetus-mother-father) sequencing results revealed no pathogenic variants in the genes related to SHFM. Whole-genome low-coverage mate-pair sequencing (WGL-MPS), breakpoint PCR, and Sanger sequencing identified the breakpoints disrupting TP63 in the patients, but not in healthy family members. CONCLUSION: This study firstly reports that a translocation breakpoint disrupting TP63 contributes to the SHFM in a Chinese family, which expands our knowledge of genetic risk and counseling underlying SHFM. It provides a basis for genetic counseling and prenatal diagnosis (preimplantation genetic diagnosis) for this family.

[Non-invasive prenatal testing and genetic analysis of a fetus with partial trisomy 21].

OBJECTIVE: To carry out prenatal diagnosis for a fetus with high risk predicted by non-invasive prenatal testing (NIPT). METHODS: Next-generation sequencing (NGS) was used to analyze free fetal DNA (ffDNA) in the maternal plasma. Chromosomal karyotyping and single nucleotide polymorphism array (SNP-array) were used to ascertain copy number variation in the fetus and its parents. RESULTS: SNP-array analysis and chromosomal karyotyping revealed that the fetus had a 15.018 Mb duplication at 4q34.1q35.2 and a 7.678 Mb duplication at 21q11.2q21.1, which were derived from a t(4;21)(q34.1;q21.1) translocation carried by its mother. CONCLUSION: NIPT is capable of detecting submicroscopic chromosomal abnormalities of the fetus. Combined use of genetic techniques, in particular SNP-array, is crucial for the diagnosis of partial trisomy 21q in this case.

[Genetic analysis of a pedigree with MECP duplication syndrome].

OBJECTIVE: To explore the genetic etiology of a pedigree with mental retardation and hypotonia by using chromosome microarray analysis (CMA), low coverage massive parallel copy number variation sequencing (CNV-seq) and quantitative PCR (qPCR). METHODS: Genomic DNA was extracted from peripheral blood samples from two male patients and healthy members from the pedigree. CNV-seq was carried out for one patient. Suspected CNV was verified by qPCR. CNV-seq or single nucleotide polymorphism array (SNP array) were carried out for another patient and his family members. RESULTS: Both patients showed severe hypotonia and global development delay, in particular language delay. CNV-seq and SNP array indicated that both patients had carried a Xq28 duplication, with spanned 0.26 Mb and 0.42 Mb, respectively. Both duplications encompassed the MECP2 gene. CNV-seq analysis of their family members confirmed that the mother and one sister had carried similar duplications, while an elder brother was normal. CONCLUSION: CNV-seq and CMA are rapid and effective tools for the diagnosis of MECP2 duplication syndrome in children with mental retardation, hypotonia and recurrent infections.

[Xq;Yq translocation in a patient with premature ovarian insufficiency].

OBJECTIVE: To explore the genetic basis for a patient with premature ovarian insufficiency. METHODS: Chromosomal G-banding and C-banding, single nucleotide polymorphism array (SNP-array), fluorescence in situ hybridization (FISH) and Y chromosome microdeletion assay were used for the analysis. RESULTS: With the combined techniques, the patient was found to carry a Xq;Yq translocation, with a karyotype of 46,X,der(X)t(X;Y)(q25;q12).ish der(X)(Tel XYp+,Tel XYq+,Yq12+). CONCLUSION: Unbalanced Xq;Yq translocation probably underlay the premature ovarian insufficiency in this patient.

Clinical and molecular characterization of 12 prenatal cases of Cri-du-chat syndrome.

