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.

In China, research on expanded carrier screening (ECS) has exhibited significant inconsistencies due to regional variations, with a lack of large-scale data analysis of the population nationwide. This study aimed to review the carrier rates of monogenic disorders throughout China comprehensively. The study was conducted in two stages, with the carrier rate of monogenic diseases as the primary outcome. In the first stage, 1449 articles were identified, of which 11 were ultimately included for data extraction. In the second stage, all 307 genes identified by the first stage were specifically investigated, and both carrier rates and allele frequencies of pathogenic or likely pathogenic (P/LP) variants were extracted. Nineteen genes were associated with monogenic disorders exhibiting carrier frequencies above 1/100, including GJB2 (7.78

Defects in the mitochondrial tRNA genes cause a group of highly clinically and genetically heterogeneous disorders, which poses a challenge for clinical identification and genetic diagnosis. Here, we present a pre-school boy with a novel MT-TD variant m.7560T>C at the heteroplasmy level of 76.53% in blood, 93.34% in urine sediments, and absent in the healthy mother's blood and urine. Besides convulsions, brain magnetic resonance imaging abnormalities and high plasma lactate, the boy presented with the prominent extra-neurologic phenotype including steroid-resistant nephrotic syndrome associated with focal segmental glomerulosclerosis characterized by abnormal mitochondria in podocytes, cortical blindness, and pancreatitis. To our knowledge, this is the unique case with MT-TD m.7560T>C-related multi-organ impairments, which expands the phenotypic and mutational spectrum of primary mitochondrial diseases.

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.

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.

BackgroundThe Protein tyrosine phosphatase receptor Q (PTPRQ) gene encodes a member of the type III receptor-like protein tyrosine phosphatase family found in the stereocilium. Mutations in PTPRQ are mostly associated with deafness, autosomal recessive type 84 (DFNB 84), which usually results in progressive familial hearing loss. MethodsA 25-year-old woman and her sister, both with postlingual-delayed progressive sensorineural hearing loss, were examined. They were from a nonconsanguineous marriage and had no family history of hearing loss. New compound heterozygous PTPRQ gene mutations, nonsense (c.90C > A, p.Y30X) and splice (c.5426 + 1G > A) mutations in two PTPRQ alleles, were identified in the two sisters and were presumably autosomal recessive. The c.90C > A (p.Y30X) mutation was mapped to exon 2 of PTPRQ (NM_001145026). ResultsThe c.90C > A mutation leads to a premature stop codon and a truncated protein. The c.5426 + 1G > A mutation leads to a truncated protein lacking the extracellular domain. Hence, both mutations were predicted to be pathogenic, leading to a deficiency of the extracellular, transmembrane, and phosphatase domains because of nonsense-mediated mRNA degradation. ConclusionsThis study increases the spectrum of PTPRQ gene mutations that might be involved in delayed progressive autosomal recessive non-syndromic hearing loss.

Objectives: Mitochondrial diabetes mellitus is caused by dysfunctional mitochondria and is often misdiagnosed because of its various clinical manifestations. It's even rarer in children, and without a clear family history of diabetes with hearing loss, it's often difficult to diagnose. Case presentation: This is a case study of a family with maternally inherited diabetes mellitus and deafness (MIDD). The proband was an adolescent girl with diabetes with a family history of type 2 diabetes (T2DM) for three generations. Family members have undetected hearing impaired. The proband could not be diagnosed with type 1 diabetes (T1DM) or T2DM. Therefore, whole exome and mitochondrial gene sequencing was performed, which identified an m.3243A>G mutation in the mitochondrial DNA. Conclusions: This suggests that we should be alert to the possibility of hereditary diabetes, especially mitochondrial diabetes in patients with atypical diabetes. A thorough physical examination is very important. What is new: (1) Mitochondrial diabetes in childhood may not be accompanied by deafness even with highly heteroplasmy levels. (2) In MIDD patients, sometimes hearing loss cannot be perceived, which requires us to conduct detailed physical examinations and related examinations. (3) The use of metformin in MIDD patients did not have adverse consequences.

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.

