Identification of four TTN variants in three families with fetal akinesia deformation sequence.

BACKGROUND: TTN is a complex gene with large genomic size and highly repetitive structure. Pathogenic variants in TTN have been reported to cause a range of skeletal muscle and cardiac disorders. Homozygous or compound heterozygous mutations tend to cause a wide spectrum of phenotypes with congenital or childhood onset. The onset and severity of the features were considered to be correlated with the types and location of the TTN variants. METHODS: Whole-exome sequencing was performed on three unrelated families presenting with fetal akinesia deformation sequence (FADS), mainly characterized by reduced fetal movements and limb contractures. Sanger sequencing was performed to confirm the variants. RT-PCR analysis was performed. RESULTS: TTN c.38,876-2 A > C, a meta transcript-only variant, with a second pathogenic or likely pathogenic variant in trans, was observed in five affected fetuses from the three families. Sanger sequencing showed that all the fetal variants were inherited from the parents. RT-PCR analysis showed two kinds of abnormal splicing, including intron 199 extension and skipping of 8 bases. CONCLUSIONS: Here we report on three unrelated families presenting with FADS caused by four TTN variants. In addition, our study demonstrates that pathogenic meta transcript-only TTN variant can lead to defects which is recognizable prenatally in a recessive manner.

An efficient molecular genetic testing strategy for incontinentia pigmenti based on single-tube long fragment read sequencing.

Incontinentia pigmenti (IP) is a rare X-linked dominant neuroectodermal dysplasia that primarily affects females. The only known causative gene is IKBKG, and the most common genetic cause is the recurrent IKBKG△4-10 deletion resulting from recombination between two MER67B repeats. Detection of variants in IKBKG is challenging due to the presence of a highly homologous non-pathogenic pseudogene IKBKGP1. In this study, we successfully identified four pathogenic variants in four IP patients using a strategy based on single-tube long fragment read (stLFR) sequencing with a specialized analysis pipeline. Three frameshift variants (c.519-3_519dupCAGG, c.1167dupC, and c.700dupT) were identified and subsequently validated by Sanger sequencing. Notably, c.519-3_519dupCAGG was found in both IKBKG and IKBKGP1, whereas the other two variants were only detected in the functional gene. The IKBKG△4-10 deletion was identified and confirmed in one patient. These results demonstrate that the proposed strategy can identify potential pathogenic variants and distinguish whether they are derived from IKBKG or its pseudogene. Thus, this strategy can be an efficient genetic testing method for IKBKG. By providing a comprehensive understanding of the whole genome, it may also enable the exploration of other genes potentially associated with IP. Furthermore, the strategy may also provide insights into other diseases with detection challenges due to pseudogenes.

Performance of noninvasive prenatal screening for fetal sex chromosome aneuploidies in a cohort of 116,862 pregnancies.

BACKGROUND: Noninvasive prenatal screening (NIPS) has shown good performance in screening common aneuploidies. However, its performance in detecting fetal sex chromosome aneuploidies (SCAs) needs to be evaluated in a large cohort. RESEARCH DESIGN AND METHODS: In this retrospective observation, a total of 116,862 women underwent NIPS based on DNA nanoball sequencing from 2015 to 2022. SCAs were diagnosed based on karyotyping or chromosomal microarray analysis (CMA). Among them, 2,084 singleton pregnancies received karyotyping and/or CMA. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of NIPS for fetal SCAs were evaluated. RESULTS: The sensitivity was 97.7% (95%CI, 87.7-99.9), 87.3% (95% CI, 76.5-94.4), 96.1% (95%CI, 86.5-99.5), and 95.7% (95% CI, 78.1-99.9), the PPV was 25.8% (95%CI, 19.2-33.2), 80.9% (95%CI, 69.5-89.4), 79.0% (95%CI, 66.8-88.3), and 53.7% (95%CI, 37.4-69.3) for 45,X, 47,XXY, 47,XXX, and 47,XYY, respectively. The specificity was 94.1% (95%CI, 93.0-95.1) for 45,X, and more than 99.0% for sex chromosome trisomy (SCT). The NPV was over 99.0% for all. CONCLUSIONS: NIPS screening for fetal SCAs has high sensitivity, specificity and NPV. The PPV of SCAs was moderate, but that of 45,X was lower than that of SCTs. Invasive prenatal diagnosis should be recommended for high-risk patients.

Prospective prenatal cell-free DNA screening for genetic conditions of heterogenous etiologies.

