[Genotype-phenotype relationship and genetics study of 115 cases with Wilson's disease].

Objective: To explore the genotype-phenotype relationship of Wilson's disease (WD) and further study the mutation spectrum in the ATP7B gene. Methods: The clinical data and genetic test results of 115 cases with WD diagnosed in the First Affiliated Hospital of Zhengzhou University from 2015 to 2022 were retrospectively analyzed. The rank sum test was used for quantitative data comparison, and χ(2) test was used for count data comparison. Multivariate logistic regression was used to analyze the relationship between patients' genotype and phenotype. Results: The onset of liver manifestations (hepatic type) accounted for 60.9%, neurological symptoms (cerebral type) for 13.0%, and mixed hepato-cerebral symptoms for 26.1%. Presymptomatic individuals (hepatic types) accounted for 62.9%. Next-generation sequencing- diagnosed WD cases accounted for 87.8%. Combined multiplex ligation-dependent probe amplification assay-diagnosed WD cases accounted for 89.6%. A single case with a detected pathogenic locus accounted for 10.4%. The diagnostic rate of WD by genetic testing combined with clinical data was 100%. A total of 76 ATP7B mutations were detected, and the top three mutation frequencies were c.2333G>T (p.Arg778Leu) (30.7%), c.2975C>T (p.Pro992Leu) (7.3%), and c.2621C>T (p.Ala874Val) (6.4%). The mutations were mainly distributed in exons 8, 11-13, and 15-18, accounting for more than 90% of the total mutations. Eight new mutations were found, including c.3724G>A (p.Glu1242Lys), c.3703G>C (p.Gly1235Arg), c.3593T>C (p.Val1198Ala), c.2494A>C (p.Lys832Gln), c.1517T>A (p.Ile506Lys), c.484G>T (p.Glu162Ter), c.1870-49A>G, and the missing of exons 10-21. Liver histopathology showed cellular edema, degeneration, inflammation, and necrosis, as well as a 42.8% copper staining positive rate. Genotype-phenotype analysis showed that the p.Arg778Leu mutation had higher alanine aminotransferase (ALT) levels than those carrying other mutations (P=0.024), while the homozygous mutation of p.Arg778Leu was associated with cerebral-type patients (P=0.027). Conclusion: Genetic testing plays an important role in the diagnosis of WD. p.Arg778Leu is the first high-frequency mutation in the Chinese population, and patients carrying it have higher ALT levels. The p.Arg778Leu homozygous mutation is prone to causing cerebral-type WD. This study expands the ATP7B gene mutation spectrum.

[Novel frameshift mutations in SALL4 in two Chinese families with Okihiro syndrome].

In the present study, clinical manifestations of two Chinese Okihiro syndrome families were analyzed, and genetic detections were performed on the two probands by exome sequencing and verified by Sanger sequencing for family members to determine the biological pathogenesis. Prenatal diagnoses were provided for three high-risk fetuses. The affected members exhibited a wildly spectrum of phenotypes, including ultrasound abnormalities of skeletal system (radius deformity and abnormal posture), and cardiac system (persistent common arterial trunk and ventricular septal defect) in the prenatal period of family 1, the severe phenotypes (grossly shortened and deformed forearm, Duane's anomaly and hearing loss), and the mild ones (usually only thenar dysplasia, or short radius styloid process). Two SALL4 variants, c.844delC p.(Q282Kfs*8) and c.2210delG p.(G737Vfs*23), have been identified respectively in two probands, and c.2210delG of SALL4 gene was unreported previously. The two variants were verified in all affected individuals, not in normal family members. Genotyping results of three fetuses indicated that one fetus was normal, and the two fetuses with heterozygous variation were affected. The two variants of SALL4 gene, c.844delC p.(Q282Kfs*8) and c.2210delG p.(G737Vfs*23), were the molecular pathological cause of Okihiro syndrome in the present study and enriched the spectrum of SALL4 variants. Our study provides accurate prenatal genetic diagnosis for the two families to avoid the birth of affected children.

[Analysis of the factors influencing positive predictive value of noninvasive prenatal testing for chromosome aneuploidies].

