OBJECTIVES:To evaluate the value of the thickness of the frontal lobe (TFL) and foramen magnum-to-cranium distance (FCD) for predicting poor neurodevelopmental outcomes in fetuses with a small head circumference (HC). METHODS:This retrospective observational study included 39 fetuses with HC < -2 standard deviations (SD) and 592 prospectively collected controls with normal growth. TFL was defined as the shortest distance between the upper outer border of the corpus callosum and the upper inner cranial border parallel to the FCD. Reference ranges for FCD and TFL were established in 592 healthy fetuses between 21 and 38 weeks of gestation and applied to fetuses with suspected microcephaly. Diagnostic performance was assessed using sensitivity, specificity, and SD-based thresholds. RESULTS:Normal reference ranges for FCD and TFL across gestation were established. All fetuses with normal postnatal outcomes had TFL values within the normal range, whereas reduced TFL was observed only in fetuses with poor neurodevelopmental outcomes. For predicting adverse outcomes, TFL <-2 SD showed a specificity of 100%, compared with 88% for FCD <-2 SD. Combining HC <-2 SD with TFL <-2 SD improved identification of fetuses with poor outcomes, including cases with borderline HC measurements (-2 to -3 SD). CONCLUSIONS:When combined with HC, reduced TFL may serve as a highly specific prenatal marker for identifying fetuses at risk for adverse neurodevelopmental outcomes in suspected microcephaly.

BackgroundThe advent of next-generation sequencing (NGS) has enhanced the diagnostic efficacy for monogenic diseases, while presenting challenges in achieving consistent diagnoses.MethodWe retrospectively analyzed the concordance rate and reasons for the inconsistency between the original diagnostic result from the genetic testing laboratory and the variant validation result from the prenatal diagnostic center. The validation procedure comprised three stages: validation of variant detection, reevaluation of variant classification, and assessment of recurrence risk, which involved verifying the mode of inheritance and parental carriage.ResultIn total, 17 (6%) of the 286 families affected by rare monogenic diseases showed different results during the variant validation procedure. These cases comprised four (23.5%) with variant detection errors, 12 (70.5%) with inconsistent interpretation, and one (6%) with non-Mendelian inheritance patterns. False-positive NGS results confirmed by Sanger sequencing were related to pseudogenes and GC-rich regions. The classification of the 17 variants was altered in the 12 cases owing to various factors. The case with an atypical inheritance pattern was originally considered autosomal recessive inheritance, but was diagnosed as maternal uniparental disomy after additional genetic analysis.ConclusionWe underscored the significance of variant validation by prenatal diagnostic centers. Families affected by monogenic diseases with reproductive plans should be referred to prenatal genetic centers as early as possible to avoid different results that may postpone subsequent prenatal diagnosis. What's already known about this topic?The inconsistency in variant interpretation among genetic testing laboratories or between clinicians and laboratories is non-neglectable.What does this study addThe study pinpointed three types of inconsistency between genetic testing laboratories and prenatal diagnostic laboratory, and underscored the importance of timely referral to prenatal genetic centers.

To explore the relationship between ultrasound signs of suspected fetal malformation of cortical development (MCD) and genetic MCD.The retrospective study involved fetuses with one of the following 10 neurosonography (NSG) signs: (A) abnormal development of the Sylvian fissure; (B) delayed achievement of cortical milestones; (C) premature or aberrant appearance of sulcation; (D) irregular border of the ventricular wall or irregular shape of the ventricle; (E) abnormal shape or orientation of the sulci; (F) hemispheric asymmetry; (G) non-continuous cerebral cortex; (H) intraparenchymal echogenic nodules; (I) persistent ganglionic eminence (GE) or GE cavitation; (J) abnormal cortical lamination.95 fetuses were included in the study. Chromosomal microarray (CMA) combined with exome sequencing (ES) was available in 40 fetuses, CMA was abnormal in nine and ES in 22. Sign C (7/7, 100%), sign H (2/2, 100%), sign A (18/19, 94.7%), and sign B (12/13, 92.3%) were the signs leading to the highest probability of genetic MCD. The incidence of genetic MCD for sign E, sign I, and sign D was 66.7-73.7%. Only one or none of the fetuses with sign J, sign F, or sign G underwent CMA+ES. The signs in the fetuses with FGFR3, CCND2, FLNA, or TSC2 mutations had the expected features. The other fetuses with different gene mutations showed several non-specific NSG signs.Several reliable signs for genetic MCD can be detected by NSG, and the probability varies with different signs. Most signs are not associated with a specific gene. Therefore, CMA combined with ES is preferred.

