Molecular spectrum and prevalence of thalassemia investigated by third-generation sequencing in the Dongguan region of Guangdong Province, Southern China.

BACKGROUND: PCR, Sanger sequencing and NGS are often employed for carrier screening of thalassemia but all of these methods have limitations. In this study, we evaluated a new third-generation sequencing-based approach termed comprehensive analysis of thalassemia alleles (CATSA) to explore the prevalence of thalassemia in the Dongguan region of southern China. METHODS: 19,932 subjects were recruited for thalassemia screening and hemoglobin testing was performed for each of them. Routine PCR was performed for all the hemoglobin testing-positive subjects and CATSA was conducted for randomly selected subjects from hemoglobin testing-positive and negative subjects. RESULTS: In the 2716 subjects tested both by PCR and CATSA, 2569 had the same results and 147 had discordant results between the two methods. Sanger sequencing, specially designed PCR and MLPA confirmed the results of CATSA were all correct. In total, CATSA correctly detected 787 subjects with variants while routine PCR correctly detected 640 subjects with variants. CATSA yielded a 5.42% (147 of 2716) increment compared with routine PCR. In the 447 hemoglobin testing-negative subjects, CATSA identified pathogenic variants in 12 subjects. Moreover, CATSA identified a novel deletion (chr16:171262-202032) in the α-globin gene cluster. As a result, the deduced carrier frequency of α-thalassemia,ß-thalassemia and α-/ß-thalassemia was 5.62%, 3.85% and 0.93%, respectively. CONCLUSIONS: Our study demonstrated CATSA was a more comprehensive and precise approach than the routine PCR in a large scale of samples, which is highly beneficial for carrier screening of thalassemia. It provided a broader molecular spectrum of hemoglobinopathies and a better basis for a control program in Dongguan region.

Novel KCNJ16 variants identified in a Chinese patient with hypokalemic metabolic acidosis.

BACKGROUND: Biallelic pathogenic variants in the KCNJ16 gene result in hypokalemic tubulopathy and deafness (HKTD) (MIM #619406), which is a rare autosomal recessive disease characterized by hypokalemic tubulopathy with renal salt wasting, disturbed acid-base homeostasis, and sensorineural deafness. Currently, nine individuals with HKTD have been reported, and seven pathogenic variants in KCNJ16 have been revealed. METHODS: A 5-year-6-month-old Chinese female patient displayed hypokalemic metabolic acidosis, salt wasting, renin-angiotensin-aldosterone system (RAAS) activation, arrhythmia, myocardial damage, cardiogenic shock and secondary diffuse brain oedema. Trio-based whole-exome sequencing (WES) was applied to detect the genetic cause. RESULTS: Novel compound heterozygous variants, c.190A>C (p.Thr64Pro) and c.628C>G (p.His210Asp), in KCNJ16 were detected in the patient, and these variants were inherited from the patient's mother and father, respectively. Then, we systematically reviewed the available clinical manifestations of individuals with HKTD. We found that HKTD patients are at risk of cardiogenic shock and secondary diffuse brain oedema, which urges clinicians to make early diagnoses with prompt treatments. CONCLUSION: These findings expand the variant spectrum of KCNJ16, enrich the clinical characteristics of HKTD, and provide a solid base for the genetic counseling, diagnosis and treatment of this condition.

[Results of carrier screening for Spinal muscular atrophy among 35 145 reproductive-aged individuals from Dongguan region].

OBJECTIVE: To carry out carrier screening for Spinal muscular atrophy (SMA) in reproductive-aged individuals from Dongguan region and determine the carrier frequency of SMN1 gene mutations. METHODS: Reproductive-aged individuals who underwent SMN1 genetic screening at the Dongguan Maternal and Child Health Care Hospital from March 2020 to August 2022 were selected as the study subjects. Deletions of exon 7 and 8 (E7/E8) of the SMN1 gene were detected by real-time fluorescence quantitative PCR (qPCR), and prenatal diagnosis was provided for carrier couples by multiple ligation-dependent probe amplification (MLPA). RESULTS: Among the 35 145 subjects, 635 were found to be carriers of SMN1 E7 deletion (586 with heterozygous E7/E8 deletion, 2 with heterozygous E7 deletion and homozygous E8 deletion, and 47 with sole heterozygous E7 deletion). The carrier frequency was 1.81% (635/35 145), with 1.59% (29/1 821) in males and 1.82% (606/33 324) in females. There was no significant difference between the two genders (χ² = 0.497, P = 0.481). A 29-year-old woman was found to harbor homozygous deletion of SMN1 E7/E8, and was verified to have a SMN1∶SMN2 ratio of [0∶4], none of her three family members with a [0∶4] genotype had clinical symptoms. Eleven carrier couples had accepted prenatal diagnosis, and one fetus was found to have a [0∶4] genotype, and the pregnancy was terminated. CONCLUSION: This study has determined the SMA carrier frequency in Dongguan region for the first time and provided prenatal diagnosis for carrier couples. The data can provide a reference for genetic counseling and prenatal diagnosis, which has important clinical implications for the prevention and control of birth defects associated with SMA.