Objective: To investigate the influence of Z-score and different risk factors on positive predictive value (PPV) of noninvasive prenatal testing (NIPT) for chromosome aneuploidies. Methods: A total of 81 838 NIPT samples from January 1, 2016 to May 31, 2021 in the First Affiliated Hospital of Zhengzhou University were retrospectively analyzed. Invasive prenatal diagnosis was applied to verify the diagnosis of NIPT-positive results and the corresponding PPV was calculated. The PPV of the samples with different Z-score were compared. The women were divided into high-risk group and non-high-risk group: high-risk group (n=39 114) included those with ultrasound soft index abnormalities, advanced maternal age or high risk for maternal serum screening, while non-high-risk group (n=42 724) included those with intermediate risk for maternal serum screening or no indications. The differences of the PPV between these two groups were compared. Finally, the comprehensive influence of Z-score and different risk factors on PPV were analyzed. Results: A total of 471 high-risk cases were detected by NIPT results, including 362 cases of trisomy 21, 77 cases of trisomy 18 and 32 cases of trisomy 13. For trisomy 21, trisomy 18 and trisomy 13, there were 226 cases, 46 cases and 6 cases which were confirmed via invasive prenatal diagnosis respectively. The corresponding PPV were 79.3% (226/285), 82.1% (46/56) and 27.3% (6/22), respectively. PPV of trisomy 21 and trisomy 18 were positively correlated with the corresponding Z-score (r=0.92, 0.62, all P<0.05), while trisomy 13 could not be analyzed due to the small sample size. The PPV of high-risk group was 85.2% (207/243), which was higher than that of the non-high-risk group with PPV of 59.2%(71/120, χ2=30.30, P<0.01). When the Z-score was between 3-<4 and 4-<5, the PPV of the high-risk group were 46.2%(12/26)and 62.5%(15/24) respectively, which were higher than those of the non-high-risk group [16.0%(4/25) and 14.3%(3/21), χ2=4.10, 8.90, all P<0.05]. With the increase of Z-score, there was no significant difference in PPV between the two groups (all P>0.05). Conclusions: The PPV of trisomy 21 and trisomy 18 are positively correlated with Z-score. The PPV of high-risk group is higher than that of non-high-risk group. The combination of Z-score and other risk factors may provide more accurate genetic counseling for those with NIPT positive results.

[Analysis of genetic variation in patients with Waardenburg syndrome type â ¡ by next generation sequencing].

Objective: To detect gene mutation sassociated with deafness in four Waardenburg syndrome (WS) type Ⅱ patients, and to explore the possible mechanism of molecular genetics. Methods: All patients with WS were identified at the genetic and prenatal diagnosis center of the First Affiliated Hospital of Zhengzhou University from August 2015 to December 2018.Clinical materials and peripheral blood were collected from patients and family members. The genes associated with deafness of the patients were tested by next generation sequencing(NGS). And suspected mutations were verified by Sanger sequencing. Results: All patients carried heterozygous mutations in SOX10, they were c.355_356insTCAGGCAGCGC, c.1106_1107insTGGGGCCCCCCACACTA, c.511T>C (p.Y171H), c.91_100del. According to the guidelines for genetic variation of the Amercian College of Medical Genetics and Genomics (ACMG), three frameshift mutations were pathogenic mutations, one missense mutation was likely pathogenic mutation. Conclusion: Application of next generation sequencing technologies make gene diagnosis of Waardenburg syndrome efficiently and accurately.

[Confirmation and analysis of 2 398 positive results of cell-free fetal DNA].

Objective: To explore the clinical application value and accuracy of cell-free fetal DNA (cff-DNA) technique in prenatal screening. Methods: The results of quantitative fluorescent PCR (QF-PCR) and karyotype of amniotic fluid cells were analyzed retrospectively in 2 398 monocyesis pregnant women who had been amniocentesis at the First Affiliated Hospital of Zhengzhou University from May 2013 to December 2019, and the results of 359 cases who had been examined by single-nucleotide polymorphism array (SNP array). Results: Cff-DNA test of 2, 398 cases indicated 987 cases of trisomy 21, 351 cases of trisomy 18, 135 cases of trisomy 13, 566 cases of sex chromosome abnormality, and 359 cases of other chromosome abnormality. Chromosome karyotype analysis detected 826 cases of trisomy 21, 213 cases of trisomy 18, 17 cases of trisomy 13, 221 cases of sex chromosome abnormality, and 26 cases of other chromosome abnormality. The detection rate were 83.69% (826/987), 60.68% (213/351), 12.59% (17/135), 39.04% (221/566) and 7.24% (26/359), respectively. QF-PCR detected 1 046 cases of trisomy and 188 cases of sex chromosomes abnormality, and the detection rate was 99.05% (1 046/1 056) and 85.07% (188/221), respectively. Compared with the abnormal number detected by chromosome karyotype analysis, 10 cases of trisomeric chimerism and 24 cases of sex chromosome were missed by QF-PCR. Among the 359 other chromosomal abnormalities detected by SNP array, 64 cases were consistent with the results of cff-DNA, and the detection rate was 17.83% (64/359), which was 10.59% higher than the karyotype result. Conclusions: Karyotype analysis is the gold standard for diagnosing chromosomal abnormalities. QF-PCR could diagnose common chromosome aneuploidy rapidly and accurately, and it could be used as an auxiliary detection technique for karyotype analysis. The incidence of sex chromosome chimerism is high, so missed diagnosis should be warned. SNP array could be given priority to verify chromosome microdeletion or microduplication detected by cff-DNA.

