Result of a Pilot External Quality Assessment Scheme for Clinical Diagnosis of Inherited Metabolic Disorders in China.

BACKGROUND: We aimed to evaluate the diagnostic capabilities of Chinese laboratories for inherited metabolic disorders (IMDs) using gas chromatography-mass spectrometry (GC-MS) on urine samples. Meanwhile, based on the result of the pilot external quality assessment (EQA) scheme, we hope to establish a standardized and reliable procedure for future EQA practice. METHODS: We recruited laboratories that participated in the EQA of quantitative analysis of urinary organic acids with GC-MS before joining the surveys. In each survey, a set of five real urine samples was distributed to each participant. The participants should analyze the sample by GC-MS and report the "analytical result", "the most likely diagnosis", and "recommendation for further tests" to the NCCL before the deadline. RESULTS: A total of 21 laboratories participated in the scheme. The pass rates were 94.4% in 2020 and 89.5% in 2021. For all eight IMDs tested, the analytical proficiency rates ranged from 84.7% - 100%, and the interpretational performance rate ranged from 88.2% - 97.0%. The performance on hyperphenylalaninemia (HPA), 3-methylcrotonyl-CoA carboxylase deficiency (MCCD), and ethylmalonic encephalopathy (EE) samples were not satisfactory. CONCLUSIONS: In general, the participants of this pilot EQA scheme are equipped with the basic capability for qualitative organic acid analysis and interpretation of the results. Limited by the small size of laboratories and samples involved, this activity could not fully reflect the state of clinical practice of Chinese laboratories. NCCL will improve the EQA scheme and implement more EQA activities in the future.

[Clinical and genetic analysis of two pedigrees affected with Carnitine-acylcarnitine translocase deficiency due to variant of SLC25A20 gene].

OBJECTIVE: To analyze the clinical phenotype and genotypes of two children with Carnitine-acylcarnitine translocase deficiency (CACTD). METHODS: Two children diagnosed with CACTD at the Gansu Provincial Maternal and Child Health Care Hospital respectively on January 3 and November 19, 2018 were selected as the study subjects. Trio-whole exome sequencing (trio-WES) was carried out, and candidate variants were validated through Sanger sequencing and pathogenicity analysis. RESULTS: Both children were males and had manifested mainly with hypoglycemia. Trio-WES and Sanger sequencing showed that child 1 had harbored compound heterozygous variants of the SLC25A20 gene, namely c.49G>C (p.Gly17Arg) and c.106-2A>G, which were inherited from his father and mother, respectively. Child 2 had harbored homozygous c.199-10T>G variants of the SLC25A20 gene, which were inherited from both of his parents. Among these, the c.106-2A>G and c.49G>C variants were unreported previously. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.49G>C (p.Gly17Arg), c.106-2A>G, and c.199-10T>G variants were classified as likely pathogenic (PM2_supporting+PP3+PM3_strong+PP4), pathogenic (PVS1+PM2_supporting+PM5+PP3), and pathogenic (PVS1+PM2_supporting+PP3+PP5), respectively. CONCLUSION: Combined with their clinical phenotype and genetic analysis, both children were diagnosed with CACTD. Above finding has provided a basis for their treatment as well as genetic counseling and prenatal diagnosis for their families.

[Analysis of a patient with Retinitis pigmentosa due to a novel variant of IMPDH1 gene].

OBJECTIVE: To explore the genetic basis for a patient with autosomal dominant retinitis pigmentosa (RP). METHODS: A male patient with RP treated at Gansu Provincial Maternal and Child Health Care Hospital in September 2019 was selected as the study subject. Clinical data was collected. Peripheral blood samples of the patient and his parents were subjected to whole exome sequencing (WES). Candidate variant was validated by Sanger sequencing and bioinformatic analysis. RESULTS: The patient, a 29-year-old male, developed night blindness, amblyopia, visual field defects and optic disc abnormalities since childhood. Gene sequencing revealed that he has harbored a heterozygous c.942G>C (p.Lys314Asn) variant of the IMPDH1 gene, which was inherited from his mother, whilst his father was of the wild type. Based on the guidelines from the American College of Medical Genetics and Genomics, the c.942G>C variant was predicted as likely pathogenic (PM1+PM2_Supporting+PP3+PP1). CONCLUSION: The c.942G>C (p.Lys314Asn) variant in the IMPDH1 gene probably underlay the RP in this patient.