A stepwise haematological screening and whole-exome sequencing reveal multiple mutations from SUPT5H causing an elevation of Hb A2 from a cohort of 47336 individuals.

INTRODUCTION: Though an increase in Hb A2 is one of the most key markers of ß-thal carriers, a few independent cases are reported to show elevated Hb A2 levels caused by mutations in other genes beyond ß-globin gene. METHODS: We reviewed the haematological indices of 47336 individuals to analyse the phenotype-genotype correlation and identified 1439 individuals (3.04%) positive in the elevation of Hb A2 . Globin and KLF1 genes analysis was performed, and further whole-exome sequencing was carried to dissect the genetic causes of those positive samples without ß-thalassemic or KLF1 mutations. RESULTS: Of these 1439 individuals with elevated Hb A2 , 1381 had a molecular defect in globin genes, and most were ß-thalassemic mutation; 10 had a molecular defect in KLF1 gene. Finally, among the 38 individuals without ß-thalassemic or KLF1 mutations, 7 were identified to carried a loss-of-function mutation in SUPT5H. CONCLUSION: This study has provided a mutation spectrum of SUPT5H in a cohort screening leading to the elevation of Hb A2 . According to the previous observations that individuals with a combination of ß-thal mutation and a SUPT5H variant might present moderate ß-thaelassemia, these findings emphasized the importance of comprehensive molecular diagnosis to prevent birth defects of ß-thaelassemia caused by rare mutations from modifier genes.

Case report: A de novo RASopathy-causing SHOC2 variant in a Chinese girl with noonan syndrome-like with loose anagen hair.

Pathogenic variants in the RASopathy-causing SHOC2 gene have been suggested to cause Noonan syndrome-like with loose anagen hair (NS/LAH). This condition is characterized by facial features resembling Noonan syndrome (NS), short stature, growth hormone deficiency (GHD), cognitive deficits, cardiac defects, and ectodermal abnormalities, including easily pluckable, sparse, thin, slow-growing hair, hyperpigmented skin and hypernasal voice. The mutation spectrum of SHOC2 is narrow, and only 8 pathogenic variants have been identified. Here, we report a 5-year-3-month-old Chinese female who displays characteristics typical of NS and has normal neurodevelopment. Trio-based whole-exome sequencing (WES) revealed a de novo variant (c.1231A>G, p.Thr411Ala) in SHOC2. This variant has been recently reported in one subject in the literature who displayed facial features typical of NS and also presented with significant speech delays, moderate intellectual disabilities, epilepsy, bilateral sensorineural deafness and renal dysplasia. The differential phenotypes between these subjects deserve to be further investigated. Next, we reviewed the clinical pictures of NS/LAH and noticed that a recurrent SHOC2 Ser2Gly variant was more likely to result in delayed neurodevelopment and short stature, compared to other SHOC2 variants. And growth hormone (GH) therapy could improve height prognosis. It was noticed that the slight sleep problems and friendly and relatively mature personality observed in our patient may be a novel phenotype of NS/LAH. Our study reconfirms the pathogenic nature of the SHOC2 Thr411Ala variant. It also provides insights into the genotype-phenotype relationship in NS/LAH and a foundation for its genetic counseling, diagnosis and treatment.

Three Novel De Novo ZEB2 Variants Identified in Three Unrelated Chinese Patients With Mowat-Wilson Syndrome and A Systematic Review.