[The efficacy of immunoadsorption with Infliximab theraepy on the modulation of disease activity in patients with severe rheumatoid arthritis].

Objective: To evaluated the efficacy of additional immunoadsorption therapy (2 times) besides infliximab (IFX) ondisease remission in patients with severe rheumatoid arthritis (RA). Methods: 90 patients with serve RA were included in this study.There were 43 patients in the control group who were treated with IFX 3 mg/kg+ methotrexate (MTX) therapy, and other 47 patients were experimental group, who were previous given 2 times additional immunoadsorption therapy before IFX 3 mg/kg+ MTX therapy.IFX 3 mg/kg was infused at weeks 0, 2, 6, 14, 22 and 30.Age, sex ration, mean disease duration and core index of disease activity in two treatment groups were collected at weeks 0, 2, 6 and 30 weeks to compare the efficacy and safety of combined immunosorbent therapy in the treatment of severe RA. Results: The baseline age, sex ration and core indexes of disease activity were comparable between the two treatment groups (P>0.05). After treatment, the core indexes of disease activity of all patients decreased significantly compared with their baseline levels (P<0.05) and the difference of sustainable maintenance to 30 weeks (P<0.05). After 2 and 6 weeks of treatment, patients' ACR20 remission rates of the experimental group were 46.81% and 68.08%, significantly higher than the control group; after 30 weeks of treatment, patients' ACR20 remission rates of the experimental group was more than 90%, while the number was 79.07% in the control group.At the same time DAS28-ESR clinical remission and low disease activity also reached 72.34% in the experimental group, higher than the control group(P<0.05). Conclusion: Additional immunoadsorption therapy can rapid relive the disease activity of serve RA patients, and the remission rate of 30W was significantly higher than only IFX treatment.

[Application of single nucleotide polymorphism array in prenatal diagnosis for fetuses with abnormal ultrasound findings].

Objective: To investigate the value of single nucleotide polymorphism array (SNP-array) for fetuses with abnormal ultrasound findings. Method: A total of 904 fetuses with abnormal ultrasound findings were enrolled in this study from May 2015 to November 2017, and 434 (48.0%) cases received conventional karyotyping analysis at the same time. According to different abnormal ultrasound category, 904 cases were divided into 5 groups: 280 cases (31.0%) in single system structural anomalies, 31 cases (3.4%) in multiple system structural anomalies, 331 cases (36.6%) in single ultrasound soft marker abnormalities without structural anomalies, 107 cases (11.8%) in multiple soft marker abnormalities and 155 cases (17.2%) in structural abnormalities combined with soft markers abnormalities. Abnormal detection rates by SNP-array among 5 groups of abnormal ultrasound category were calculated. Result: (1) Total SNP-array results: 171 (19.0%) cases out of 904 cases analyzed by SNP-array, presented chromosomal abnormalities. Pathogenic copy number variants were detected in 27 cases (3.0%) and variants of unknown significance were detected in 81 cases (7.8%) . In addition, 7 cases (26.0%) were found with new mutation by parental validation. (2) SNP-array of 5 groups: among the 5 groups of abnormal ultrasound category, chromosomal abnormalities were identified by SNP-array in 19.3% (54/280) with single system structural abnormalities, 25.8% (8/31) with multiple system structural abnormalities, 13.9% (46/331) with single nonstructural anomalies, 19.6% (21/107) with multiple nonstructural anomalies and 27.1% (42/155) with structural abnormalities combined with nonstructural anomalies. The differences were significant (P=0.010) . No chromosome abnormalities was identified in single soft marker abnormalities, such as choroid plexus cysts, echogenic foci in the heart, single umbilical artery and pyelectasis. (3) Chromosomal abnormalities: the abnormal detection rate of aneuploidy chromosomal abnormalities by SNP-array increased with the maternal age, decreased with the gestational weeks (all P<0.05) . However, the pathogenic copy number variants and variants of unknown significance rates did not change with maternal age and gestational weeks (all P>0.05) . (4) SNP-array and karyotyping: 434 cases were analyzed by conventional karyotyping and SNP-array respectively, 10.3% (43/419) of which presented chromosomal abnormalities by conventional karyotyping and 18.7% (81/434) of which presented chromosomal abnormalities by SNP-array. Conclusions: SNP-array could be a useful genetic analysis method in prenatal diagnosis for fetuses with abnormal ultrasound findings. For different abnormal ultrasound category, SNP-array has different detection rate. Compared with conventional karyotyping analysis, SNP-array can improve the detection rates for chromosomal abnormalities and find the chromosome abnormalities which can't be detected by conventional karyotyping analysis. In clinical prenatal genetic counseling, SNP-array should be selected rationally in combination with the various abnormal ultrasound category.