Analysis of spinal muscular atrophy carrier screening results in 32,416 pregnant women and 7,231 prepregnant women.

Objectives: Spinal muscular atrophy (SMA) is an autosomal recessive disease that is one of the most common in childhood neuromuscular disorders. Our screenings are more meaningful programs in preventing birth defects, providing a significant resource for healthcare professionals, genetic counselors, and policymakers involved in designing strategies to prevent and manage SMA. Method: We screened 39,647 participants from 2020 to the present by quantitative real-time PCR, including 7,231 pre-pregnancy participants and 32,416 pregnancy participants, to detect the presence of SMN1 gene EX7 and EX8 deletion in the DNA samples provided by the subjects. To validate the accuracy of our findings, we also utilized the Multiplex Ligation-dependent Probe Amplification (MLPA) to confirm the reliability of screening results obtained by quantitative real-time PCR. Result: Among the 39,647 participants who were screened, 726 participants were the carriers of SMN1. The overall carrier rate was calculated to be 1.83% (95% confidence interval: 0.86-2.8%). After undergoing screening, a total of 592 pregnancy carriers were provided with genetic counseling and only 503 of their spouses (84.97, 95% confidence interval: 82.09-87.85%) voluntarily underwent SMA screening. Conclusion: This study provides crucial insights into the prevalence and distribution of SMA carriers among the female population. The identification of 726 asymptomatic carriers highlights the necessity of comprehensive screening programs to identify at-risk individuals and ensure appropriate interventions are in place to minimize the impact of SMA-related conditions.

[Genetic analysis and prenatal diagnosis for a Chinese pedigree affected with co-morbid Ornithine carbamoyl transferase deficiency and MECP2 duplication syndrome].

OBJECTIVE: To explore the genetic basis for a Chinese pedigree affected with co-morbid Ornithine carbamoyl transferase deficiency (OTCD) and MECP2 duplication syndrome. METHODS: A proband who was admitted to the Neonatal Intensive Care Unit of Gansu Provincial Maternal and Child Health Care Hospital on December 19, 2017 was selected as the study subject. High-throughput sequencing and multiplex ligation-dependent probe amplification (MLPA) were carried out for her pedigree, and short tandem repeat-based linkage analysis and chromosome copy number variation sequencing (CNV-seq) were used for the prenatal diagnosis. RESULTS: The proband, a 3-day-old female, was found to harbor heterozygous deletion of exons 7-9 of the OTC gene. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as likely pathogenic (PVS1+PM2_Supporting+PP4). The proband was diagnosed with OTCD , which was in keeping with her acute encephalopathy and metabolic abnormalities (manifesting as hyperammonemia, decreased blood citrulline, and increased urine orotic acid). Prenatal diagnosis was carried out for the subsequent pregnancy. The fetus did not harbor the exons 7-9 deletion of the OTC gene, but was found to carry a duplication in Xq28 region (which encompassed the whole region of MECP2 duplication syndrome) and was positive for the SRY sequence. The same duplication was also found in the proband and her mother. Considering the possible existence of X-chromosome inactivation, the proband was diagnosed with two X-linked recessive disorders including OTCD and MECP2 duplication syndrome, and the fetus was determined as a male affected with the MECP2 duplication syndrome. CONCLUSION: Discoveries of the pathogenic variants underlying the OTCD and MECP2 duplication syndrome have enabled clinical intervention, treatment, genetic counseling and prenatal diagnosis for this pedigree.

Combined Amniotic Membrane and Self-Powered Electrical Stimulator Bioelectronic Dress Promotes Wound Healing.

Human amniotic membranes (hAMs) are widely used as wound management biomaterials, especially as grafts for corneal reconstruction due to the structure of the extracellular matrix and excellent biological properties. However, their fragile nature and rapid degradation rate hinder widespread clinical use. In this work, we engineered a novel self-powered electronic dress (E-dress), combining the beneficial properties of an amniotic membrane and a flexible electrical electrode to enhance wound healing. The E-dress displayed a sustained discharge capacity, leading to increased epidermal growth factor (EGF) release from amniotic mesenchymal interstitial stem cells. Live/dead staining, CCK-8, and scratch-wound-closure assays were performed in vitro. Compared with amniotic membrane treatment alone, the E-dress promoted cell proliferation and migration of mouse fibroblast cells and lower cytotoxicity. In a mouse full-skin defect model, the E-dress achieved significantly accelerated wound closure. Histological analysis revealed that E-dress treatment promoted epithelialization and neovascularization in mouse skin. The E-dress exhibited a desirable flexibility that aligned with tissue organization and displayed maximum bioactivity within a short period to overcome rapid degradation, implying great potential for clinical applications.