Background: ZEB2 gene mutations or deletions cause Mowat-Wilson syndrome (MWS), which is characterized by distinctive facial features, global developmental delay, intellectual disability, epilepsy, friendly and happy personalities, congenital heart disease, Hirschsprung disease and multiple congenital anomalies. Currently, more than 300 MWS patients have been described in the literature, and nearly 280 variants in ZEB2 have been identified. Methods: In this study, we report three unrelated Chinese patients presenting multiple congenital anomalies that were consistent with those of MWS. Whole-exome sequencing (WES) was used to identify the causative variants. Results: WES identified two novel de novo frameshift variants in ZEB2 (NM_014795.4:c.2136delC, p. Lys713Serfs*3 and c.2740delG, p. Gln914Argfs*16) in patients 1 and 2, respectively, and a novel de novo splicing variant in ZEB2 (NM_014795.4:c.808-2delA) in patient 3, all of which were confirmed by Sanger sequencing. Next, we systematically reviewed the clinical characteristics of Chinese and Caucasian MWS patients. We revealed a higher incidence of constipation in Chinese MWS patients compared to that previously reported in Caucasian cohorts, while the incidence of Hirschsprung disease and happy demeanor was lower in Chinese MWS patients and that epilepsy in Chinese MWS patients could be well-controlled compared to that in Caucasian MWS individuals. Conclusion: Our study expanded the mutation spectrum of ZEB2 and enriched our understanding of the clinical characteristics of MWS. Definitive genetic diagnosis is beneficial for the genetic counseling and clinical management of individuals with MWS.

Analysis of tissue from pregnancy loss and aborted fetus with ultrasound anomaly using subtelomeric MLPA and chromosomal array analysis.

OBJECTIVE: To evaluate the incidence and types of chromosomal abnormalities in pregnancy loss and aborted fetuses with anomaly and compare the performance of subtelomeric MLPA and chromosomal microarray analysis (CMA) in these specimens. METHODS: Samples were collected from spontaneous miscarriages, stillbirths and aborted fetuses with anomaly between January 2015 and April 2019. Chromosomal abnormalities were detected using subtelomeric MLPA and CMA. RESULTS: Among the 172 miscarriage samples, CMA detected pathogenic chromosomal abnormalities in 88 cases. MLPA could identified all aneuploidies and most pathogenic CNVs, missing all polyploidies; Of the 30 stillbirths, one pathogenic CNV and two VOUS were identified by CMA, all of which were missed from MLPA; Of the 135 aborted fetuses with anomaly, CMA identified pathogenic chromosomal abnormalities in 32 fetuses (23.7%); 18.95% in fetuses with isolated, and 35% in fetuses with multiple anomalies. MLPA can identify all aneuploidies but missing most pathogenic CNVs. CONCLUSION: Our systematical comparison of subtelomeric MLPA and CMA for chromosomal analysis of tissue from pregnancy loss and aborted fetuses with anomaly is useful for assessing clinical utility of these techniques. MLPA screening, coupled with CMA analysis, is a cost-effective approach to detect chromosomal abnormalities in miscarriage and anomalous fetuses. However, MLPA might not be appropriate for chromosome analysis in stillbirth without structural anomaly; further research with more samples is needed.

Polymorphisms of α-Globin Genes Compromise Polymerase Chain Reaction-Based α-Thalassemia Genotyping in Three Chinese Families.

In practice, gap-polymerase chain reaction (gap-PCR) and reversed dot-blot are the two most frequently used molecular diagnostic methods for α-thalassemia (α-thal) genotyping. Here, we describe three Chinese individuals from three unrelated families in whom a polymorphism on the α-globin gene cluster led to diagnostic pitfalls. During general molecular diagnosis of thalassemia, three individuals with unexplained results were found. Blood or chorionic villus samples were collected from these three individuals and their family members. Hematological investigations and genetic tests were performed. In Family 1, a polymorphism of HBA2: c.301-24delinsCTCGGCC at the annealing site of the forward primer used in the PCR-reverse dot-blot assay was identified, leading to allele drop-out during the PCR amplification process. In Family 2, a synonymous mutation of C>T substitution at codon 125 of the α2 gene (HBA2: c.376C>T) was identified, leading to the failure of PCR-reversed dot-blot for the HBA2: c.377T>C (Hb Quong Sze or Hb QS) mutation. In Family 3, the size of the PCR fragment from the α2-globin allele carrying the HBA2: c.-771_-428del mutation was smaller and nearly equal to the size of the fragment corresponding to the -α4.2 (leftward) deletion; we also found that the HBA2: c.-771_-428del mutation was linked to a known HBA1: c.-673A>G mutation in this family. In conclusion, diagnostic errors may be caused by technical pitfalls or inherent properties of the DNA sample. All logical steps should be taken to monitor and thus preclude such events.