[Analysis of non-invasive prenatal screening detection in fetal chromosome aneuploidy].

Objective: To evaluate the efficacy of non-invasive prenatal screening (NIPS) in the detection of fetal aneuploidies. Methods: Cell free DNA was sequenced in 5 566 pregnant women to identify the fetal aneuploidies in the First Affiliated Hospital of Zhengzhou University from January 1(st), 2015 to March 15(th), 2016. Among them, 5 230 (93.96%, 5 230/5 566) were singleton pregnancies and 336 (6.04%, 336/5 566) were twin pregnancies. In singleton pregnancies, 1 809 (34.59%, 1 809/5 230) were women with advanced maternal age, and 3 421 (65.41%, 3 421/5 230) were young women. The positive results of NIPS were validated by karyotyping through invasive procedures and neonatal outcomes were followed up by telephone. Results: Among the 5 566 women, 69 (1.24%, 69/5 566) got positive NIPS results, with 66 in singleton pregnancies and 3 in twin pregnancies. Two were monochorionic diamniotic twins and 1 was dichorionic twin pregnancy. The positive predictive value of NIPS for trisomy 21, 18 and 13 were 100.0%, 90.9% and 100.0%, and was 55.6% for sex chromosome aneuploidies. There was no false negative case found during the follow-up. In the advanced maternal age group and young women group, the prevalence rates of fetal chromosomal aneuploidies were 1.11%(20/1 809) and 0.94%(32/3 421), respectively. In the young women with soft markers in fetal ultrasound, the prevalence of fetal chromosomal aneuploidies was 1.44% (7/487), and in serum high risk women, it was 0.94% (7/747). In women with the serum screening risk with cut-off value, 0.89%(9/1 016) had fetal aneuploidies, and the prevalence was 0.77%(9/1 171) in volunteers. There was no statistically significant difference among these groups (P=0.636). Conclusions: There is no difference in the detection rate of fetal aneuploidies between high-risk women in serum screening and volunteers in NIPS. NIPS is more suitable as a first line screening test for women without fetal ultrasound abnormalities. It should be used carefully when there is ultrasound abnormalities.

[Mutation analysis for two hypophosphatasia families with targeted next-generation sequencing].

Objective: To detect the mutations in alkaline phosphatase (ALPL) gene of two Chinese families with perinatal hypophosphatasia (HPP), in order to explore the mechanism of this condition. Methods: Next-generation sequencing (NGS) of osteology system panel was carried out for exome sequencing in the mothers of 2 HPP fetuses, who visited Prenatal Diagnosis Center of the First Affiliated Hospital of Zhengzhou University. Further polymerase chain reaction (PCR) and Sanger sequencing validation was performed in the parents, affected fetuses and 200 unrelated healthy individuals to verify the mutation sites. Results: The mother and father of No.1 family carried ALPL gene c. 333delC (p.Gly112AlafsX10) and c. 568_570delAAC (p.190delAsn) base deletions, respectively. The affected fetus carried compound heterozygotes of the two mutations. Two mutations in ALPL gene known to be associated with hypophosphatasia were found in No.2 family, c. 1250A>G (p.Asn417Ser) in the mother and c. 1166C>A (p.Thr389Asn) in the father, while the fetus was a compound heterozygote carrying both of the two mutations. Both families met the pattern of autosomal recessive inheritance. ALPL gene c. 333delC (p.Gly112AlafsX10) was a novel mutation, and it was not found in the 200 unrelated healthy individuals. Conclusions: The mutations in ALPL gene may be the cause of HPP in the 2 families. NGS technology combined with Sanger sequencing could be an efficient and accurate diagnostic method.

[Mutation screening of 433 families with Duchenne/Becker muscular dystrophy].