Background To analyze the genetic characteristics and long-term outcomes of fetuses with dysplasia of the corpus callosum (DCC) or partial agenesis of the corpus callosum (PACC). Methods A total of 42 fetuses with DCC ( n = 36) or PACC ( n = 6) were retrospectively analyzed from January 2016 to December 2022 at the Peking University First Hospital. The cohort was categorized into isolated (15/42, 36%) and nonisolated groups (27/42, 64%), and differences in the genetic abnormalities and long-term outcomes between the two groups were analyzed. DCC was subdivided into short CC, thin CC, and thick CC. The outcomes of the three different types of DCC were analyzed and discussed. Results (1) Thirty-nine of the 42 cases underwent CMA (chromosomal microarray analysis) and CMA + WES (whole exome sequencing), with 13/15 cases in isolated group and 26/27 cases in nonisolated group. Only pathogenic or likely pathogenic (P/LP) variants were considered, identifying P/LP variants in 2/13 cases in isolated group and 12/26 cases in nonisolated group. There was no significant difference between the two groups (χ² = 3.566, P = 0.05897). (2) In the isolated group, 8 cases were terminated, and 7 cases were delivered. Postnatal follow-up detected 1 case of gross motor development delay one year after birth; no obvious abnormalities were found in the other six cases. In the nonisolated group, 21 cases were terminated, and 6 cases were delivered. Postnatal follow-up detected 4 cases of children with different degrees of language, motor and intelligence abnormalities; 1 case died 10 days after birth. No obvious abnormalities were observed in one case. Six cases (86%, 6/7) in the isolated group showed normal development, compared with 1 case (17%, 1/6) in the nonisolated group, with a significant difference (χ² = 6.198, P = 0.01279). (3) In DCC, the delivery rates of short CCs (18 cases), thin CCs (13 cases), and thick CCs (5 cases) were 17% (3/18), 54% (7/13), and 20% (1/5), respectively, with good outcomes observed in 0% (0/3), 71% (5/7), and 0% (0/1), respectively. P/LP variants were found in 6/17 cases of short CC, 3/12 cases of thin CC, and 2/5 cases of thick CC. Conclusions Fetuses with DCC or PACC combined with other structural abnormalities had a poor long-term prognosis compared with the isolated group. Patients with thin CCs had a higher probability of a good prognosis than those with short or thick CCs.

Objective: To analyse the genetic cause of a proband with mitochondrial disease caused by FASTKD2 gene variation and uniparental disomy. Methods: Detailed medical history of a child suspected "mitochondrial disease" were inquired in Peking University First Hospital on November 23, 2017. c.810_820dup homozygous variation in FASTKD2 gene was found by high-throughput sequencing, and her mother had heterozygous variation, but her father didn't have such variation, which didn't conform to the genetic law of variation. Further clinical examinations and molecular genetic tests were carried out. The venous blood of the child and her parents was drawn, and genomic DNA was extracted. Sanger sequencing, polymerase chain reaction (PCR) testing, short tandem repeat (STR) analysis, chromosome microarray analysis and loss of heterozygosity (LOH) genetic relationship analysis were performed on the proband and the parents to determine the variation. Results: The clinical manifestations, physical examination and laboratory examination of the child supported the diagnosis of mitochondrial disease. c.810_820dup(p.Ser274Phefs*8) homozygous variant in FASTKD2 gene was identified. Sanger sequencing indicated that the mother was a heterozygote of the variant, while the father had no such variation, which did not conform to the genetic law. PCR testing and Sanger sequencing review to eliminate sampling errors, PCR amplification and sequencing errors. Non-biological father was excluded by STR analysis. Three large segmental LOH of FASTKD2 gene were found by chromosome microarray analysis, then the LOH relative analysis verified the child was a mixed maternal uniparental disomy of chromosome 2. The child was diagnosed as mitochondrial disease caused by oxidative phosphorylation coupling defect of type 44. Conclusions: In this study, an autosomal recessive mitochondrial disease which does not conform to the genetic law was found, and it was confirmed that this mitochondrial disease family had both pathogenic variation and uniparental disomy phenomenon. It was diagnosed as mitochondrial disease caused by type 44 oxidative phosphorylation coupling defect.

In the postgenomic era, variant interpretation is crucial for diagnosing monogenic diseases, which is the premise of precision medicine. The bottleneck and difficulty of genetic disease diagnosis have switched from the inaccessibility of detection technology to the interpretation of sequencing results. Multiple studies have suggested that the inconsistency rate of interlaboratory variant interpretation is approximately 10~40%. However, many clinicians have not paid enough attention to this area at present. In this review, we summarized the reasons for inconsistency, including classification methodology, information obtained by the interpreter, evidence application, and expert judgement. For clinicians, genetic counsellors, and molecular pathologists, it is necessary to reevaluate genetic reports, especially those supported by old literature and databases in clinical practice. For unresolvable cases, pedigree analysis, collaboration with research labs for functional experiments, and long-term follow-up to combine advanced clinical presentations with updated data and literature are needed.