Hb H Disease Results from Compound Heterozygosity of - -SEA and -αMAL3.5 in a Chinese Family.

The α+-thal deletion of 3.557 kb (NG_000006.1: g.32745_36301del, -αMAL3.5), involving the entire α2-globin gene, was identified in a Chinese family by multiplex ligation-dependent probe amplification (MLPA) followed by gap-polymerase chain reaction (gap-PCR) and sequencing. The proband, a compound heterozygote for this mutant gene and the Southeast Asian (- -SEA; NG_000006.1: g.26264_45564del19301) deletion, had a phenotype of Hb H disease [hemoglobin (Hb) 7.6 g/dL, mean corpuscular volume (MCV) 60.0 fL, Hb H (ß4) 0.7%, Hb Bart's (γ4) 2.4% and Hb A2 1.1%]; one of her sisters with same genotype showed a similar phenotype. Another two family members, who were carriers of this mutant gene, had a hematological phenotype of a silent α-thal. The 5' and 3' breakpoints of this deletion are located at the Y2 and Y1 boxes, respectively, therefore, it probably originated from an unequal crossover between these two homologous boxes. This mutation constitutes an additional heterogeneous defect causing α-thal in the Chinese population and would be valuable for elucidating the arrangement in the human α-globin gene cluster.

[Genetic mutation screening of glucose-6-phosphate dehydrogenase deficiency in Dongguan district].

OBJECTIVE: To determine the incidence and genotypes of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Dongguan region of Guangdong Province and assess the efficacy and feasibility of flow-through hybridization. METHODS: Peripheral blood samples were randomly selected and detected by modified G6PD/6PGD ratio method. Flow-through hybridization was used to detect 14 G6PD mutations among all samples. RESULTS: In total 1005 samples were collected, the detection rate for modified G6PD/6PGD ratio method and flow-through hybridization were 2.79% and 20.90%, respectively. The consistency of the two methods was poor(Kappa=0.187). When c.1311C>T mutation is excluded, the consistency of the two methods was good for males (Kappa=0.952) but still poor for females (Kappa=0.194). The most common mutations were c.1376G>T, c.1388G>A and c.95A>G. No G6PD deficiency was found among those only carrying the c.1311C>T mutation. CONCLUSION: Flow-through hybridization can simultaneously detect 14 loci, covering over 90% of common mutations in Chinese population, and can be easily expanded. The routine method may miss many females carrying homozygous, compound heterozygous and heterozygous mutations, but the detection rate for male hemizygous mutation was much higher.

[Analysis of the cause of pregnancy failure with combined MLPA assay for subtelomeric regions and ultrasonography].

OBJECTIVE: To explore the value of multiplex ligation-dependent probe amplification (MLPA) for the detection of chromosome abnormalities in miscarriage tissues, and to correlate the result with ultrasound findings. METHODS: A total of 421 cases of spontaneous abortions and fetal deaths were detected with the MLPA method. RESULTS: Among the 421 samples, 232 (55.11%) had an abnormal MLPA result. For the 286 cases derived from < 13 weeks pregnancy, 206 (72.03%) were abnormal. For the 49 cases from 14-19 weeks pregnancy, 14 (28.57%) were abnormal. For the 86 cases derived after 20 weeks pregnancy, 12 (13.95%) were abnormal. Among the 117 cases with abnormal ultrasound findings, 33 cases (28.21%) had an abnormal MLPA result, 28 out of the 33 cases were numerical chromosome abnormality, 4 cases were chromosome microdeletion and/or micro duplication, 1 case had both numerical abnormality and microduplication. For those with abnormal ultrasound findings for the neck region, fetal edematous syndrome, multiple malformations and digestive system, the detection rates for MLPA were 71.4%, 58.8%, 37.8%, and 9.1%, respectively. For those with abnormal finding of cardiac system, nervous system, face, skeletal system and urinary system, none was found with positive results of MLPA. CONCLUSION: Numerical chromosomal abnormalities account for the majority of cases with spontaneous abortion. With the increase of gestational age, the occurrence of chromosomal abnormalities gradually declines. Combined ultrasound and MLPA assay can improve the detection rate and accuracy for chromosomal abormalities.