BACKGROUND: This study aimed to define the molecular basis for 12 prenatal cases of Cri-du-chat syndrome (CdCS) and the potential genotyping-phenotyping association. METHODS: Karyotyping and single nucleotide polymorphism array analyses for copy number variants were performed. RESULTS: Nine cases had 5p terminal deletions and three had 5p interstitial deletions, and these cases had variable deletion sizes with partial overlapping. Phenotypically, besides intrauterine growth restriction (IUGR) and brain as well as heart abnormalities, hypospadias, and lung dysplasia were observed. Potential genetic causes for specific phenotypes in these cases were identified. CONCLUSION: This study defined the molecular bases for the patients of CdCS, which is important for genetic counseling for these families. The findings of present study expand the clinical features of CdCS in the fetal period, and provided important information for further refining the genotypic-phenotypic correlations for this syndrome.

Identification of a novel gross deletion of TCOF1 in a Chinese prenatal case with Treacher Collins syndrome.

BACKGROUND: Treacher Collins syndrome (TCS) is the most common mandibulofacial dysostosis with an autosomal dominant or rarely recessive manner of inheritance. It is still challenging to make a definite diagnosis for affected fetuses with TCS only depending on the ultrasound screening. Genetic tests can contribute to the accurate diagnosis for those prenatal cases. METHODS: Targeted exome sequencing was performed in a fetus of a Chinese family, who presenting an abnormal facial appearance by prenatal 2D and 3D ultrasound screening, including micrognathia, nasal bridge pit, and abnormal auricle. The result was validated with multiplex ligation-dependent probe amplification (MLPA) and real-time quantitative PCR (qPCR). RESULTS: A novel 2-6 exons deletion of TCOF1 gene was identified and confirmed by the MLPA and qPCR in the fetus, which was inherited from the affected father with similar facial anomalies. CONCLUSION: The heterozygous deletion of 2-6 exons in TCOF1 results in the TCS of this Chinese family. Our findings not only enlarge the spectrum of mutations in TCOF1 gene, but also highlight the values of combination of ultrasound and genetics tests in diagnosis of craniofacial malformation-related diseases during perinatal period.

Variants in CAPZA2, a member of an F-actin capping complex, cause intellectual disability and developmental delay.

The actin cytoskeleton is regulated by many proteins including capping proteins that stabilize actin filaments (F-actin) by inhibiting actin polymerization and depolymerization. Here, we report two pediatric probands who carry damaging heterozygous de novo mutations in CAPZA2 (HGNC: 1490) and exhibit neurological symptoms with shared phenotypes including global motor development delay, speech delay, intellectual disability, hypotonia and a history of seizures. CAPZA2 encodes a subunit of an F-actin-capping protein complex (CapZ). CapZ is an obligate heterodimer consisting of α and ß heterodimer conserved from yeast to human. Vertebrate genomes contain three α subunits encoded by three different genes and CAPZA2 encodes the α2 subunit. The single orthologue of CAPZA genes in Drosophila is cpa. Loss of cpa leads to lethality in early development and expression of the human reference; CAPZA2 rescues this lethality. However, the two CAPZA2 variants identified in the probands rescue this lethality at lower efficiency than the reference. Moreover, expression of the CAPZA2 variants affects bristle morphogenesis, a process that requires extensive actin polymerization and bundling during development. Taken together, our findings suggest that variants in CAPZA2 lead to a non-syndromic neurodevelopmental disorder in children.

Prenatal genetic analysis and differential pregnancy outcomes of two de novo cases showing mosaic isodicentric Y chromosome.

BACKGROUND: Fetal cells collected from the amniotic fluid of two pregnant women indicated sex chromosome abnormalities. Therefore, we performed G-banded chromosome karyotype analysis, single nucleotide polymorphism array (SNP array), fluorescence in situ hybridization (FISH), and sequence-tagged sites (STS) analysis of the Y chromosome to determine the rare molecular genetics of the two fetuses. CASE PRESENTATION: The karyotypes of the fetuses from patients 1 and 2 were mos 45,X[92]/46,X,+idic(Y)(q11.21)[8] and mos 45,X[20]/46,X,+idic(Y)(q11.223)[80], respectively. Fetus 1 had a 7.76 Mb deletion in Yq11.222q11.23 and a 15.68 Mb duplication in Yp11.2q11.21. Fetus 2 had 21 Mb of repetitive segments in Yp11.3q11.223. Azoospermia factor (AZF) detection by STS analysis revealed a missing AZFb+c region in fetus 1 and three functional AZF regions in fetus 2. The isodicentric Y chromosome (idic (Y)) in both fetuses arose de novo. The pregnancy of patient 1 was terminated, whereas the fetus of patient 2 was delivered and is now 10 months old with normal appearance and growth. CONCLUSION: A combination of technologies such as chromosome karyotyping, FISH, SNP arrays, and STS analysis of the Y chromosome is important in prenatal diagnosis to reduce birth defect rates and improve the health of the Chinese population.