OBJECTIVE: Mutation analysis of unrelated families with Duchenne/Becker muscular dystrophy (DMD/BMD) was performed to investigate the characteristic of DMD gene mutation, especially the distribution pattern of point mutation of DMD gene in Chinese population. METHODS: A total of 433 unrelated DMD/BMD families were collected at the Center of Prenatal Diagnosis of the First Affiliated Hospital of Zhengzhou University from March 2010 to December 2014. The deletions or duplications in 79 exons of DMD gene were screened using multiplex ligation-dependent probe amplification (MLPA). Any single-exon deletion detected by MLPA was further validated by PCR amplification. In the 117 unrelated Chinese families in which large-scale deletions and duplications had been excluded by MLPA, the point mutation in 79 exons of DMD gene were tested in the propositus using next-generation sequencing (NGS), and further verified the point mutation using Sanger sequencing. RESULTS: In the 433 unrelated DMD/BMD families, 316 families with DMD deletions/duplications were identified by MLPA. Out of 57 single-exon deletions detected by MLPA, 3 were found as point mutations by PCR and Sanger sequencing, including 2 nonsense mutation (c.1729G>T [p.Glu577X], c. 3346A>T [p.Lys1116X]) and 1 frame-shift mutation (c.8605_8606delGT [p.Val2869ThrfsX25]). Direct sequencing with Ion PGM and Sanger sequencing in 117 families with negative results in MLPA detected 92 different point mutations in 96 families, including 46 novel mutations, 42 previously reported ones, and 4 possible polymorphisms (rs189143447, rs202008454, rs200213555, rs187617705). The 46 novel mutations consisted of 16 nonsense mutations (c.100A>T [p.Lys34X], c. 1201C>T [p.Gln401X], c. 1707C>A [p.Cys569X], c. 1831G>T [p.Glu611X], c. 1912C>T [p.Gln638X], c. 2213C>G [p.Ser738X], c. 3673_3673delA [p.Ile1225X], c. 3774C>A [p.Cys1258X], c. 4858G>T [p.Glu1620X], c. 5764A>T [p.Lys1922X], c. 6035T>G [p.Leu2012X], c. 6408G>A [p.Trp2136X], c. 7717C>T [p.Gln2573X], c. 7864G>T [p.Glu2622X], c. 8184_8185insT [p.Lys2729X], c. 8215C>T [p.Gln2739X]), 5 missense mutations (c.139G>A [p.Gly47Arg], c. 238G>C [p.Ala80Pro], c. 335G>T [p.Trp112Leu], c. 804A>C [p.Leu268Phe], c. 1149G>T [p.Glu383Asp]), 6 splice-site mutations (c.2293-3C>A, c. 2380+ 1G>T, c. 3277-1G>C, c.4519-7A>G, c. 5740-15G>T, c. 7661-1G>C), 16 small deletions (c.688_688delA [p.Met230CysfsX14], c.1760_1791del32 [p.Thr587IlefsX37], c. 2271_2271delA [p.Asp774ThrfsX22], c. 2281_2285delGAAAA [p.Glu761SerfsX10], c. 2527_2527delG [p.Glu843SerfsX3], c. 3405_3405delC [p.Asn1135LysfsX18], c. 4450_4450delC [p.His1484ThrfsX14], c. 4770_4770delA [p.Thr1590ThrfsX5], c. 4937_4937delA [p.Glu1646GlyfsX11], c. 5253_5256delATTA [p.Lys1751LysfsX2], c. 5654_5654delA [p.Gln1885ArgfsX6], c. 7441_7441delG [p.Glu2481AsnfsX13], c. 7860_7860delC [p.Ile2620IlefsX18], c. 8668-8668delG /c.8668+ 1-8668+ 1delG, c. 9009_9009delC [p.Thr3003ThrfsX18], c. 9021_9021delT [p.Ile3007IlefsX14]), and 3 small insertions (c.305_306insG [p.Gly102GlyfsX4], c. 3116_3117insA [p.His1039GlufsX11], c. 9197_9198insATCTC [p.Ser3066SerfsX25]). And 87.4% (83/95) of the pathologic point mutations disrupted the translational reading frame (46 nonsense mutations, 24 frame-shift mutations, and 13 splice-site mutations). CONCLUSIONS: Inexpensive and efficient genetic/prenatal diagnosis of DMD/BMD may be plausible by MLPA analysis, NGS, and Sanger sequencing. Most of the mutations identified in this study led to a predictable premature stop codon or splicing defects, resulting in defective function of dystrophin.

