[Analysis of a Chinese pedigree affected with van der Woude syndrome due to variant of IRF6 gene].

OBJECTIVE: To explore the genetic characteristics of a Chinese pedigree affected with van der Woude syndrome (VWS). METHODS: A proband who had visited the Drum Tower Hospital Affiliated to Nanjing University Medical School in May 2020 for "two previous pregnancies with cleft lip and palate" was selected as the study subject. Trio-whole exome sequencing (trio-WES) was carried out for the patient. Candidate variants were verified by Sanger sequencing of her pedigree members (8 individuals from four generations) and bioinformatic analysis. Chromosomal microarray analysis (CMA) was used to rule out copy number variations in the fetuses. RESULTS: Trio-WES revealed that the proband and her father had both harbored a heterozygous c.742G>T (p.G248C) missense variant of the IRF6 gene, for which her mother was of the wild type. The variant was located in a region with important functions and has not been reported previously. Prediction with several software suggested that it is likely to have a significant impact on the protein structure/function and is highly correlated with the specific phenotypes in this pedigree. Sanger sequencing confirmed co-segregation of the genotypes and phenotypes in the pedigree. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), this variant was rated as likely pathogenic (PM1+PM2_Supporting+PP1+PP3+PP4). Based on the above results, pre-implantation genetic diagnosis was carried out for the proband, which has led to birth of a healthy offspring with normal results for both site testing and CMA. CONCLUSION: The IRF6: c.742G>T (p.G248C) heterozygous variant probably underlay the VWS in this pedigree. Above finding has also enabled reproductive guidance for the proband.

Fetal congenital gastrointestinal obstruction: prenatal diagnosis of chromosome microarray analysis and pregnancy outcomes.

OBJECTIVE: The aim of this study was to investigate the incidence of chromosome anomalies in different types of congenital gastrointestinal obstruction and assess pregnancy outcomes of fetuses with congenital gastrointestinal obstruction. METHODS: A total of 64 cases with gastrointestinal obstruction between January 2014 and December 2020 were enrolled in this study. They were divided into three groups according to sonographic images. Group A: isolated upper gastrointestinal obstruction; Group B: isolated lower gastrointestinal obstruction; Group C: non-isolated gastrointestinal obstruction. The rate of chromosome anomalies in different groups was calculated. Pregnant women with amniocentesis were followed up by medical records and telephone. The follow-up included pregnancy outcomes and development of the live born infants. RESULT: From January 2014 to December 2020, there were 64 fetus with congenital gastrointestinal obstruction underwent chromosome microarray analysis(CMA), the overall detection rate of CMA testing was 14.1%(9/64). The detection rate of Group A, B and C were 16.2%, 0 and 25.0% respectively. 9 fetuses with abnormal CMA results were all terminated. Among 55 fetuses with normal chromosomes, 10(18.2%) fetuses were not found to have any gastrointestinal obstruction after birth. 17(30.9%) fetuses were diagnosed with gastrointestinal obstruction and underwent surgical treatment after birth, one of which had lower gastrointestinal obstruction combined with biliary obstruction and died due to liver cirrhosis. 11(20.0%) pregnancy were terminated due to multiple abnormalities. 5(9.1%) fetuses were intrauterine death. 3(5.5%) fetuses were neonatal deaths. 9(16.4%) fetuses were lost to follow-up. CONCLUSION: It is crucial to understand whether the gastrointestinal tract abnormality is isolated or associated to other findings. The risk of chromosomal abnormalities in fetuses with isolated lower gastrointestinal obstruction is lower than upper gastrointestinal obstruction. While genetic abnormalities excluded, a promising prognosis is expected for fetuses with congenital gastrointestinal obstruction.

[Follow-up of fetuses with de novo copy number variations of unknown significance detected by chromosomal microarray analysis].