The Epilepsy of Infancy With Migrating Focal Seizures: Identification of de novo Mutations of the KCNT2 Gene That Exert Inhibitory Effects on the Corresponding Heteromeric KNa1.1/KNa1.2 Potassium Channel.

The epilepsy of infancy with migrating focal seizures (EIMFS; previously called Malignant migrating partial seizures of infancy) are early-onset epileptic encephalopathies (EOEE) that associate multifocal ictal discharges and profound psychomotor retardation. EIMFS have a genetic origin and are mostly caused by de novo mutations in the KCNT1 gene, and much more rarely in the KCNT2 gene. KCNT1 and KCNT2 respectively encode the KNa1.1 (Slack) and KNa1.2 (Slick) subunits of the sodium-dependent voltage-gated potassium channel KNa. Functional analyses of the corresponding mutant homomeric channels in vitro suggested gain-of-function effects. Here, we report two novel, de novo truncating mutations of KCNT2: one mutation is frameshift (p.L48Qfs43), is situated in the N-terminal domain, and was found in a patient with EOEE (possibly EIMFS); the other mutation is nonsense (p.K564*), is located in the C-terminal region, and was found in a typical EIMFS patient. Using whole-cell patch-clamp recordings, we have analyzed the functional consequences of those two novel KCNT2 mutations on reconstituted KNa1.2 homomeric and KNa1.1/KNa1.2 heteromeric channels in transfected chinese hamster ovary (CHO) cells. We report that both mutations significantly impacted on KNa function; notably, they decreased the global current density of heteromeric channels by ~25% (p.K564*) and ~55% (p.L48Qfs43). Overall our data emphasize the involvement of KCNT2 in EOEE and provide novel insights into the role of heteromeric KNa channel in the severe KCNT2-related epileptic phenotypes. This may have important implications regarding the elaboration of future treatment.

Analysis of balanced reciprocal translocations in patients with subfertility using single-molecule optical mapping.

PURPOSE: Approximately 1% of individuals who carry a balanced reciprocal translocation (BRT) are subfertile. Current karyotyping does not have the resolution to determine whether the breakpoints of the involved chromosomes perturb genes important for fertility. The aim of this study was to apply single-molecule optical mapping (SMOM) to patients presenting for IVF (in vitro fertilization) to ascertain whether the BRT disrupted any genes associated with normal fertility. METHODS: Nine subfertile patients with different BRTs were recruited for the study. Methyltransferase enzyme DLE1 was used to fluorescently label their genomic DNA samples at the recognition motif CTTAAG. The SMOM was performed on the Bionano platform, and long molecules aligned against the reference genome hg19 to identify the breakpoint regions. Mate-pair and PCR-Sanger sequencing were used to confirm the precise breakpoint sequences. RESULTS: Both breakpoint regions in each of the nine BRTs were finely mapped to small regions of approximately 10 Kb, and their positions were consistent with original cytogenetic banding patterns determined by karyotyping. In three BRTs, breakpoints disrupted genes known to be associated with male infertility, namely NUP155 and FNDC3A [46,XY,t(5;13)(p15;q22)], DPY19L1 [46,XY,t(1;7)(p36.3;p15), and BAI3 [46,XY,t(3;6)(p21;q16)]. CONCLUSIONS: The SMOM has potential clinical application as a rapid tool to screen patients with BRTs for underlying genetic causes of infertility and other diseases.