[PTPS gene analysis and prenatal diagnosis in patients with 6-pyruvoyl-tetra hydropterin synthase deficiency].

Objective: To analyze the variations of PTPS gene in patients with suspected 6-pyruvoyl-tetra hydropterin synthase deficiency (PTPSD) and to make prenatal diagnosis in high-risk families. Methods: Chemiluminescence was used for phenylalanine detection in blood or dried blood spots.Patients with phenylalanine concentration over 120 µmol/L were detected by urine pterin analysis, and the activity of dihydropteridine reductase (DHPR) was detected. tetrahydrobiopterin loading tests were performed in suspected patients with abnormal urinary pterin profiles. PTPS gene variation analysis was performed by direct Sanger sequencing based on PCR amplification. Prenatal diagnosis in 7 high-risk families was performed by chorionic villus sampling when the genotype was identified. Results: In 656 patients with hyperphenylalanine, 22 cases were diagnosed as PTPSD clinically. 16 variations were detected in the 22 PTPSD cases. The 5 variations, p.Lys77Arg, p.Ile84Phe, c.315-2A>G, c.244-2A>T, c.187-1G>T, were identified as novel variations. Two fetuses carried the same mutation with the proband and therefore were thought to be PTPSD fetuses. Three fetuses carried only one mutant allele and thus were thought to be PTPSD carriers. The other 2 fetuses carried no mutations and were presumed normal. Conclusions: PTPS gene variation analysis is necessary to confirm the diagnosis. Prenatal diagnosis could help avoiding the defect birth in PTPSD families.

Analysis and application of ATP7B gene mutations in 35 patients with hepatolenticular degeneration.

We investigated the genetic mutations involved in Wilson's disease to improve prenatal genetic diagnosis and presymptomatic diagnosis. The polymerase chain reaction (PCR) was used to amplify the exons and exon-intron boundaries of the ATP7B gene in 35 Wilson's disease pedigrees. The PCR products were further analyzed by Sanger sequencing. Prenatal genetic diagnoses were performed by chorionic villus sampling after the genotypes of parents of the probands were identified. The overall mutation detection frequency was 92.9%. A total of 24 distinct mutations were detected, seven of which are novel: A1291T (c.3871G>A), c.2593_2594insGTCA, c.2790_2792delCAT, c.3661_3663delGGG, c.3700delG, c.4094_4097delCTGT, and IVS6+1G>A. Three mutations, R778L (c.2333G>T) (45.7%), A874V (c.2621C>T) (7.1%), and P992L (c.2975C>T) (7.1%) are relatively frequent. Two presymptomatic patients were detected through familial screening, and they began taking medicine after diagnosis. Of the subjects with Wilson's disease pedigrees who had received a prenatal genetic diagnosis, three fetuses were normal and one was a carrier. Twenty-four distinct mutations were identified, and our knowledge of the population genetics of Wilson's disease in China has therefore improved. For pedigrees with the Wilson's disease, genetic counseling, prenatal diagnosis, and presymptomatic diagnosis by Sanger sequencing and haplotype analysis are feasible.

Mutation analyses and prenatal diagnosis in families of X-linked severe combined immunodeficiency caused by IL2Rγ gene novel mutation.

We investigated the feasibility of interleukin-2 receptor gamma (IL2Rγ) gene based on gene mutation analysis and pre-natal diagnosis of X-linked severe combined immunodeficiency (X-SCID). Blood samples of patients and their parents of X-SCID (family 1) and X-SCID (family 2) were collected. IL2Rγ gene sequences of the 2 families were analyzed using bi-directional direct sequencing by polymerase chain reaction. DNA sequence changes in the IL2Rγ gene exon region and shear zone were also analyzed. We also sequenced the IL2Rγ gene in 100 healthy individuals. Prenatal genetic diagnoses for a high-risk fetus in family 1 were performed by chorionic villus sampling after determining each family's genotypes. The suspect fe-male in family 1 underwent carrier detection. Two novel mutations of IL2Rγ gene were identified, including c.361-363delGAG (p.E121del) in the patient and his mother in family 1, and c.510-511insGAACT (p.W173X) heterozygous mutation in the proband's mother in family 2. These mutations were absent in the 100 controls. Prenatal diagnosis of early pregnancy in the female fetus of family 1 was performed; the fetus was heterozygous, which was confirmed at postnatal follow-up. The suspect female in family 1 showed no mutation in carrier detection. The novel p.E121del and p.W173X mutations in IL2Rγ may have been the primary causes of disease in 2 families with X-SCID. In couples with an X-SCID reproductive history, prenatal gene mutation analysis of IL2Rγ can effectively prevent the birth of children with X-SCID and carrier detection for suspected females.