[Genetic analysis of eighteen patients from Gansu Province with Tetrahydrobiopterin deficiency].

OBJECTIVE: To explore the genetic basis of eighteen patients with Tetrahydrobiopterin deficiency (BH4D) from Gansu Province. METHODS: Eighteen patients diagnosed with BH4D at Gansu Provincial Maternal and Child Health Care Hospital from January 2018 to December 2021 were selected as the study subjects. Whole exome sequencing was carried out, and candidate variants were verified by Sanger sequencing. RESULTS: All of the thirty-six alleles of the eighteen patients were successfully determined by molecular genetic testing. Sixteen patients were found to harbor variants of the PTS gene, and two had harbored variants of the QDPR gene. Ten variants were detected in the PTS gene, with the most common ones being c.259C>T (34.38%) and c.286G>A (15.63%). Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.259C>T was classified as a pathogenic variant, whilst the c.286G>A, c.166G>A, c.200C>T, c.272A>G, c.402A>C, c.421G>T, c.84_291A>G and c.317C>T were classified as likely pathogenic variants. A novel c.289_290insCTT variant was classified as likely pathogenic (PM1+PM2_Supporting+PM3+PP3+PP4). The two variants (c.478C>T and c.665C>T) detected in the QDPR gene were both classified as variants of uncertain significance (PM1+PM2_Supporting+PP3+PP4). CONCLUSION: Genetic testing has clarified the pathogenic variants in these BH4D patients, which has enabled timely and accurate clinical intervention and treatment, and provided a reference for genetic counseling and reproductive guidance for their families.

[Disease spectrum and pathogenic genes of inherited metabolic disorder in Gansu Province of China]. / ç”˜è‚ƒåœ°åŒºé—ä¼ ä»£è°¢ç— ç–¾ç— è°±åŠè‡´ç— åŸºå› åˆ†æž.

OBJECTIVES: To investigate the disease spectrum and pathogenic genes of inherited metabolic disorder (IMD) among neonates in Gansu Province of China. METHODS: A retrospective analysis was conducted on the tandem mass spectrometry data of 286 682 neonates who received IMD screening in Gansu Provincial Maternal and Child Health Hospital from January 2018 to December 2021. A genetic analysis was conducted on the neonates with positive results in tandem mass spectrometry during primary screening and reexamination. RESULTS: A total of 23 types of IMD caused by 28 pathogenic genes were found in the 286 682 neonates, and the overall prevalence rate of IMD was 0.63 (1/1 593), among which phenylketonuria showed the highest prevalence rate of 0.32 (1/3 083), followed by methylmalonic acidemia (0.11, 1/8 959) and tetrahydrobiopterin deficiency (0.06, 1/15 927). In this study, 166 variants were identified in the 28 pathogenic genes, with 13 novel variants found in 9 genes. According to American College of Medical Genetics and Genomics guidelines, 5 novel variants were classified as pathogenic variants, 7 were classified as likely pathogenic variants, and 1 was classified as the variant of uncertain significance. CONCLUSIONS: This study enriches the database of pathogenic gene variants for IMD and provides basic data for establishing an accurate screening and diagnosis system for IMD in this region.

Immune skeletal dysplasia with neurodevelopmental abnormalities caused by a novel variant of EXTL3 gene in a Chinese family.

