Exome-based preconception carrier testing for consanguineous couples in China.

OBJECTIVE: To evaluate the utility of clinical exome sequencing (ES)-based carrier screening in Chinese consanguineous couples. METHODS: Consanguineous couples were screened for autosomal recessive (AR) disorders using the clinical ES of 5000 genes associated with human diseases. RESULTS: We recruited 14 couples who elected to have sequencing. One couple was related as first cousins and 13 as second cousins. Both partners carrying the same pathogenic variant were detected in four couples. One couple was found in which one partner carried a splice variant, and the other had a missence variant of the same gene. These five couples were identified as being at risk of having a child affected by an AR disorder. CONCLUSION: Our study demonstrates that ES-based preconception screening yields a clinical value for Chinese consanguineous couples. It enables to detect at-risk couples for rare AR diseases.

Tetrasomy 18p Case Report.

BACKGROUND: Tetrasomy 18p is a rare disorder. It is known to affect about 250 families worldwide. Tetrasomy 18p is also the most common type of isochromosome. Here we report a de novo tetrasomy 18p. METHODS: The copy number variation of the patient was detected by microarray. Whether the abnormal gene was inherited from the parents was detected by karyotype analysis. Then the source of the chromosome was located by fluorescence in situ hybridization. Finally, we used MLPA technology to validate the results of patient testing. RESULTS: Microarray detection found that patients with 18p11.32p11.21 had duplication, with a copy number of four, which was tetrasomy 18 syndrome. The karyotype results showed 48,XY,+2mar?. Chromosome 18 telomere probe FISH experimental results: 48,XY,+i(18)(p10),+mar.ish. MLPA results showed that the number of chromosome 18 short arm copies is increased. Karyotype analysis results of his mother were 47,XX,+mar. Microarray results showed normal. Karyotype results of his father were normal. CONCLUSIONS: This case is de novo case, the patient's marker chromosome may be inherited from his mother, which does not rule out the influence of his mother's marker chromosome on his isochromosome 18.

Molecular and Hematological Characterization of a Novel Translation Initiation Codon Mutation of the α2-Globin Gene (ATG>ATC or HBA2: c.3G>C).

Although mutations causing α-thalassemia (α-thal) are mainly larger deletions involving one or both of the duplicated α-globin genes, point mutations are not rare. We have identified a novel mutation of the translation initiation codon of the α2-globin gene with DNA sequencing and allele-specific multiplex ligation-dependent probe amplification (MLPA) in a Chinese family. RNA analysis was performed with reverse transcription-MLPA (RT-MLPA). A novel mutation at the translation initiation codon of the α2-globin gene (HBA2: c.3G>C) was identified. The proband and his father, who were both carriers of this mutation, had a hematological phenotype of mild α+-thalassemia (α+-thal) trait with low-normal limit of mean corpuscular volume (MCV) and normal Hb A2. RNA analysis showed markedly decreased levels of α-globin mRNA and the presence of a small amount of mutant mRNA. The HBA2: c.3G>C mutation most likely caused α-thal by lowering levels of wild α-globin chain. Our study increases the mutation spectrum of α-thal.

Fetal Anemia and Hydrops Fetalis Associated with Homozygous Hb Constant Spring (HBA2: c.427T > C).

Hb Constant Spring (Hb CS, HBA2: c.427T > C) is a common nondeletional α-thalassemia (α-thal) that results from a nucleotide substitution at the termination codon of the α2-globin gene. Homozygosity for Hb CS (α(CS)α/α(CS)α) is relatively rare, and generally characterized with mild hemolytic anemia, jaundice, and splenomegaly. In this report we present a fetus with cardiomegaly, pericardial effusion, enlarged placenta and increased middle cerebral artery-peak systolic velocity (MCA-PSV) at 24 weeks' gestation. Fetal blood sampling revealed the severe anemia [hemoglobin (Hb) level being 4.8 g/dL] and Hb H (ß4) disease-like hematological findings with Hb Bart's (γ4) level of 17.9%. DNA sequencing of the α-globin genes found that both partners were Hb CS carriers and the fetus was an Hb CS homozygote. Therefore, this was a rare case of homozygous Hb CS which demonstrated an unusual and serious anemia and hydrops fetalis in utero.