OBJECTIVE: To analyze the prognosis of fetuses identified with de novo variants of unknown significance (VOUS) by chromosome microarray analysis (CMA). METHODS: A total of 6 826 fetuses who underwent prenatal CMA detection at the Prenatal Diagnosis Center of Drum Tower Hospital from July 2017 to December 2021 were selected as the study subjects. The results of prenatal diagnosis, and outcome of fetuses identified with VOUS of de novo origin were followed up. RESULTS: Among the 6 826 fetuses, 506 have carried VOUS, of which 237 were detected for the parent-of-origin and 24 were found to be de novo. Among the latters, 20 were followed up for 4 to 24 months. Four couples had opted elective abortion, 4 had developed clinical phenotypes after birth, and 12 were normal. CONCLUSION: Fetuses with VOUS should be continuously follow-up, in particular those carrying de novo VOUS, in order to clarify their clinical significance.

[Value of chromosomal microarray analysis for the diagnosis of fetuses with anomalies of central nervous system].

OBJECTIVE: To assess the value of chromosomal microarray analysis (CMA) for the diagnosis of fetuses with anomalies of the central nervous system (CNS) and summarize the outcome of the pregnancies and follow-up. METHODS: A total of 636 fetuses from June 2014 to December 2020 who were referred to the Prenatal Diagnosis Center of Nanjing Drum Tower Hospital due to abnormal CNS prompted by ultrasound were selected as the research subjects. Based on the ultrasound findings, the fetuses were divided into ventricular dilatation group (n = 441), choroid plexus cyst group (n = 41), enlarged posterior fossa group (n = 42), holoprosencephaly group (n = 15), corpus callosum hypoplasia group (n = 22), and other anomaly group (n = 75). Meanwhile, they were also divided into isolated (n = 504) and non-isolated (n = 132) groups based on the presence of additional abnormalities. Prenatal samples (amniotic fluid/chorionic villi/umbilical cord blood) or abortus tissue were collected for the extraction of genomic DNA and CMA assay. Outcome of the pregnancies and postnatal follow-up were summarized and subjected to statistical analysis. RESULTS: In total 636 fetuses with CNS anomalies (including 89 abortus tissues) were included, and 547 cases were followed up. The overall detection rate of CMA was 11.48% (73/636). The detection rates for the holoprosencephaly group, ACC group, choroid plexus cyst group, enlarged posterior fossa group, ventricular dilatation group and other anomaly group were 80% (12/15), 31.82% (7/22), 19.51% (8/41), 14.29% (6/42), 7.48% (33/441) and 9.33% (7/75), respectively. Compared with the isolated CNS anomaly group, the detection rate for the non-isolated CNS anomaly group was significantly higher (6.35% vs. 31.06%) (32/504 vs. 41/132) (χ² = 62.867, P < 0.001). Follow up showed that, for 52 fetuses with abnormal CMA results, 51 couples have opted induced labor, whilst 1 was delivered at full term with normal growth and development. Of the 434 fetuses with normal CMA results, 377 were delivered at full term (6 had developmental delay), and 57 couples had opted induced labor. The rate of adverse pregnancy outcome for non-isolated CNS abnormal fetuses was significantly higher than that of isolated CNS abnormal fetuses (26.56% vs. 10.54%) (17/64 vs. 39/370) (χ² = 12.463, P < 0.001). CONCLUSION: Fetuses with CNS anomaly should be tested with CMA to determine the genetic cause. Most fetuses with negative CMA result have a good prognosis, but there is still a possibility for a abnormal neurological phenotype. Fetuses with CNS abnormalities in conjunct with other structural abnormalities are at increased risk for adverse pregnancy outcomes.

[Analysis of PDK1 gene variants and prenatal diagnosis for eight pedigrees affected with autosomal dominant polycystic kidney disease].

OBJECTIVE: To detect potential variants in eight Chinese pedigrees affected with autosomal dominant polycystic kidney disease (ADPKD) and provide prenatal diagnosis for two of them. METHODS: Whole exome sequencing and high-throughput sequencing were carried out to detect variants of PKD1 and PKD2 genes in the probands. Sanger sequencing was used to validate the variants, and their pathogenicity was predicted by searching the ADPKD and protein variation databases. RESULTS: Eight PKD1 variants were detected, which have included five nonsense mutations and three missense mutations. Among these, four nonsense variants (PKD1: c.7555C>T, c.7288C>T, c.4957C>T, c.11423G>A) were known to be pathogenic, whilst one missense variant (PKD1: c.2180T>G) was classified as likely pathogenic. Three novel variants were detected, which included c.6781G>T (p.Glu2261*), c.109T>G (p.Cys37Gly) and c.8495A>G (p.Asn2832Ser). Prenatal testing showed that the fetus of one family has carried the same mutation as the proband, while the fetus of another family did not. CONCLUSION: PKD1 variants, including three novel variants, have been identified in the eight pedigrees affected with ADPKD. Based on these results, prenatal diagnosis and genetic counseling have been provided.