IntroductionNext generation sequencing results in an explosive identification of rare variants of RYR1, making the correlation between phenotype and genotype complicated. We analyzed the data of 33 patients with RYR1-related myopathy, attempting to elucidate correlations between phenotype, genotype, and protein structure of RyR1.MethodsClinical, histopathologic, and genetic data were evaluated, and variants were mapped to the cryo-EM RyR1 structure. The three-dimensional structure of the variant on RyR1 was analyzed.ResultsThe clinical spectrum was highly variable regardless of the mode of inheritance. Recessive variations were associated with more severe feeding problems and respiratory insufficiency in infancy (p < 0.05). Forty pathogenic and likely pathogenic variations were identified, and 14 of them were novel. Missense was the most common variation type regardless of inheritance mode. Arginine (15/45) was the most frequently involved residue. All but one dominant variation clustered in Pore forming and pVSD domains, while recessive variations enriched in Bsol (7/25) and SPRYs (6/25) domains. Analysis of the spatial structure of variants showed that dominant variants may impact RyR1 mainly by breaking down hydrogen or electrovalent bonds (10/21); recessive variants located in different domains may impact the function of RyR1 through different pathways. Variants located in RyR1 coupling sites (PY1&2 and the outermost of Bsol) may cause the most severe clinical manifestation.ConclusionClinical diversity of RYR1-related myopathy was impacted by the inheritance mode, variation type, and variant location. Dominant and recessive variants have different sensitive domains impacting the function of RyR1 through different pathways.

Abstract Background Congenital myopathies are a group of rare neuromuscular diseases characterized by specific histopathological features. The relationship between the pathologies and the genetic causes is complex, and the prevalence of myopathy-causing genes varies among patients from different ethnic groups. The aim of the present study was to characterize congenital myopathies with infancy onset among patients registered at our institution. Method This retrospective study enrolled 56 patients based on the pathological and/or genetic diagnosis. Clinical, histopathological and genetic features of the patients were analysed with long-term follow-up. Results Twenty-six out of 43 patients who received next-generation sequencing had genetic confirmation, and RYR1 variations (12/26) were the most prevalent. Eighteen novel variations were identified in 6 disease-causing genes, including RYR1, NEB, TTN, TNNT1, DNM2 and ACTA1. Nemaline myopathy (17/55) was the most common histopathology. The onset ages ranged from birth to 1 year. Thirty-one patients were followed for 3.83 ± 3.05 years (ranging from 3 months to 11 years). No patient died before 1 year. Two patients died at 5 years and 8 years respectively. The motor abilities were stable or improved in 23 patients and deteriorated in 6 patients. Ten (10/31) patients developed respiratory involvement, and 9 patients (9/31) had mildly abnormal electrocardiograms and/or echocardiograms. Conclusion The severity of congenital myopathies in the neonatal/infantile period may vary in patients from different ethnic groups. More concern should be given to cardiac monitoring in patients with congenital myopathies even in those with static courses.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease characterized by the degeneration of motor neurons and progressive muscle atrophy. Accurate detection of SMN1 and SMN2 copy numbers is essential for SMA diagnosis, carrier screening, disease severity prediction, therapy, and prognosis. However, a method for SMN1 and SMN2 copy number determination that is simultaneously accurate, simple, rapid, multitargeted, and applicable to various samples has not previously been reported. Here, we developed a single-tube multiplex digital polymerase chain reaction (dPCR) assay for simultaneous determination of the copy numbers of SMN1 exons 7 and 8 and SMN2 exons 7 and 8. A total of 317 clinical samples, including peripheral blood, amniotic fluid, chorionic villus, buccal swabs, and dried blood spots, were collected to evaluate the performance of this dPCR-based assay. The test results were accurate for all the clinical samples. Our assay is accurate, rapid, easy to handle, and applicable to many types of samples and uses a small amount of DNA; it is a powerful tool for SMA molecular diagnosis, large-scale screening, and disease severity assessment.

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 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.

Next-generation sequencing has resulted in an explosion of rare de novo TTN variants. The clinical interpretation of these de novo variants in patients with recessive titinopathy is very difficult. Here, we provided a useful way to identify compound heterozygous mutations with a de novo one.

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.