AbstractBackgroundVariants in the ATP binding cassette protein subfamily D member 1 (ABCD1) gene are known to cause X‐linked adrenoleukodystrophy (X‐ALD). This study focused on the characteristics of ABCD1 variants in Chinese X‐ALD families and elucidated the value of genetic approaches for X‐ALD.Methods68 male probands diagnosed as X‐ALD were screened for ABCD1 variants by the Sanger sequencing of polymerase chain reaction (PCR) products and multiplex ligation‐dependent probe amplification (MLPA) combined with long‐range PCR. Prenatal diagnosis was performed in 20 foetuses of 17 probands’ mothers. Descriptive statistics were used to summarise the gene variants and prenatal diagnosis characteristics and outcomes.ResultsThis study allowed the identification of 61 variants occurring in 68 families, including 58 single nucleotide variants or small deletion/insertion variants and 3 large deletions. Three probands with no variants detected by next‐generation sequencing were found to have variants by PCR‐sequencing. Prenatal diagnosis found that 10 of the 20 foetuses had no variants in ABCD1.ConclusionPCR primers that do not amplify the pseudogenes must be used for PCR‐sequencing. MLPA combined with long‐range PCR can detect large deletions and insertions, which are usually undetectable by PCR‐sequencing. Prenatal diagnosis could help to prevent the birth of infants with X‐ALD.

Background Developmental and epileptic encephalopathies (DEEs) are a heterogeneous group of severe disorders that are characterized by early-onset, refractory seizures and developmental slowing or regression. Genetic variations are significant causes of these changes. De novo variants in an increasing number of candidate genes have been found to be causal. The YWHAG gene is one such gene that has been reported to cause developmental and epileptic encephalopathy 56 (DEE56). Here, we report a heterozygous missense variant, c.170G > A (p.R57H), in the YWHAG gene that caused early-onset epilepsy and developmental delay in a Chinese family. Methods We described the clinical manifestations of the proband and his mother in detail. Then, we use trio-based whole-exome sequencing to search the etiology of this family. Results Both the proband and his mother exhibited early-onset seizures, intellectual disability, and developmental delay. While the proband attained seizure control with sodium valproate, his mother's seizures were not well controlled. Trio-based whole-exome sequencing revealed a heterozygous missense variant, c.170G > A (p.R57H), in the YWHAG gene, which was considered as the cause of early-onset epilepsy and developmental delay in this family. Conclusions Our report further confirmed that YWHAG haploinsufficiency results in developmental and epileptic encephalopathy 56.

With the development of gene detection technology, more and more rare diseases can be diagnosed prenatally, and a growing number of related case reports have been submitted. This article aims to provide guidance for clinicians who are considering writing a case report on the prenatal diagnosis of monogenic diseases from the following aspects: the option of genetic testing methods for prenatal diagnosis, essential components of the report, the importance of phenotype, and discussion of the related case.

Background In China, it is estimated that there are 15,000-30,000 patients with β Thalassemia Major (β-TM). Our recent Cross-sectional survey of β-TM patients’ conditions in China found that only 7% of patients with β-TM expect to live beyond age 18, and only a fraction of patients have received allogeneic hematopoietic stem cell transplantation. Higher level of fetal hemoglobin (HbF) by downregulation of BCL11A during erythroid differentiation has been reported to ameliorate the clinical severity of the patients with β-TM. Disruption of GATAA motif in DHS h+58 region of BCL11A erythroid enhancer (BEE) by gene editing in CD34+ cells has been shown to significantly induce the production of HbF in patients. An adjacent motif, i.e., the CTG motif within TAL1-GATA1 binding site could potentially also be such a gene-editing target due to naturally occurring SNPs associated with elevated HbF levels (eg rs1427407). ET-01 is an investigational cell therapy product comprised of autologous CD34+ cells that have undergone CRISPR/Cas9-mediated ex vivo editing targeting this motif (Fig 1). Methods The EDI-001 trial (NCT04390971) aimed to evaluate the safety and efficacy of ET-01 in patients with transfusion dependent thalassemia (TDT). Patients (aged 6 to 35 years) with TDT whose total hemoglobin level fell within the range of 4.5g/dL and 7g/dL were eligible. One patient was enrolled and CD34+ cells were collected from the patient by apheresis following mobilization with lenograstim and plerixafor. ET-01 was manufactured by editing at BEE with Cas9 mRNA and a specific single-guide RNA via electroporation. The ET-01 product was infused following myeloablative conditioning on date 18 Nov 2020 (patient age at 12). Afterwards, the patient was followed up per schedule defined in the Protocol, monitoring safety and efficacy endpoints. Results cGMP manufacturing process at clinical scale was developed for ET-01. High editing rate was achieved with stemness well maintained (Fig 2A). Indels pattern analysis showed that in ET-01 four indel types were most prevalent (Fig 2B). Moreover, the percentage of HbF+ cells were significantly increased compared with the unedited CD34+ cells in erythroid differentiation assay (Fig 2C). IND-enabling studies showed that no tumorigenicity nor other toxicities associated with ET-01 were observed in over 400 mice at up to 40 weeks of observation. One patient (β0/β+) was enrolled, for which ET-01 was manufactured under cGMP process and infused at a dose of 23.19 X 10^6 CD34+ cells/kg. Successful hematopoietic reconstitution was observed, in which neutrophil engraftment occurred on day 24 and platelet engraftment on day 37 post ET-01 infusion. The patient experienced a significant increase of HbF production and total Hb, in which the levels of HbF increased from 3.35 g/L at baseline to 89.87 g/L, and the total hemoglobin level reached 110 g/L at month 18, respectively (Fig 3). The patient received her last pRBCs transfusion on day 87 after ET-01 infusion, and achieved transfusion-free for over 15 months. Moreover, we observed that the prevalence of indels changed during the initial few weeks and remained stable in both peripheral blood and bone marrow (Fig 4A). NGS analysis of 42 potential off-target sites, discovered by in silico prediction and DiGenome-seq, showed editing efficiencies below 1%, and no significant change was observed in any of the 42 sites throughout visits (Fig 4B). The patient experienced 62 cases of adverse events, most of which were Grade 1 or 2 in severity. Of these, only one Grade 1 AE was attributed to the ET-01 drug product, caused by the ET-01 preparation component DMSO during infusion, and was gradually relieved by adjusting the infusion rate. Two Grade 3 serious adverse events (SAE) unrelated to ET-01 were reported: enterocolitis and febrile neutropenia, caused by inappropriate diet after apheresis and busulfan conditioning, respectively. Conclusions These data demonstrated that a single-dose of ET-01 composed of autologous CRISPR/Cas9 mediated CD34+ cells led to the timely hematopoietic engraftment, significant and durable increases in HbF production and total Hb, and transfusion-independent for 15 months at the time of data cut-off. Safety profile of ET-01 is consistent with that of autologous HSC transplant including busulfan myeloablative conditioning. These preliminary results support further experimental testing of ET-01 to treat patients with β-TM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