[Pathological variant of OFD1 gene identified in a pedigree affected with oral-facial-digital syndrome type 1].

OBJECTIVE: To detect pathological variant in a Chinese pedigree affected with oral-facial-digital syndrome type 1 (OFD1). METHODS: Whole-exome sequencing was used to scan the whole exome of the proband. Potential variant of the OFD1 gene was also detected in all members of the pedigree and 100 healthy controls by Sanger sequencing. X chromosome inactivation analysis was performed. With the determination of the genotype, prenatal diagnosis was carried out by amniotic fluid sampling. RESULTS: A c.1189_1192delAATC (p. Q398Lfs*2) variant was identified in the OFD1 gene of the proband, other patients from this pedigree, as well as the fetus. The same variant was not found among healthy members from this pedigree and the 100 healthy controls. X chromosome inactivation analysis identifies the pregnant woman and her younger sister both had a non-random inactivation, other women patients had a random inactivation. CONCLUSION: The c.1189_1192delAATC (p. Q398Lfs*2) variant of the OFD1 gene probably underlies the pathogenesis in this case. The new variant has enriched pathological spectrum of the OFD1 gene. The reason of intrafamilial clinical variability still need to be further confirmed.

Cell-free DNA test for pathogenic copy number variations: A retrospective study.

OBJECTIVE: To evaluate the detection rate (DR) by prenatal cell-free DNA test for pathogenic copy number variations (CNVs)＞2 Mb among pregnancies with fetal ultrasound abnormalities. MATERIALS AND METHODS: This was a retrospective study on 29 pregnant women with fetuses diagnosed as microdeletion/microduplication syndromes by prenatal chromosome microarray analysis (CMA). Cell-free DNA from the maternal plasma was sequenced on the NextSeq CN500 sequencer. The quality standard of unique map reads in a single sample was greater than 10 M and only gains and losses of more than 2 Mb were reported. RESULTS: A total of 24 CNVs were identified by cell-free DNA test among the 21 fetuses with pathogenic CNVs identified by prenatal CMA, including 20 consistent CNVs and 4 inconsistent CNVs. Overall, the DR of cell-free DNA test for pathogenic CNVs ＞2 Mb was 69%. Microdeletions or microduplications at 22q11.2 were the most common CNVs, with a DR of 4/5 (80%) and 3/4 (75%) respectively. CONCLUSION: Cell-free DNA test exhibited a moderate DR for pathogenic CNVs ＞2 Mb among fetuses with ultrasound abnormalities. Cell-free DNA test could provide an opportunity for early screening before the appearance of abnormalities on fetal ultrasound, while further clinical data and cost-effectiveness assessment are needed.

[Analysis of PHEX gene variant and prenatal diagnosis for a Chinese pedigree affected with X-linked hypophosphatemia].

OBJECTIVE: To detect pathological variant in a Chinese pedigree affected with X-linked hypophosphatemia (XLH). METHODS: Whole-exome sequencing was carried out to screen genetic variants in the proband and her parents. Candidate variant of the phosphate regulating gene with homologies to endopeptidases on the X chromosome (PHEX) was verified by Sanger sequencing of all members of the pedigree and the 100 healthy controls. Prenatal diagnosis was carried out on chorionic villi sample derived from the fetus of the proband. RESULTS: A c.1256G>A (p. Gly419Glu) variant was identified in the PHEX gene of the proband and all other patients from this pedigree. The same variant was not found among healthy members from this pedigree and the 100 healthy controls. Prenatal diagnosis suggested that the fetus also carried the c.1256G>A (p. Gly419Glu) variant. CONCLUSION: The c.1256G>A (p. Gly419Glu) variant of the PHEX gene probably underlay the pathogenesis of XLH in this family. Discovery of the novel variant has enriched the mutational spectrum of the PHEX gene.