Delineation of the molecular basis of borderline hemoglobin A2 in Chinese individuals.

BACKGROUND: The "gray zone" of borderline hemoglobin A2 (Hb A2) may be present in a large section of the population, especially in countries where thalassemia is common. However, very little is currently known of the molecular basis of borderline Hb A2 in Chinese individuals. METHOD: In this study, we performed a comprehensive analysis of the globin genotypes and KLF1 gene mutations associated with borderline Hb A2 in 165 Chinese subjects. RESULT: Fifteen (9.1%) were positive for a molecular defect in the α-,ß-globin genes, of whom, α-thalassemia mutations and α-globin gene triplication were found in eleven cases, accounting for about 73.3% of these globin gene defects. Twenty (12.1%) were positive for a molecular defect in the KLF1 gene. Eight different mutations were identified, six of which are here reported for the first time. The most common is the G176AfsX179 mutation, accounting for 50% of the total. CONCLUSIONS: The molecular characterization of borderline Hb A2 in Chinese individuals is significantly different than in Italian population. Our data is conductive to provision of genetic counseling for Chinese individuals with borderline Hb A2.

Screening and diagnosis of Hb Quong Sze [HBA2: c.377T > C (or HBA1)] in a prenatal control program for thalassemia.

Hb Quong Sze [Hb QS, HBA2: c.377T > C (or HBA1)] is a common nondeletional thalassemia in southern China. It is one of the major alleles causing nondeletional Hb H (ß4) disease in the Chinese population. There is no strategy currently in place that aims to screen using hematological index cutoffs for this variant. This study was carried out to evaluate whether it is effective to use mean corpuscular hemoglobin (MCH) <27.0 pg as a screening test in the first step of screening for Hb QS carriers in southern China. The data of hematological testing in the Hb QS carriers obtained from couples who underwent prenatal thalassemia screening, regardless of the red blood cell (RBC) indices, were retrospectively reviewed. A total of 51 Hb QS carriers were identified, giving a prevalence rate of 0.2%; among these, 45 were Hb QS heterozygotes. The values of hemoglobin (Hb), MCV and mean corpuscular Hb (MCH) in the 45 Hb QS heterozygotes were 13.2 ± 1.8 g/dL, 75.2 ± 3.3 fL and 24.5 ± 0.5 pg, respectively. Eight heterozygotes (17.8%) had an MCV value of >80.0 fL, ranging from 80.9 to 84.1 fL, and would not be detected using the cutoff value of MCV <80.0 fL as a criterion for thalassemia screening. However, if screening had been based on the MCH <27.0 pg value, all 45 Hb QS heterozygotes would have been detected. Using a cutoff value of MCH <27.0 pg in nondeletional thalassemia screening would greatly decrease the DNA diagnosis burden.

Prevalence and molecular characterization of structural hemoglobin variants in the Dongguan region of Guangdong province, southern China.

The aim of the present study was to find the most prevalent structural hemoglobin (Hb) variants in southern China and to present hematological and molecular data of abnormal Hbs in the population from southern China. The type and frequency of structural Hb variants and their hematological and molecular characteristics were identified in 131 individuals from 30,848 unrelated partners who were referred to the prenatal clinic of Dongguan Maternal & Children Health Hospital, Dongguan, Guangdong, People's Republic of China (PRC) from 2011 to 2013. α-Globin or ß-globin chain variants were screened using a capillary electrophoresis (CE) system, and α-globin or ß-globin gene mutations were confirmed using sequencing techniques. The gene frequency of Hb variants was 0.4% (131/30,848). The most common α-globin variants were Hb Constant Spring (Hb CS, HBA2: c.427T > C) (0.2%), followed by Hb Q-Thailand (HBA1: c.223G > C) and Hb G-Honolulu (HBA2: c.91G > C). The most common ß-globin variant was Hb E (HBB: c.79G > A) (0.09%), followed by Hb New York (HBB: c.341T > A). Our results provide a detailed prevalence and molecular characterization of abnormal Hbs in the population of the Dongguan region. These findings have important implications for a region with a high frequency of α- and ß-thalassemias.