患儿 　女，3岁，36 +3周顺产，出生后因"反应低下"入住NICU。患儿出生后表现为严重的喂养困难，查体：身长47 cm(第50百分位)，体重1900 g(第3百分位)。肌张力低下、皮肤花纹、腕下垂、足内翻，特殊面容包括长头、前额突出、高发际线、宽眼距、低耳位、招风耳(幼儿期明显)、长人中、高腭弓、小下颌。颅脑磁共振检查发现鼻中隔偏曲(图1)。新生儿期黄疸消退延迟。新生儿溶血病检查除外新生儿ABO溶血，甲状腺功能三项FT4 20.52 pmol/L(11.5~22.7 pmol/L)，FT3 3.53 pmol/L(3.5~6.5 pmol/L)，TSH 16.699 mIU/L(0.27~4.2 mIU/L)，口服左甲状腺素钠替代治疗。治疗后患儿可脱离箱内吸氧，但仍存在吸吮力弱等情况，家属随后携患儿出院。患儿出生时父亲31岁，母亲32岁。患儿有一哥哥，体健。母亲既往有两次药物流产史，此次妊娠产前检查未见明显异常。结合患儿喂养困难、生长缓慢、黄疸消退延迟、皮肤花纹、特殊面容、多发畸形等特点，在征得其父母知情同意后，抽取患儿及父母外周血样送广州金域医学检验中心进行染色体核型分析，患儿核型为46，XX，add(13)(p10)，母亲核型为46，XX，t(10；13)(q10；q10)，父亲核型未见异常。为明确患儿13p未知片段的来源，抽取患儿的外周血样送北京大学第一医院实验中心进行微阵列比较基因组杂交分析，结果发现10p15.3-p11.22区重复约33.79 Mb(图2)，判断为致病性，另发现Xq28区0.44 Mb缺失(仅男性致病)。故诊断患儿为10p三体综合征。本研究通过了本院医学伦理委员会的审查(QYFYWZLL26145)。

Objective:To explore the role of parental origin verification in chromosomal microarray analysis (CMA) on the determination of the clinical significance of copy number variations (CNVs).Methods:This retrospective study collected clinical information from 73 core families who underwent prenatal diagnosis at Peking University First Hospital from November 2017 to December 2019. Indications for prenatal diagnosis included ultrasound abnormality in 54 cases (including 12 with thickened nuchal translucency (≥2.5 mm), four with fetal growth restriction, seven with abnormal pregnancy history, and 31 with isolated ultrasound abnormality), NIPT indicated high-risk in four cases, advanced age in nine cases, abnormal pregnancy history alone in three cases, intrauterine death in two cases and one with maternal mental retardation. Genomic DNA of amniotic fluid sample, chorionic villi, cord blood, fetal tissues, and fetal heart blood were extracted using genomic DNA extraction kit. The CNVs of prenatal samples in 73 subjects were analyzed using array-based comparative genomic hybridization (array-CGH) analysis and single nucleotide polymorphism array (SNP-array). Peripheral blood DNA of the couples, and relevant families if necessary, were collected and analyzed in the same way. The results of parental origin detection in CMA were summarized.Results:A total of 76 CNVs were detected in these 73 samples, out of which nine were pathogenic and parental origin detection revealed that six were de novo, two were maternally, and one was paternally inherited; six CNVs were likely pathogenic, including three de novo, two maternally inherited and one paternally inherited; 20 CNVs were variants of uncertain significance, including five paternally inherited, three maternally inherited and 12 de novo; 41 CNVs were likely benign, among which 38 were inherited from parents with normal phenotype. Conclusions:Parental origin verification plays an important role in explaining the clinical significance of detected fetal CNVs and thereby can help to analyze its clinical effect and reproductive risk.