Prenatal diagnosis of Chinese families with phenylketonuria.

The aim of this study is to investigate the ability to prenatally diagnose phenylketonuria (PKU) by using phenylalanine hydroxylase (PAH) gene mutation analysis combined with short tandem repeat (STR) linkage analysis in 118 fetuses from 112 Chinese families. Genomic DNA was extracted from the peripheral blood from members of 112 families and the exons and exon-intron boundaries of the PAH gene were amplified by PCR. PCR products were analyzed by bi-directional Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). The three variable number of tandem repeat (VNTR) markers PAH-1, PAH-26, PAH-32 were used in the prenatal diagnosis for the PKU families. We identified a spectrum of 63 different mutations, including 61 point mutations and indels, two large exon deletion mutations, and five novel mutations. A substantial proportion of mutant alleles were accounted for by p.R243Q (15.62%), EX6-96AG (9.82%), p.V399V (7.59%), p.Y356X (6.70%), and p.R413P (5.36%). The same mutations were identified in 31 prenatally genotyped fetuses. We identified 58 fetuses that carried only one mutant allele and 29 fetuses that carried no mutations of PAH and were presumed normal. PAH gene mutation analysis combined with STR linkage analysis can provide rapid and accurate prenatal diagnosis for PKU families.

A novel 3-base pair deletion of the CRYAA gene identified in a large Chinese pedigree featuring autosomal dominant congenital perinuclear cataract.

Congenital cataract is caused by reduced transparency of the lens resulting from metabolic disorders during the fetal period. The disease shows great heterogeneity both clinically and genetically. We identified a 4-generation ethnic Han Chinese family affected by autosomal dominant congenital perinuclear cataract. The patients underwent full clinical and ophthalmologic examinations to rule out any concomitant disorders. Blood samples were collected and genomic DNA was extracted. Potential mutations in the candidate gene alpha A crystallin (CRYAA) were screened. Prenatal diagnosis was then provided for a fetus of the affected proband by chorionic villus sampling. In all patients, DNA sequencing of the CRYAA gene revealed a novel 3-bp deletion mutation in exon 3 (c.246_248delCGC), which led to deletion of codon 117 encoding arginine (p.117delR) in the peptide chain. The same mutation was not found among unaffected and healthy individuals. Bioinformatic analysis revealed that although the c.246_248delCGC is an 'in-frame' mutation, removal of arginine resulted in a significant change in the protein structure. The fetus did not possess this mutation and was confirmed to be healthy at 1-year follow-up. A novel disease-causing mutation, c.246_248delCGC (p.117delR), of the CRYAA gene has been identified in a Chinese family with autosomal-type perinuclear congenital cataracts. This is also the first report of prenatal diagnosis of this type of congenital cataract.

Prenatal diagnosis based on HPRT1 gene mutation in a Lesch-Nyhan family.

We explored the feasibility of applying gene diagnosis in prenatal diagnosis by analysis of hypoxanthine-guanine phosphoribosyltransferase-1 (HPRT1) gene mutation in a Chinese Lesch-Nyhan family. A homozygous mutation of p.R170X (c.508C＞T) in HPRT1 gene was detected in the proband, and a heterozygous mutation of p.R170X was detected in his mother. This mutation failed to be found in the 50 unrelated healthy individuals. Prenatal diagnosis indicated that the foetus was male and also carried p.R170X (c.508C＞T) mutation, same as the proband. Parents of the foetus decided termination of pregnancy, and the result of gene analysis for the aborted tissue was consistent with that of prenatal diagnosis. We can see that Lesch-Nyhan syndrome (LNS) is caused by non-sense mutation p.R170X（c.508C＞T）in HPRT1 gene in this family. Prenatal gene diagnosis is a valid strategy to prevent LNS because it can avoid the birth of LNS foetuses.

Mutation analysis and prenatal diagnosis for three families affected by isolated methylmalonic aciduria.