Prenatal cell-free DNA (cfDNA) screening uses extracellular fetal DNA circulating in the peripheral blood of pregnant women to detect prevalent fetal chromosomal anomalies. However, numerous severe conditions with underlying single-gene defects are not included in current prenatal cfDNA screening. In this prospective, multicenter and observational study, pregnant women at elevated risk for fetal genetic conditions were enrolled for a cfDNA screening test based on coordinative allele-aware target enrichment sequencing. This test encompasses the following three of the most frequent pathogenic genetic variations: aneuploidies, microdeletions and monogenic variants. The cfDNA screening results were compared to invasive prenatal or postnatal diagnostic test results for 1,090 qualified participants. The comprehensive cfDNA screening detected a genetic alteration in 135 pregnancies with 98.5% sensitivity and 99.3% specificity relative to standard diagnostics. Of 876 fetuses with suspected structural anomalies on ultrasound examination, comprehensive cfDNA screening identified 55 (56.1%) aneuploidies, 6 (6.1%) microdeletions and 37 (37.8%) single-gene pathogenic variants. The inclusion of targeted monogenic conditions alongside chromosomal aberrations led to a 60.7% increase (from 61 to 98) in the detection rate. Overall, these data provide preliminary evidence that a comprehensive cfDNA screening test can accurately identify fetal pathogenic variants at both the chromosome and single-gene levels in high-risk pregnancies through a noninvasive approach, which has the potential to improve prenatal evaluation of fetal risks for severe genetic conditions arising from heterogenous molecular etiologies. ClinicalTrials.gov registration: ChiCTR2100045739 .

Case report: A compound heterozygous mutations in ASNS broadens the spectrum of asparagine synthetase deficiency in the prenatal diagnosis.

Asparagine synthetase deficiency (ASNSD) is a rare congenital disorder characterized by severe progressive microcephaly, global developmental delay, spastic quadriplegia, and refractory seizures. ASNSD is caused by variations of the ASNS gene. The present study showed a Chinese family with a fetus suffering microcephaly. Whole-exome sequencing and Sanger sequencing were used to identify the disease-associated genetic variants. Compound heterozygous variants c.97C>T p. (R33C) and c.1031-2_1033del were identified in the ASNS gene and the variants were inherited from the parents. The mutation site c.97C>T was highly conserved across a wide range of species and predicted to alter the local electrostatic potential. The variant c.1031-2_1033del was classified pathogenic. However, there is no case report of prenatal diagnosis of ASNSD. This is the first description of fetal compound mutations in the ASNS gene leading to ASNSD, which expanded the spectrum of ASNSD.

Limb girdle muscular dystrophy 23 caused by compound heterozygous mutations of LAMA2 gene.

Introduction: Mutations of LAMA2 gene are associated with congenital muscular dystrophy (CMD). The LAMA2-related CMD mainly consists of two diseases, merosin deficient congenital muscular dystrophies type 1A (MDC1A) and limb girdle muscular dystrophy 23 (LGMD23). LGMD23 is characterized by slowly progressive proximal muscle weakness, which primarily affects the lower limbs and results in gait difficulties. Additional clinical features include increased serum creatine kinase, abnormal electromyography with or without white matter abnormalities on brain imaging. Methods: Clinical data were collected from a Chinese Han family. Whole-exome sequencing, Sanger sequencing, RT-PCR and TA clone sequencing were performed on the family members. Results: Compound heterozygous mutations of LAMA2: c.1693C > T (p. Q565*) (maternally inherited) and c.9212-6T > G (paternally inherited) were identified and confirmed in the proband. The mutation c.1693C > T (p. Q565*) was classified as pathogenic according to American College of Medical Genetics and Genomics (ACMG) guidelines. By performing RT-PCR and TA clone sequencing, an insertion of 40-bp intronic sequence (intron 64) was found in the transcripts of the proband and her father, which resulted in a frameshift and premature truncation codon of the LAMA2. In particular, the variant truncated the LamG domain of the LAMA2. Therefore, the c.9212-6T>G was classified as likely pathogenic according to American College of Medical Genetics and Genomics (ACMG) guidelines. Discussion: Our findings described two novel mutations in a girl with LGMDR23, which contributes to the genetic counseling of the family and expands the clinical and molecular spectrums of the rare disease.

Integrated Bioinformatics Analysis to Screen Hub Gene Signatures for Fetal Growth Restriction.

Background: Fetal growth restriction (FGR) is the impairment of the biological growth potential of the fetus and often leads to adverse pregnancy outcomes. The molecular mechanisms for the development of FGR, however, are still unclear. The purpose of this study is to identify critical genes associated with FGR through an integrated bioinformatics approach and explore the potential pathogenesis of FGR. Methods: We downloaded FGR-related gene microarray data, used weighted gene co-expression network analysis (WGCNA), differentially expressed genes (DEGs), and protein-protein interaction (PPI) networks to screen hub genes. The GSE24129 gene set was used for validation of critical gene expression levels and diagnostic capabilities. Results: A weighted gene co-expression network was constructed, and 5000 genes were divided into 12 modules. Of these modules, the blue module showed the closest relationship with FGR. Taking the intersection of the DEGs and genes in the blue module as pivotal genes, 277 genes were identified, and 20 crucial genes were screened from the PPI network. The GSE24129 gene set verified the expression of 20 genes, and CXCL9, CXCR3, and ITGAX genes were identified as actual pivotal genes. The expression levels of CXCL9, CXCR3, and ITGAX were increased in both the training and validation sets, and ROC curve validation revealed that these three pivotal genes had a significant diagnostic ability for FGR. Single-gene GSEA results showed that all three core genes activated "hematopoietic cell lineage" and "cell adhesion molecules" and inhibited the "cGMP-PKG signaling pathway" in the development of FGR. CXCL9, CXCR3, and ITGAX may therefore be closely associated with the development of FGR and may serve as potential biomarkers for the diagnosis and treatment of FGR.