Objective To determine changes in mitochondrial DNA (mtDNA) copy number in peripheral blood in Rett syndrome caused by methyl-CpG-binding protein-2 (MECP2) variants and explore the mechanism of mitochondrial dysfunction in Rett syndrome. Study design Female patients who were diagnosed with Rett syndrome and had an MECP2 variant (n = 142) were recruited in this study, along with the same number of age- and sex-matched healthy controls. MtDNA copy number was quantified by real-time quantitative polymerase chain reaction with TagMan probes. The differences in mtDNA copy number between the Rett syndrome group and the control group were analyzed using the independent-samples t test. Linear regression, biserial correlation analysis, and one-way ANOVA were applied for the correlations between mtDNA copy number and age, clinical severity, variant types, functional domains, and hot-spot variants. Results MtDNA copy number was found to be significantly increased in the patients with Rett syndrome with MECP2 gene variants compared with the control subjects. Age, clinical severity, variant types, functional domains, and hot-spot variants were not related to mtDNA copy number in patients with Rett syndrome. Conclusions MtDNA copy number is increased significantly in patients with Rett syndrome, suggesting that changes in mitochondrial function in Rett syndrome trigger a compensatory increase in mtDNA copy number and providing new possibilities for treating Rett syndrome, such as mitochondria-targeted therapies.

Objective:To summarize the characteristics of genetic variation and prenatal diagnosis in pedigrees with X-linked adrenoleukodystrophy (X-ALD) and elucidate the value of prenatal diagnosis in preventing the birth of children with X-ALD.Methods:Twenty pedigrees, clinically diagnosed with X-ALD in Peking University First Hospital from November 2012 and March 2019, were included in this retrospective study. Genomic DNA was extracted from peripheral blood and amniotic fluid or chorionic villi samples of probands and their families for detecting variants in ATP-binding cassette subfamily D member 1 ( ABCD1) gene using polymerase chain reaction (PCR)-Sanger sequencing. Linkage analysis was also performed on five microsatellite markers near ABCD1 gene to exclude maternal contamination. Characteristics of ABCD1 gene variants and prenatal diagnosis of X-ALD pedigrees were summarized by descriptive statistics. Results:Twenty ABCD1 gene variants were identified in the 20 pedigrees. The variants in three probands that were not detected by next-generation sequencing were identified by PCR-Sanger sequencing. Among the mothers of the 20 probands, 17 carried ABCD1 variants and three did not. We performed 24 prenatal diagnoses on 20 pregnancies (24 fetuses) and identified eight fetuses with variants who were finally terminated. The 16 cases without variants were born alive. The validation results obtained after termination or delivery were consistent with those performed prenatally. Conclusions:No hotspot variants in ABCD1 gene are detected in these X-ALD patients and most variants are maternally inherited. PCR-Sanger sequencing is an effective method for detecting ABCD1 variants. Prenatal diagnosis for mothers who had a body with X-ALD could prevent another one from birth.

Objective:To explore the genetic basis for a child with mental and motor retardation, language impairment, facial dysmorphism and epilepsy.Methods:Whole exome sequencing was carried out to detect pathogenic variant in the proband, and candidate variant was selected based on his phenotype. Sanger sequencing was used to verify the variant in the proband, his parents and other family members.Results:The proband was found to carry a frameshifting mutation of MBD5 gene, namely c. 2217delT (p.F739Lfs*6), which was inherited from his mother and unreported previously. Sanger sequencing confirmed that his brother carried the same mutation with a similar phenotype. His mother also had poor language expression when she was young, in addition with poor academic performance, though she could do some housework and had no history of convulsion. Conclusion:A novel pathogenic variant of the MBD5 gene was discovered, which has enriched the mutational spectrum of the MBD5 gene. Above discovery has enabled genetic counseling and prenatal diagnosis for the family.

Objective:To study the value of chromosome microarray analysis (CMA) application in children with developmental delay (DD), intellectual disability (ID), autistic spectrum disorder (ASD) and multiple congenital anomalies (MCA).Methods:Genomic DNA was extracted from peripheral blood samples. Array-based comparative genomic hybridization (array-CGH) analysis and single nucleotide polymorphism array (SNP-array) were performed in 1 320 children with DD/ID, ASD, with or without epilepsy and MCA who were admitted to Peking University First Hospital from 2014 to 2019. The results of genetic etiology test of CMA in children with mental retardation or global DD was summarized.Results:Of 1 320 samples, there were 10 cases of aneuploid abnormality, 6 cases of uniparental disomy and one case of mosaicism, respectively. Pathogenic copy number variations (CNVs) were found in 320 cases and pathogenic CNVs were detected in 23 cases, with a combined detection rate of 26% (343/1 320). CNVs of uncertain clinical significance occurred in 107 cases, accounting for 8.1% (107/1 320). There were 25 cases of possible benign CNVs, accounting for 2% (25/1 320), while benign CNVs were reported in 20 cases, accounting for 1.5% (20/1 320). The detection rate of MCA with DD/ID was 39.8% (130/327).Conclusions:CMA has the advantages of high resolution and covering the whole genome. It can detect the chromosomal abnormalities, microdeletions and duplications seen under the microscope, thus the genetic etiology of children with mental retardation or global DD can be diagnosed.