BACKGROUND: Immune skeletal dysplasia with neurodevelopmental abnormalities (ISDNA) is an extremely rare, autosomal recessive genetic disorder characterized by various skeletal abnormalities, neurodevelopmental deficits, and abnormal immune system function. ISDNA is caused by variation in the exostosin-like 3 (EXTL3) gene, located on chromosome 8p21.2, whose primary function is the biosynthesis of heparan sulfate (HS) skeleton structure. Only a few variations in the EXTL3 gene have been discovered so far. In these years of development, many pathogenic variants in genetic diseases with genetic and phenotypic heterogeneity have been investigated using whole-exome sequencing (WES) technology. METHODS: In this research, a novel EXTL3 variant was first detected in a patient using WES, which was validated from Sanger sequencing in this family. Family history and clinical information were then collected through comprehensive medical examinations and genetic counseling. In silico prediction was then utilized to confirm the pathogenicity of the variant. RESULTS: A novel homozygous variant, NM_001440: c.2015G>A (p.Arg672Gln) in the EXTL3 gene, was identified using WES, which has never been reported before. Sanger sequencing was performed to confirm that the variant segregated with the disease within the family. CONCLUSION: This research identified a novel pathogenic variant in the EXTL3 gene responsible for ISDNA in a Chinese family. It showed the potential diagnostic role of WES in ISDNA, expanded the EXTL3 gene variation spectrum, and demonstrated that the diagnosis of ISDNA using WES is feasible and effective. More comprehensive genetic counseling and precise prenatal diagnosis for the next pregnancy can also be provided to families with genetic disorders.

Genetic analysis and literature review of a Poirier-Bienvenu neurodevelopmental syndrome family line caused by a de novo frameshift variant in CSNK2B.

BACKGROUND: Poirier-Bienvenu neurodevelopmental syndrome (POBINDS) is a rare autosomal dominant neurologic disorder caused by a heterozygous variant of CSNK2B, which is characterized by early onset epilepsy, hypotonia, varying degrees of intellectual disability (ID), developmental delay (DD), and facial dysmorphism. This study clarifies the molecular diagnosis and causative factors of a Chinese boy with POBINDS. METHODS: The clinical phenotypes and ancillary laboratory tests were collected and analyzed by trio whole exome sequencing (WES) and copy number variant sequencing (CNV-seq) in the follow-up proband's families. The candidate variant was validated by Sanger sequencing and bioinformatics software was used to further explore the effect of the de novo frameshift variant on the protein structure. RESULTS: The proband carries a de novo frameshift variant c.453_c.454insAC (p.H152fs*76) in CSNK2B. According to the ACMG genetic variant classification criteria and guidelines, the locus is a pathogenic variant (PVS1+PS2+PM2) and the associated disease was POBINDS. Protein structure prediction suggests significant differences in amino acid sequences before and after mutation. CONCLUSION: A rare case of POBINDS caused by a novel frameshift variant in CSNK2B was diagnosed. The novel variant extends the variation spectrum of CSNK2B, which provides guidance for early clinical diagnosis, genetic counseling and treatment of this family. A review of the currently reported cases of POBINDS further enriches and summarizes the relationship between genotype and phenotype of POBINDS.

Novel de novo mutation in ZBTB20 in a Chinese Primrose syndrome family and a review of the literature.

BACKGROUND: Primrose syndrome is an autosomal dominant disorder characterized by craniofacial dysmorphism, mental retardation, developmental delay, progressive muscle atrophy and calcification of the earlobe due to a mutation in the ZBTB20. METHOD: We reported a case of a Chinese boy with clinical symptoms resembling Primrose Syndrome, and performed genetic etiology analysis of the proband's family through Trio whole exome sequencing. RESULT: A novel missense variant c.1927T>A(p.F643I) in exon 14 of the ZBTB20 (NM_001348803) was identified in the proband. This is the first report case of primrose syndrome in China, and our case extends the variant spectrum of ZBTB20 and further strengthens the understanding of primrose syndrome. CONCLUSION: However, there are no formal clinical guidelines for the management of this disease, and research on treatment and prognosis remains a challenge and focus in future.

Accuracy and depth evaluation of clinical low pass genome sequencing in the detection of mosaic aneuploidies and CNVs.