Detection of Hb anti-Lepore Hong Kong (NG_000007.3: g.63154_70565dup) in Chinese individuals.

We have identified four Chinese individuals from three unrelated families with raised Hb A2 levels. The anti-Lepore hybrid hemoglobin (Hb) variant was amplified using a pair of primers, 5' to the ß-globin gene Cap site and 3' to the δ-globin gene polyadenylation site (polyA) region, respectively. Direct sequencing of the ßδ fusion products confirmed the anti-Lepore Hong Kong (NG_000007.3: g.63154_70565dup) variant. We found that this anti-Lepore variant is positioned in zone 3 on the capillary electrophoresis system. It may help in differential diagnosis of Hb variants and providing better information in clinical counseling.

[Analysis of clinical phenotypes of compound heterozygotes of Hb J-Bangkok and ß-thalassemia].

OBJECTIVE: To analyze hematological characteristics of compound heterozygotes of Hb J-Bangkok and ß-thalassemia, and to explore the influence of Hb J-Bangkok on the phenotype of ß-thalassemia. METHODS: Peripheral blood samples from a patient carrying Hb J-Bangkok and a ß-thalassemia mutation, her family members and three sporadic Hb J-Bangkok carriers were collected. RBC analysis and hemoglobin electrophoresis were performed. Genotypes of α- and ß-globin genes were analyzed. RESULTS: The father of the proband and the three sporadic cases were single carriers of Hb J-Bangkok. All of them were asymptomatic and have normal hematological parameters except for an abnormal hemoglobin band detected on hemoglobin electrophoresis. The proband was a compound heterozygote for Hb J-Bangkok and ß-thalassemia mutation IVS-Ⅱ-654. She presented typical ß-thalassemia trait, featuring hypochromic microcytic anemia and increased Hb A2 level. An abnormal hemoglobin band was also detected. CONCLUSION: Carriers of Hb J-Bangkok alone are asymptomatic. Co-existence of Hb J-Bangkok and ß-thalassemia may not aggravate the phenotype. Therefore, couples with one carrying Hb J-Bangkok and another carrying a ß-thalassemia mutation do not require prenatal diagnosis.

Can cell-free DNA testing be used in pregnancies with isolated fetal omphalocele? Preliminary evidence from cytogenetic results of prenatal cases.

Objective: To evaluate whether cell-free DNA (cfDNA) testing could replace an invasive procedure in pregnancies with isolated fetal omphalocele.Study design: This was a retrospective study of all pregnancies with sonographically detected fetal omphalocele at three tertiary referral centers between 2012 and 2016. Invasive diagnostic testing was performed for genetic investigations using conventional karyotyping or chromosomal microarray. cfDNA testing was assumed to be offered to patients with isolated fetal omphalocele for screening for common aneuploidies.Results: Invasive genetic testing was performed in a total of 107 pregnancies with a fetal omphalocele. Abnormal karyotype was found in 66% (31/47) of nonisolated omphalocele cases and in 1.7% (1/60) of isolated omphalocele cases. No pathogenic copy number variations (CNVs) were detected in 59 cases with isolated omphalocele and normal karyotype. If cfDNA screening was used in cases with isolated omphalocele, the affected fetus with trisomy 18 would be detected, and no rare chromosomal aberrations or submicroscopic pathogenic CNVs would be missed.Conclusions: cfDNA testing could be recommended for prenatal genetic evaluation in pregnancies with isolated fetal omphalocele after thorough pretest counseling.Key Message: A very low percentage of aneuploidies and rare chromosomal/subchromosomal abnormalities are found in prenatal cases of isolated omphalocele. It seems that for pregnancies with isolated omphalocele, cfDNA testing represents an alternative for patients who choose to continue the pregnancies and are reluctant to undertake invasive diagnostic testing.

Identification of the linkage of a 1.357 KB beta-globin gene deletion and A gamma-globin gene triplication in a Chinese family.