To the Editor: Phenylketonuria (PKU) is an autosomal recessive genetic disease caused by pathogenic variants in the phenylalanine hydroxylase (PAH) gene encoding phenylalanine hydroxylase, a key enzyme in the metabolism of phenylalanine. Early low-phenylalanine diet improves most of the neuropsychological disorders, but it is difficult to be maintained for a long period of time.[1] To date, 1184 variants in PAH gene, including missense, splicing, nonsense, insertion and deletion variants, have been identified. The distribution of the variants is quite variable in ethnic groups. Genetic testing and prenatal diagnosis are effective to prevent PKU families from transmitting the pathogenic PAH alleles to their progeny. However, only a few reports about the prenatal diagnosis of PKU from north China have been found in the literature. Here we summarized the results of variant detection in 157 probands and their parents, and prenatal diagnosis of 103 fetuses from 95 PKU families. This study was approved by the Research Ethics Committee of Peking University First Hospital. Informed consent was obtained from the probands or their guardians and their family members. A total of 157 probands with their parents were examined for the variants in PAH gene during the period from May 2012 to December 2018. The age of the probands ranged from 1 month to 17 years, the male to female ratio was 1:0.92. Most of them lived in north China. All the probands had higher levels of plasma phenylalanine (>2 mg/dL), and the diagnosis of tetrahydrobiopterin (BH4) deficiency was excluded by a BH4-loading test. Genomic deoxyribonucleic acid (DNA) was isolated from peripheral lymphocytes of the probands and their parents by a QuickGene DNA Whole Blood Kit (KURABO, Osaka, Japan). The 13 exons and their flanking sequences of the probands were amplified by polymerase chain reaction (PCR) that contained 50 ng DNA, 2.5 mmol/L each deoxy-ribonucleoside triphosphates (dNTPs) 2 μL, 10× reaction buffer 5 μL, 10 μmol/L each primers 1 μL, and 2.5 units of Taq DNA polymerase in a total volume of 50 μL. PCR products were purified and sequenced in an ABI 3130XL DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Sequencing results were compared with the transcript (NM_000277) of PAH gene and its genomic sequence (GRCh38/hg38). Detected variants were further searched in the three databases PAHvdb (www.biopku.org/pah/), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), and HGMD (http://www.hgmd.cf.ac.uk/ac/). A novel variant not found in these databases was evaluated by the online predictive tools of sorting tolerant from intolerant (SIFT) (http://provean.jcvi.org/index.php), PROVEAN (http://provean.jcvi.org/index.php), and PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/) to predict pathogenic effect of the mutant protein. Variants found in the probands were then examined in their respective father and mother. For probands without pathogenic variants or only one pathogenic variant found, the DNA samples were subjected to multiplex ligation-dependent probe amplification (MLPA; MLPA P055 kit, MRC-Holland, Amsterdam, Netherlands) to detect large insertions, deletions, or duplications in PAH gene. MLPA products were separated in ABI 3130XL Genetic Analyzer and analyzed by Coffalyser. Net (MRC-Holland). In the 157 families, prenatal diagnosis was performed for 95 pregnant mothers, in which eight mothers were pregnant twice with prenatal diagnosis twice. DNA samples were extracted from chorionic villi, amniotic fluid, or abortion tissues (for verification of affected fetuses after abortion) using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) and subjected to the same PCR-direct sequencing and/or MLPA procedures as described above. In addition, six short tandem repeats (STR) markers nearby PAH were amplified by PCR and separated on an ABI 3130XL Genetic Analyzer. Genotypes of the six STR markers were compared between mother and the fetus to exclude false results due to maternal blood contamination. Among the 157 probands, 145 probands have two pathogenic alleles, including one proband with two pathogenic variants in one allele and one pathogenic variant in another allele, ten probands have only one pathogenic allele, and no pathogenic alleles were found in two probands. There were total 80 kinds of pathogenic variants, including 71 point nucleotide substitutions, seven small insertions/deletions, and two large deletions, resulting in 52 missense variants in 159 alleles, 13 splicing variants in 82 alleles, seven premature terminations in 46 alleles, five frame shifts in six alleles, two large deletions in four alleles, and one amino acid deletion in four alleles. The spectrum of the 301 variants listed in Supplementary Table 1, https://links.lww.com/CM9/A525, in which the variant of 163_164insATAT is a novel variant not stored in the above three databases. The most prevalent variants were R243Q, splicing variant due to c.611A>G and splicing variant due to c.1197A>T, accounting for 17.9% (54/301), 9.0% (27/301), and 8.3% (25/301) of the variant alleles, respectively. The highest frequency of exon and its flanking sequences in which pathogenic variants (excluding large deletions/duplications) locate was exon 7, followed by exon 11, exon 6, exon 12, and exon 3. MLPA was performed in 13 probands and found one large deletion of exon 1 and its upstream region in two probands and one large deletion of exon 4/exon 5 in the other two probands. Prenatal diagnosis of PKU was performed in 103 fetuses in 95 of the 157 families. Thirty fetuses (29.1%, 30/103) were identified as PKU (carrying two pathogenic variants); all of the families chose abortion, and the pathogenic variants were confirmed by testing the abortion tissues. Fifty-two (50.5%, 52/103) fetuses were identified as PKU carriers (carrying one pathogenic variant), and 21 (20.4%, 21/103) as normal fetuses (no pathogenic variant found). Most of the carrier fetuses and normal fetuses were born (a few of them were aborted by other reasons), and their genotypes of PAH gene were confirmed by testing peripheral blood after birth. Figure 1 showed the importance of molecular diagnosis and prenatal diagnosis for PKU in a family as an example. After the molecular diagnosis of PKU in the proband (III1) and her parents (II1, II2), the proband's aunt (II4) and uncle (II3) were also at the risk to have a PKU baby. Molecular diagnosis revealed that both II3 and II4 were the carriers of PKU. Genetic counseling was then provided to prevent the two pairs of couples from delivery of another PKU case.Figure 1: Pedigree of a phenylketonuria family. After the molecular diagnosis of PKU in the proband (III1) and her parents (II1, II2), the proband's aunt (II4) and uncle (II3) were also at the risk to have a PKU baby. Molecular diagnosis revealed that both II3 and II4 were the carriers of PKU.PAH gene locates in human chromosome 12q23.2, consisting of 13 exons that encode a polypeptide of 452 amino acid residues. Mutant phenylalanine hydroxylase blocks the metabolism of phenylalanine to tyrosine. The accumulation of phenylalanine leads to the alterations of cerebral myelination and protein synthesis and reduced levels of serotonin, dopamine, and noradrenaline in the brain.[2] Eventually, severe mental retardation and neurobehavioral abnormalities are present in these children. Neonatal screening for PKU is only useful for the early treatment of PKU. During the period from 2014 to 2017 in the Haidian District of Beijing city, screened for PKU among 176,340 newborns, in which 33 newborns were confirmed to have PKU with the incidence of 1/5344.[3] In this cohort of PKU probands, the most prevalent variants of R243Q, splicing variants of c.611A>G and c.1197A>T accounted for 35.2% (106/301) of the variants, similar to the reports from other regions in China and Korea.[4,5] In contrast in Japan, the most prevalent variant was R413P.[6] The R243Q variant causes a mutant phenylalanine hydroxylase which has only <10% normal activity in the eukaryotic cell expression system.[7] The novel variant of 163_164insATAT we found causes frameshift and premature termination of the polypeptide, which is a definite pathogenic variant. Two large deletions, exon 1 and its upstream region in two cases and exon 4/exon 5 in other two cases, were identified by MLPA in the 13 probands in which two variant alleles were not found by PCR-Sanger sequencing. Chen et al[8] reported that three large deletion alleles (exon 1 and its upstream region, exon 4/exon 5, and exon 5) were disclosed in 17 PKU families without two pathogenic variants. Yan et al[9] examined 43 PKU patients with none or only one variant allele by MLPA and identified that 22 PKU patients had 24 (51.1%) large deletion/duplication alleles, of which Ex1del3758 was detected in ten cases and Ex4_5del in four cases, similar to our findings. Therefore, the large deletions of exon 1 and exon 4/exon 5 may be relatively common in Chinese PKU patients. No variant hotspot in PAH gene exists in this cohort of PKU patients. The variants were distributed in all 13 exons. The highest frequency of exon and its flanking sequences in which variants locate was exon 7, followed by exon 11, exon 6, exon 12, and exon 3. Zhang et al[10] examined the variants in exons 3, 5, 6, 7, 10, 11, and 12 of PAH gene in 40 PKU families and demonstrated that most variants concentrated in exon 7, followed by exons 6, 11, and 3, which was similar to our results. Therefore, these exons can be chosen first for variant screening. The next-generation sequencing technology has become a powerful tool for the diagnosis of genetic diseases.[4,11] Regular PCR-Sanger sequencing and MLPA could detect 95.6% (301/314) pathogenic alleles in PAH gene in this cohort of PKU patients, suggesting that the classic methods are still effective for the genetic diagnosis of PKU. In families that both the husband and wife who carrying a pathogenic variant in PAH, the possibility of giving birth of a PKU baby is 25%, theoretically. Prenatal diagnosis is the unique way for PKU families to prevent the birth of infant with PKU case. Technologically, genotyping of several STR markers must be included to prevent misdiagnosis due to maternal blood contamination in fetal samples.[12] The six highly polymorphic STR markers that we used for linkage analysis were located around the PAH gene, two upstream, three downstream, and one in intron 3 of PAH gene. In case the fetal samples was contaminated by maternal blood, DNA extracted from cultured amniotic fluid cells or chorionic villi cells must be used to obtain accurate results. Prenatal diagnosis using chorionic villi is usually performed at 11 to 13th week of gestation, and the earlier molecular diagnosis of the fetus is obtained, the less physical and psychological damages to the pregnant woman when the fetus is affected and the pregnancy is terminated. However, abortion due to the manipulation of chorionic villi sampling is relatively high, and the presence of placental chimerism may affect the accuracy of the results. In contrast, amniocentesis is usually performed at 16 to 23rd week of gestation and is relatively safe. However, the later the molecular diagnosis of the fetus, the higher the risk of abortion when the fetus is affected. Here we present the spectrum of variants in PAH gene in PKU patients in north China. No variant hotspot in PAH gene was found. The variants were frequently detected in exon 7. Prenatal diagnosis is the unique way to prevent the progeny of heterozygous couples from PKU. Conflicts of interest None.