BACKGROUND: Low-pass genome sequencing (LP GS) has shown distinct advantages over traditional methods for the detection of mosaicism. However, no study has systematically evaluated the accuracy of LP GS in the detection of mosaic aneuploidies and copy number variants (CNVs) in prenatal diagnosis. Moreover, the influence of sequencing depth on mosaicism detection of LP GS has not been fully evaluated. METHODS: To evaluate the accuracy of LP GS in the detection of mosaic aneuploidies and mosaic CNVs, 27 samples with known aneuploidies and CNVs and 1 negative female sample were used to generate 6 simulated samples and 21 virtual samples, each sample contained 9 different mosaic levels. Mosaic levels were simulated by pooling reads or DNA from each positive sample and the negative sample according to a series of percentages (ranging from 3 to 40%). Then, the influence of sequencing depth on LP GS in the detection of mosaic aneuploidies and CNVs was evaluated by downsampling. RESULTS: To evaluate the accuracy of LP GS in the detection of mosaic aneuploidies and CNVs, a comparative analysis of mosaic levels was performed using 6 simulated samples and 21 virtual samples with 35 M million (M) uniquely aligned high-quality reads (UAHRs). For mosaic levels > 30%, the average difference (detected mosaic levels vs. theoretical mosaic levels) of 6 mosaic CNVs in simulated samples was 4.0%, and the average difference (detected mosaic levels vs. mosaic levels of Y chromosome) of 6 mosaic aneuploidies and 15 mosaic CNVs in virtual samples was 2.7%. Furthermore, LP GS had a higher detection rate and accuracy for the detection of mosaic aneuploidies and CNVs of larger sizes, especially mosaic aneuploidies. For depth evaluation, the results of LP GS in downsampling samples were compared with those of LP GS using 35 M UAHRs. The detection sensitivity of LP GS for 6 mosaic aneuploidies and 15 mosaic CNVs in virtual samples increased with UAHR. For mosaic levels > 30%, the total detection sensitivity reached a plateau at 30 M UAHRs. With 30 M UAHRs, the total detection sensitivity was 99.2% for virtual samples. CONCLUSIONS: We demonstrated the accuracy of LP GS in mosaicism detection using simulated data and virtual samples, respectively. Thirty M UAHRs (single-end 35 bp) were optimal for LP GS in the detection of mosaic aneuploidies and most mosaic CNVs larger than 1.48 Mb (Megabases) with mosaic levels > 30%. These results could provide a reference for laboratories that perform clinical LP GS in the detection of mosaic aneuploidies and CNVs.

[Clinical features and genetic analysis of a child with 3-methylglutenedioic aciduria type VII due to novel variants of CLPB gene].

OBJECTIVE: To explore the clinical features and genetic basis for a child with 3-methylglutaconic aciduria type VII. METHODS: A child who was diagnosed at the Gansu Provincial Maternity and Child Health Care Hospital on August 9, 2019 was selected as the study subject. Clinical data of the child, including urine gas chromatography and mass spectrometry, were collected. The child and her parents were subjected to whole exome sequencing. RESULTS: The child, a female neonate, had presented mainly with intermittent skin cyanosis, convulsions, hypomagnesemia, apnea, neutropenia after birth. Her urine 3-methylpentenedioic acid has increased to 17.53 µmol/L. DNA sequencing revealed that she has harbored compound heterozygous variants of the CLPB gene, namely c.1016delT (p.L339Rfs*5) and c.1087A>G (p.R363G), which were respectively inherited from her mother and father. Both variants were unreported previously. Based on the standards from the American College of Medical Genetics and Genomics (ACMG), the variants were respectively predicted to be pathogenic and likely pathogenic. CONCLUSION: The child was diagnosed with 3-methylglutenedioic aciduria type VII. Discovery of the c.1016delT and c.1087A>G variants has enriched the mutational spectrum of the CLPB gene.

Evaluation of the clinical effects of non-invasive prenatal screening for diseases associated with aneuploidy and copy number variation.