The 1.357 kb beta-globin gene deletion was identified in a Chinese family by multiplex ligation-dependent probe amplification (MLPA) followed by gap-polymerase chain reaction (gap-PCR) and sequencing. Interestingly, this form of the deletion was linked to a -(G)gamma-(AG)gamma-(A)gamma triplication. The proband, a compound heterozygote for this linked mutant gene and a beta-globin gene [-28 (A>G)] mutation, had a phenotype of beta-thalassemia intermedia (beta-TI). She was not transfusion dependent and had the following parameters: a Hb level of 5.3 g/dL, 72.8% Hb F and 55.1% (G)gamma chain in Hb F. Four members of the family, who were carriers of this linked mutant gene, had a hematological phenotype of beta(0)-thalassemia (beta(0)-thal) with high Hb F and low (G)gamma chain values. RNA analyses showed decreased levels of beta-globin mRNA and increased levels of gamma-globin mRNA in heterozygotes. Haplotype analyses indicated that the unusual form of the beta-globin gene deletion and gamma-globin gene triplication in cis were linked to halotype [+ - - - - - -].

[Analysis of the phenotype-genotype relationship of Hb Constant Spring].

OBJECTIVE: To analyze the genotype-phenotype correlations in the Hb Constant Spring (HbCS) carriers, and to investigate the effect of HbCS on hematologic parameters. METHODS: Complete blood cell count and hemoglobin electrophoresis analyses were performed in 125 HbCS cases. The α-and ß-thalassemia mutations were determined by reverse dot-blotting and Gap-PCR. RESULTS: The presence of the SEA deletion or Hb Quong Sze (HbQS) with HbCS leads to HbH-CS disease. There was significant difference between HbH-CS and αCSα/-α, HbH-CS and αCSα/αα in the hematological parameters. The genotype of αCSα/-α or αα/αCSα had slight effect on hematological parameters. When the Hb Constant Spring mutation co-existed with heterozygous ß-thalassemia, the hematological characteristics of ß-thalassemia was presented. Only 57.6% of carriers with HbCS were detected by hemoglobin electrophoresis. CONCLUSION: The cases with co-existence of HbCS trait and other α-thalassemia trait, or ß-thalassemia trait, showed variation in their red blood cell parameters. For such compound heterozygotes for HbCS and other α- or ß-thalassaemia mutations, which were usually misdiagnosed in clinical screening by hemoglobin electrophoresis, accurate diagnose can be made by molecular diagnosis.

[Analysis of hematological characteristics on the 79 co-inheritance of α-thalassemia and ß-thalassemia carriers in Guangxi].

OBJECTIVE: To investigate the hematological characteristics of co-inheritance of α-thalassemia (α-thal) and ß-thalassemia (ß-thal) and to survey the incidence of co-inheritance of α-thal and ß-thal in Guangxi. METHODS: DNA samples from 370 primary and middle school students who were ß-thal carriers in Guangxi were further processed for the α-goblin gene mutation screening, and were grouped based on the genotype of ß- and α-goblin gene. The hematological indexes to the different groups were compared by One-way ANOVA. RESULTS: Of the total 370 ß-thal carriers, 79 were found to carry α-thal, which gave a frequency of 21.35% for ß-thal carriers and 1.36% for coincidence of these two common disorders in the local population. As expected, the 79 patients presented very variable α-globin alterations in combination with ß-globin mutations, showing 31 genotype combined with the coincidence of both Hb disorders. Except the genotypes of 3 ß-thal heterozygotes combined with ααα(anti3.7) triplication and 2 ß-thal carriers with IVS-II-654(C→T)/N combined-α(3.7)/αα presented the phenotype of thalassemia intermedia, and other 74 carriers with co-inheritance of α-thal and ß-thal all presented the phenotype of ß-thal trait. There were significant differences between ß-thal heterozygotes and the carriers with a co-inheritance of both ß+α(0) thal in MCH, MCV and Hb. In addition, there existed significant difference between the carriers with a co-inheritance of both ß+α(+) thal and a co-inheritance of both ß+α(0) thal in MCV, MCH and Hb. CONCLUSION: Compared to that of ß-thal heterozygotes, the carriers with a co-inheritance of α-thal and ß-thal had slighter phenotype with hematological characteristics. It's difficult to distinguish the double heterozygotes with the co-inheritance of α-thal and ß-thal from ß-thal heterozygotes by hematological indexes, the molecular diagnosis should be performed.