Objective:To summarize the prenatal diagnostic characteristics of monogenic global developmental delay/intellectual disability(GDD/ID) pedigrees.Methods:This study retrospectively collected the prenatal molecular diagnostic results of 43 pedigrees that were affected with monogenic GDD/ID in the genetic counseling clinic of Peking University First Hospital from January 2015 to June 2019. The results of prenatal molecular tests were validated after birth or pregnancy termination. Pregnancy outcomes and healthy condition of the offspring were followed up. All data were analyzed by descriptive statistical analysis.Results:Among the 43 pedigrees, 24 were affected with autosomal recessive inheritance (AR) GDD/ID, in which six (25%) fetuses were found to carry two pathogenic variants; 13 (55%) had only one pathogenic variant; five (20%) did not harbor any variant. GDD/ID inherited in an autosomal dominant inheritance (AD) pattern was found in 13 pedigrees, in which 11 fetuses carried no variants while the other two fetuses had the same variants as the proband had (in one pedigree, a low-level variant was detected in the peripheral blood sample of the father while absent in peripheral blood samples of parents in the other pedigree, so it was suspected that the variants of these two affected fetuses were inherited from parental mosaicism). In the other six pedigrees with X-linked inheritance (XL) of GDD/ID, one male fetus was found to harbor the pathogenic variant, while no variants were detected in the others. Maternal contamination was excluded in all prenatal samples using short tandem repeat for linkage analysis. Postnatal validations were consistent with the prenatal tests. All nine affected fetuses were terminated, and the other thirty-four children were delivered and in good health.Conclusions:Prenatal molecular diagnostic test is an effective method to detect pathogenic variants during the first and second trimesters for pedigrees affected by monogenic GDD/ID. For pedigrees affected with AD or XL patterns caused by de novo mutations, potential parental mosaicism should be noted and prenatal diagnostic tests are also recommended.