[Analysis of related phenotype of prenatal cases with copy number variations in various region of 22q11.2].

OBJECTIVE: To analyze the prenatal ultrasound phenotypes of copy number variations (CNVs) in different regions of 22q11.2, their parental original, and pregnancy outcome. METHODS: Prenatal phenotypes of 25 cases with CNVs of the 22q11.2 region detected by chromosomal microarray analysis (CMA) was reviewed, which including There were 13 deletions and 12 duplications. Multiplex ligation-dependent probe amplification(MLPA) was carried out to determine their parental origin. All cases were followed up for their pregnancy outcome and postnatal growth. RESULTS: Among the 25 cases, the ultrasound phenotypes of those involving the TBX1 gene were mostly cardiovascular system abnormalities, the ultrasound phenotypes of cases involving CRKL gene are mostly polycystic renal dysplasia. The ultrasound phenotypes of CNVs in the distal region (involving the SMARCB1 gene) are nervous system abnormalities. 12 cases (48%) of CNVs were de novo in origin. Five cases were lost during follow-up,12 had opted to terminate the pregnancy, 8 fetuses were born,7 with normal growth and development, 1 case with CNV in A-D region was abnormal.Prenatal ultrasound showed abnormalities in the cardiovascular system consistent with postnatal ultrasound, in addition with dysphagia and growth retardation. CONCLUSION: Prenatal phenotypes of the 22q11.2 region CNVs are diverse, which may be related to gene function. NT thickening may be used as an early ultrasound finding of proximal 22q11.2 CNV. More research is still required to delineate the nature of CNVs and gene function, so as to facilitate genetic counseling.

[Analysis of PHEX gene variant and prenatal diagnosis for a Chinese pedigree affected with X-linked hypophosphatemia].

OBJECTIVE: To detect pathological variant in a Chinese pedigree affected with X-linked hypophosphatemia (XLH). METHODS: Whole-exome sequencing was carried out to screen genetic variants in the proband and her parents. Candidate variant of the phosphate regulating gene with homologies to endopeptidases on the X chromosome (PHEX) was verified by Sanger sequencing of all members of the pedigree and the 100 healthy controls. Prenatal diagnosis was carried out on chorionic villi sample derived from the fetus of the proband. RESULTS: A c.1256G>A (p. Gly419Glu) variant was identified in the PHEX gene of the proband and all other patients from this pedigree. The same variant was not found among healthy members from this pedigree and the 100 healthy controls. Prenatal diagnosis suggested that the fetus also carried the c.1256G>A (p. Gly419Glu) variant. CONCLUSION: The c.1256G>A (p. Gly419Glu) variant of the PHEX gene probably underlay the pathogenesis of XLH in this family. Discovery of the novel variant has enriched the mutational spectrum of the PHEX gene.

[Mutation analysis for a methylmalonic acidemia pedigree without proband by high-throughput sequencing].

OBJECTIVE: To apply high-throughput sequencing for the detection of potential mutation in a methylmalonic academia pedigree for which no proband was available. METHODS: For a couple who had previously given birth to an affected child, 14 genes were re-sequenced by high-throughput sequencing. Suspected mutations were validated by Sanger sequencing. Specific mutations were tested for amniotic fluid sample from the fetus. RESULTS: High-throughput sequencing suggested that the husband has carried a heterozygous mutation of the MUT gene (Exon 3: c.729_730insTT; p.Asp244Leufs*39), while the wife also carried a heterozygous mutation of the MUT gene (Exon 5: c.914T>C; p.Leu305Ser). Both mutations were confirmed by Sanger sequencing. Testing of amniotic sample suggested that the fetus has carried neither mutation. Follow-up has found no sign of methylmalonic academia in the neonate. CONCLUSION: High-throughput sequencing is a sensitive method to screen a bunch of genes in a single test. For autosomal recessive diseases, when no proband is available, carrier testing for both parents with high-throughput sequencing can provide an alternative approach, though great caution should be taken in the setting of prenatal diagnosis.