Isolated methylmalonic acidemia (MMA) is a genetically heterogeneous disorder caused mainly by deficiency of methylmalonyl-CoA mutase. In the present study, we analyzed MUT gene mutations in 3 Chinese couples with a birth history of isolated MMA. We also provided prenatal diagnoses for the detected mutation. Exons and exon-intron boundaries of the MUT gene were analyzed by polymerase chain reaction and direct sequencing. Prenatal genetic diagnoses were performed by chorionic villus sampling after the genotypes of parents were determined. Six heterozygous mutations in the MUT gene were identified in the 3 families, including c.1880A>G (p.H627R) and IVS9-1G>A for family 1, c.1741C>T (p.R581X) and c.729insTT (p.D244fX39) for family 2, and c.616C>T (p.Q206X) and c.1280G>A (p.G427D) for family 3. Among these, c.616C>T (p.Q206X), c.1280G>A (p.G427D), IVS9-1G>A, and c.1741C>T (p.R581X) were novel mutations. These mutations were not detected in 100 normal controls. The fetus in pedigree 3 was free of the mutations carried by the parents, while the fetuses in pedigrees 1 and 2 were heterozygous mutation carriers. All 3 families decided to continue with their pregnancies and the neonates did not show any symptoms of MMA after birth. Our results indicated that mutations in the MUT gene are the primary cause of isolated MMA, and that most mutations were novel. For families with early-onset isolated MMA, direct sequencing of the MUT gene is crucial for genetic counseling, prenatal diagnosis, and identification of carriers.

[Clinical features and Y chromosome abnormalities in children with 45, X/46, XY mosaicism].

Objective: To investigate the clinical and genetic characteristics of children with 45, X/46, XY mosaicism. Methods: The retrospective study included 20 children diagnosed with 45, X/46, XY and 45, X/46, X,+mar mosaicism in the First Affiliated Hospital of Zhengzhou University from 2018 to 2022. The clinical features, gonadal pathology, treatment and follow-up were summarized. Genetic tests were performed by SRY gene test, azoospermia factor region (AZF) deletion test, copy number variation-sequencing (CNV-seq). Age at first diagnosis was compared between boys and girls using independent sample t-test. Results: The 20 patients included 3 boys and 17 girls, and the age at first diagnosis were (7.6±5.5) years, it is (2.1±1.9) years in boys, (8.7±5.4) years in girls, significantly younger for boys (t=-3.86, P=0.004). The chief complaint was external genitalia malformation for boys, and short stature (13 cases) and dysplastic external genital for girls (4 cases). Five girls presented with features of Turner syndrome. The gonadal phenotypes included mixed gonadal dysplasia (MGD, 6 cases), complete gonadal dysplasia (CGD, 10 cases), unilateral ovotestis (2 cases), possible ovaries (1 case) and undetermined gonad (1 case). One female with dysplastic genital was reassigned to male, and the gender of the remaining cases remained unchanged. Seven females were treated with recombinant human growth hormone. The height increased by (17±7) cm during the (2.9±1.2) years follow-up. No gonadal malignancy was observed. The karyotype was 45, X/46, XY in 16 cases, and 45, X/46, X,+mar in 4 cases. All of the 4 marker chromosomes were derived from Y chromosome confirmed by CNV-seq. SRY gene was detected in all 20 patients genome, and AZF deletion was found in 7 girls. Conclusions: 45, X/46, XY mosaicism presented with dysplastic external genital or female with remarkable short stature. Gonadal phenotypes included MGD, CGD and ovotestis. AZF microdeletions were found in the majority of female cases.

[Analysis of DMD gene variants in a single center].

Objective: To investigate the DMD genetic variants of the Chinese population with Duchenne (DMD) and Becker muscular dystrophies (BMD). Methods: A cross-sectional study was conducted on 2 690 unrelated patients with DMD and BMD aged 0-18 who visited the Genetic and Prenatal Diagnosis Center of the First Affiliated Hospital of Zhengzhou University from January 2005 to February 2022. The clinical data, such as gender, age, clinical manifestations, and address, were collected. Multiplex ligation-dependent probe amplification, next generation sequencing panel, Sanger sequencing, and PCR amplification were used to detect the variants of the DMD gene in the patients, whose clinical information and gene detection results were descriptively analyzed. Results: The 2 690 patients included 2 648 males and 42 females, with an age of 6.0 (4.0, 9.0) years. The serum creatine kinase increased in all patients. Pathogenic DMD gene variants were detected in the 2 618 patients, including 1 875 cases (71.6%) large deletions, 231 cases (8.8%) duplications, and 512 cases (19.6%) small variants. Among the deletion variants, the deletion of 3 exons was the most common, accounting for 15.4% (288/1 875); and hotspot deletion involved exons 45 to 50, accounting for 6.3% (119/1 875). Exon 2 was the most common type duplication region, accounting for 13.0% (30/231). Small variants were distributed in all 79 exons of the DMD gene, with no hotspots. In addition, the 46 small variants were previously unreported. Conclusion: Exon deletion is the most common type of DMD gene variant, followed by small variants and exon duplication.