Background Global developmental delay/intellectual disability (GDD/ID), used to be named as mental retardation (MR), is one of the most common phenotypes in neurogenetic diseases. In this study, we described the diagnostic courses, clinical and genetic characteristics and prenatal diagnosis of a cohort with patients presented GDD/ID with monogenic causes, from the perspective of a tertiary genetic counseling and prenatal diagnostic center. Method We retrospectively analyzed the diagnostic courses, clinical characteristics, and genetic spectrum of patients presented GDD/ID with rare monogenic causes. We also conducted a follow-up study on prenatal diagnosis in these families. Pathogenicity of variants was interpreted by molecular geneticists and clinicians according to the guidelines of the American College of Medical Genetics and Genomics (ACMG). Results Among 81 patients with GDD/ID caused by rare monogenic variants it often took 0.5–4.5 years and 2–8 referrals to obtain genetic diagnoses. Devlopmental delay typically occurred before 3 years of age, and patients usually presented severe to profound GDD/ID. The most common co-existing conditions were epilepsy (58%), microcephaly (21%) and facial anomalies (17%). In total, 111 pathogenic variants were found in 62 different genes among the 81 pedigrees, and 56 variants were novel. The most common inheritance patterns in this outbred Chinese population were autosomal dominant (AD; 47%), following autosomal recessive (AR; 37%), and X-linked (XL; 16%). SCN2A, SHANK3 and STXBP1 were important causal genes. Hot-spot variants were rarely found. By the follow-up, 33 affected families, including 15, 13 and 5 families inherited in AR, AD and XL modes respectively, had undergone prenatal diagnosis. And the recurrence rates are 26.7%, 15.4% and 20% for families inherited in AR, AD, and XL patterns. Conclusion Patients presented with GDD/ID caused by rare single gene variants are characterized by early onset, relatively severe symptoms and great clinical variability and genetic heterogeneity. Timely referrals to genetic counseling and prenatal diagnostic laboratories are important for affected families planning to have additional children.

临床资料 孕妇32 岁,孕1 产0 ,其配偶34 岁,夫妻均体健,否认不良孕产史,否认孕前和孕期用药史,否认有毒有害物质接触史,否认遗传性疾病家族史.孕妇平素月经规律,周期为28 d,于本院规律产检.