[Genetic analysis of a child with mental retardation and hypospadia].

OBJECTIVE: To carry out genetic testing for a boy presenting with mental retardation and hypoplasia. METHODS: Conventional karyotyping, fluorescence in situ hybridization (FISH) and single nucleotide polymorphism based array (SNP-array) were used to analyze the boy and his parents. RESULTS: SNP-array has detected a 25.7 Mb microduplication at 2q33.3q36.3 in the boy. Chromosomal karyotyping and FISH analysis indicated that his mother had a karyotype of 46,XX,ish ins(11;2) (p15;q33q36), and that the boy has carried an abnormal chromosome 11 derived from the maternal translocation. The karyotype of the boy was ascertained as 46,XY,ish der(11)ins(11;2) (p15;q33q36)mat. CONCLUSION: SNP-array combined with G-banding and FISH can delineate the cryptic translocation and is valuable for the assessment of recurrence risk for subsequent pregnancies.

[Analysis on the Genotype of 5018 Cases of Thalassemia in Hunan Area].

OBJECTIVE: To investigate the type and distritution of thalassemia gene mutaitons in Hunan area, so as to provide evidence for prenatal screening, diagnosis and reduction of birth defects. METHODS: A total of 5018 cases from Maternal and Children Health Hospital of Hunan from June 2017 to Dec 2018 were undergone thalassemia gene mutation analysis. The reverse dot blot hydridization was used to detect 6 kinds of genotypes of α-thalassemia and 17 kinds of point mutations of ß-thalassemia, and the detected data were analyzed statistically. RESULTS: 889 cases (55.9%) of α-thalassemia carriers were found, including 385 cases of silent α-thalassemia, 488 cases of α-thalassemia trait, 16 cases of Hb H disease. --SEA/αα was the most common genotype in α thalassemia. 664 cases (41.7%) were diagnosed as ß-thalassemia carriers, heterozygotes accounted for 99.8% (663/664), IVS-Ⅱ-654, CD41-42M and CD17M were the main genotypes, and compound heterozygote accounted for 0.2% (1/664). 38 cases were diagnosed as α-thalassemia combined ß-thalassemia. CONCLUSION: The constituent ratio of thalassemia gene mutations in Hunan has regional characteristics, --SEA/αα is the most common genotype in α-thalassemia carrier. IVS-Ⅱ-654, CD41-42 and CD17 are common ones in ß-thalassemia. The frequency of α-thalassemia combined with ß-thalassemia is high.

Molecular investigation in Chinese patients with primary carnitine deficiency.

BACKGROUND: Primary carnitine deficiency (PCD) is an autosomal recessive disorder of carnitine transportation caused by mutations in the SLC22A5 that lead to low serum carnitine levels and decreased intracellular carnitine accumulation. Characteristic clinical findings are hypoketotic hypoglycemia and skeletal and cardiac myopathy. OBJECTIVE: To genetically diagnose 24 unrelated Chinese patients with PCD, including 18 infants and six adults. METHODS: The entire coding region and the intron-exon boundaries of SLC22A5 were amplified by polymerase chain reaction (PCR). In silico analyses and reverse transcription-polymerase chain reaction (RT-PCR) were used to predict variants' impact on protein structure and function. RESULTS: Disease-causing variants in the SLC22A5 were identified in all 24 subjects, and c.288delG, c.495C>A, c.774_775insTCG, c.824+1G>A, and c.1418G>T were novel. The novel variant c.824+1G>A caused a truncated protein p.Phe276Tyrfs*8. CONCLUSIONS: We identified 13 variants in the SLC22A5 in 24 PCD patients, and five of these variants are novel mutations. c.824+1G>A was confirmed to alter mRNA splicing by reverse transcription PCR. Furthermore, our findings broaden the mutation spectrum of SLC22A5 and the understanding of the diverse and variable effects of PCD variants.