BACKGROUND: To explore and compare the clinical effects of high-resolution non-invasive prenatal screening (NIPS-Plus) for common/uncommon chromosomal aneuploidy and microdeletion/microduplication syndromes (MMS). METHODS: The current prospective study included a total of 25,380 pregnant women who performed NIPS-Plus, and amniocentesis was performed on women with MMS with the screening results to diagnose patients with suspected MMS. RESULTS: There were 415 samples with positive results for NIPS-Plus, included 275 with aneuploidy and 140 with MMS. After diagnosis by amniocentesis, 188 cases were confirmed as true positive, included46 cases of T21, 9 cases of T18, 1 case of T13, 34 cases of SCA, 41 cases of other chromosomal euploidy and 57 cases of MMS. In addition, no false negative cases were found, MMS was classified with 5 Mb with the cutoff value, and the PPV of different fragment size was counted, respectively. CONCLUSION: We found that the corresponding PPV was 44.66% with the fragment of copy number variation (CNV) being less than or equal to 5 Mb, and when it was greater than 5 Mb, the PPV was 29.73%, which suggested that NIPS-Plus was more suitable for screening the PPV of small fragment abnormalities. NIPS-Plus has a good application effect in routine aneuploidy screening and had the best detection effect for T21; moreover, it performed well in screening of MMS and had better detection effect on MMS with CNV fragment length less than 5 Mb. Based on the current results, we suggested that NIPS-Plus should be used as a comprehensive elementary prenatal screening method for all pregnant women, but for MMS caused by abnormal large fragment CNV, the detection method and efficiency still need to be improved.

Identification of deep intronic variants of PAH in phenylketonuria using full-length gene sequencing.

BACKGROUND: Phenylketonuria (PKU) is an autosomal recessive congenital metabolic disorder caused by PAH variants. Previously, approximately 5% of PKU patients remained undiagnosed after Sanger sequencing and multiplex ligation-dependent probe amplification. To date, increasing numbers of pathogenic deep intronic variants have been reported in more than 100 disease-associated genes. METHODS: In this study, we performed full-length sequencing of PAH to investigate the deep intronic variants in PAH of PKU patients without definite genetic diagnosis. RESULTS: We identified five deep intronic variants (c.1199+502A>T, c.1065+241C>A, c.706+368T>C, c.706+531>C, and c.706+608A>C). Of these, the c.1199+502A>T variant was found at high frequency and may be a hotspot PAH variant in Chinese PKU. c.706+531T>C and c.706+608A>C are two novel variants that extend the deep intronic variant spectrum of PAH. CONCLUSION: Deep intronic variant pathogenicity analysis can further improve the genetic diagnosis of PKU patients. In silico prediction and minigene analysis are powerful approaches for studying the functions and effects of deep intronic variants. Targeted sequencing after full-length gene amplification is an economical and effective tool for the detection of deep intron variation in genes with small fragments.

[Genetic analysis of a child with Pitt-Hopkins syndrome due to a novel variant of TCF4 gene derived from low percentage maternal mosaicism].

OBJECTIVE: To explore the genetic etiology of a child with Pitt-Hopkins syndrome. METHODS: A child who had presented at the Medical Genetics Center of Gansu Provincial Maternal and Child Health Care Hospital on February 24, 2021 and his parents were selected as the study subjects. Clinical data of the child was collected. Genomic DNA was extracted from peripheral blood samples of the child and his parents and subjected to trio-whole exome sequencing (trio-WES). Candidate variant was verified by Sanger sequencing. Karyotype analysis was also carried out for the child, and her mother was subjected to ultra-deep sequencing and prenatal diagnosis upon her subsequent pregnancy. RESULTS: The clinical manifestations of the proband included facial dysmorphism, Simian crease, and mental retardation. Genetic testing revealed that he has carried a heterozygous c.1762C>T (p.Arg588Cys) variant of the TCF4 gene, for which both parents had a wild-type. The variant was unreported previously and was rated as likely pathogenic based on the guidelines of the American College of Medical Genetics and Genomics (ACMG). Ultra-deep sequencing indicated that the variant has a proportion of 2.63% in the mother, suggesting the presence of low percentage mosaicism. Prenatal diagnosis of amniotic fluid sample suggested that the fetus did not carry the same variant. CONCLUSION: The heterozygous c.1762C>T variant of the TCF4 gene probably underlay the disease in this child and has derived from the low percentage mosaicism in his mother.

[Prenatal diagnosis for a fetus with Walker-Warburg syndrome].

OBJECTIVE: To explore the genetic etiology for a fetus with Walker-Warburg syndrome(WWS). METHODS: A fetus with WWS diagnosed at Gansu Provincial Maternity and Child Health Care Hospital in June 9, 2021 was selected as the study subject. Genomic DNA was extracted from amniotic fluid sample of the fetus and peripheral blood samples from its parents. Trio-Whole exome sequencing (trio-WES) was carried out. Candidate variants were verified by Sanger sequencing. RESULTS: The fetus was found to harbor compound heterozygous variants of the POMT2 gene, namely c.471delC (p.F158Lfs*42) and c.1975C>T (p.R659W), which were respectively inherited from its father and mother. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), they were respectively rated as pathogenic (PVS1+PM2_Supporting+PP4) and likely pathogenic (PM2_Supporting+PM3+PP3_Moderate+PP4). CONCLUSION: Trio-WES may be used for the prenatal diagnosis of WWS. The compound heterozygous variants of the POMT2 gene probably underlay the disorder in this fetus. Above finding has expanded the mutational spectrum of the POMT2 gene and enabled definite diagnosis and genetic counseling for the family.

The spectrum of phenylalanine hydroxylase variants and genotype-phenotype correlation in phenylketonuria patients in Gansu, China.

BACKGROUND: Phenylketonuria (PKU) is a common, congenital, autosomal recessive, metabolic disorder caused by Phenylalanine hydroxylase (PAH) variants. METHODS: 967 PKU patients from Gansu, China were genotyped by Sanger sequencing, multiplex ligation-dependent probe amplification, and whole exome sequencing. We analyzed the variants of PAH exons, their flanking sequences, and introns. RESULTS: The detection of deep intronic variants in PAH gene can significantly improve the genetic diagnostic rate of PKU. The distribution of PAH variants among PKU subtypes may be related to the unique genetic background in Gansu, China. CONCLUSION: The identification of PAH hotspot variants will aid the development of large-scale neonatal genetic screening for PKU. The five new PAH variants found in this study further expand the spectrum of PAH variants. Genotype-phenotype correlation analysis may help predict the prognosis of PKU patients and enable precise treatment regimens to be developed.

[Clinical features and genetic analysis of a child with acute form of Tyrosinemia type I due to a novel variant of FAH gene].

OBJECTIVE: To analyze the clinical phenotype and genetic basis for a child with acute form of tyrosinemia type I (TYRSN1). METHODS: A child with TYRSN1 who presented at the Gansu Provincial Maternal and Child Health Care Hospital in October 2020 was selected as the subject. The child was subjected to tandem mass spectrometry (MS-MS) and urine gas chromatography-mass spectrometry (GC-MS) for the detection of inherited metabolic disorders, in addition with whole exome sequencing (WES). Candidate variants were validated by Sanger sequencing. RESULTS: The child's clinical features included abdominal distension, hepatomegaly, anemia and tendency of bleeding. By mass spectrometry analysis, her serum and urine tyrosine and succinylacetone levels have both exceeded the normal ranges. WES and Sanger sequencing revealed that she has harbored c.1062+5G>A and c.943T>C (p.Cys315Arg) compound heterozygous variants of the FAH gene, which were inherited from her father and mother, respectively. Among these, the c.943T>C was unreported previously. CONCLUSION: Considering her clinical phenotype and result of genetic testing, the child was diagnosed with TYRSN1 (acute type). The compound heterozygous variants of the FAH gene probably underlay the disease in this child. Above finding has further expanded the spectrum of FAH gene variants, and provided a basis for accurate treatment, genetic counseling and prenatal diagnosis for her family.

[Analysis of genotypes and phenotypes of three children with Cornelia de Lange syndrome].

OBJECTIVE: To analyze the clinical phenotype and results of genetic testing in three children with Cornelia de Lange syndrome (CdLS). METHODS: Clinical data of the children and their parents were collected. Peripheral blood samples of the pedigrees were collected for next generation sequencing analysis. RESULTS: The main clinical manifestations of the three children have included growth delay, mental retardation, peculiar facies and other accompanying symptoms. Based on the criteria proposed by the International Diagnostic Consensus, all three children were suspected for CdLS. As revealed by whole exome sequencing, child 1 has harbored NIPBL gene c.5567_5569delGAA insTAT missense variant, child 2 has harbored SMC1A gene c.607A>G missense variant, and child 3 has harbored HDAC8 gene c.628+1G>A splicing variant. All of the variants were de novo in origin. CONCLUSION: All of the children were diagnosed with CdLS due to pathogenic variants of the associated genes, among which the variants of NIPBL and HDAC8 genes were unreported previously. Above finding has enriched the spectrum of pathogenic variants underlying CdLS.