Validation and depth evaluation of low-pass genome sequencing in prenatal diagnosis using 387 amniotic fluid samples.

BACKGROUND: Low-pass genome sequencing (LP GS) is an alternative to chromosomal microarray analysis (CMA). However, validations of LP GS as a prenatal diagnostic test for amniotic fluid are rare. Moreover, sequencing depth of LP GS in prenatal diagnosis has not been evaluated. OBJECTIVE: The diagnostic performance of LP GS was compared with CMA using 375 amniotic fluid samples. Then, sequencing depth was evaluated by downsampling. RESULTS: CMA and LP GS had the same diagnostic yield (8.3%, 31/375). LP GS showed all copy number variations (CNVs) detected by CMA and six additional variant of uncertain significance CNVs (>100 kb) in samples with negative CMA results; CNV size influenced LP GS detection sensitivity. CNV detection was greatly influenced by sequencing depth when the CNV size was small or the CNV was located in the azoospermia factor c (AZFc) region of the Y chromosome. Large CNVs were less affected by sequencing depth and more stably detected. There were 155 CNVs detected by LP GS with at least a 50% reciprocal overlap with CNVs detected by CMA. With 25 M uniquely aligned high-quality reads (UAHRs), the detection sensitivity for the 155 CNVs was 99.14%. LP GS using samples with 25 M UAHRs showed the same performance as LP GS using total UAHRs. Considering the detection sensitivity, cost and interpretation workload, 25 M UAHRs are optimal for detecting most aneuploidies and microdeletions/microduplications. CONCLUSION: LP GS is a promising, robust alternative to CMA in clinical settings. A total of 25 M UAHRs are sufficient for detecting aneuploidies and most microdeletions/microduplications.

Noninvasive Prenatal Screening for Common Fetal Aneuploidies Using Single-Molecule Sequencing.

Amplification biases caused by next-generation sequencing (NGS) for noninvasive prenatal screening (NIPS) may be reduced using single-molecule sequencing (SMS), during which PCR is omitted. Therefore, the performance of SMS-based NIPS was evaluated. We used SMS-based NIPS to screen for common fetal aneuploidies in 477 pregnant women. The sensitivity, specificity, positive predictive value, and negative predictive value were estimated. The GC-induced bias was compared between the SMS- and NGS-based NIPS methods. Notably, a sensitivity of 100% was achieved for fetal trisomy 13 (T13), trisomy 18 (T18), and trisomy 21 (T21). The positive predictive value was 46.15% for T13, 96.77% for T18, and 99.07% for T21. The overall specificity was 100% (334/334). Compared with NGS, SMS (without PCR) had less GC bias, a better distinction between T21 or T18 and euploidies, and better diagnostic performance. Overall, our results suggest that SMS improves the performance of NIPS for common fetal aneuploidies by reducing the GC bias introduced during library preparation and sequencing.

Cytosine-guanine-guanine repeats of FMR1 gene negatively affect ovarian reserve and response in Chinese women.

RESEARCH QUESTION: Do cytosine-guanine-guanine (CGG) repeats of the FMR1 gene affect ovarian function, ovarian response and assisted reproductive technology (ART) outcomes in Chinese women? DESIGN: A retrospective cohort study of 5869 women who underwent 8932 ART cycles at Women's Hospital, School of Medicine, Zhejiang University between January 2018 and June 2021. Basic hormone level, oocyte yield, embryo quality and the rate of live birth were considered as main outcome measures to evaluate the effects of CGG repeats on ovarian function, ovarian response and ART outcomes. RESULTS: The CGG repeats were negatively related to serum anti-Müllerian hormone (AMH), oestradiol, antral follicle count (AFC) and oocyte yield. A significant association was found between serum AMH, oestradiol and AFC even after age was controlled for. No statistically significant association, however, was found between CGG repeats and embryo quality or live birth rate. Ovarian function mediated the association between CGG repeats and ovarian response. CONCLUSION: Increased CGG repeats on the FMR1 gene were associated with diminished ovarian function and poor ovarian response, and ovarian function played an intermediary role in the relationship between CGG repeats and ovarian response.

Case report: Prenatal diagnosis of fetal intracranial hemorrhage due to compound mutations in the JAM3 gene.

Intracranial hemorrhage is a common complication in preterm infants but occasionally occurs in fetuses. Disruptions of the genes, such as the COL4A1 and COL4A2 genes, are common genetic causes identified in fetal intracranial hemorrhage; however, the disruptions of the JAM3 gene are rarely reported. In the current investigation, fetal intracranial hemorrhage and dilated lateral ventricles were observed in three consecutive siblings in a pedigree. The pregnancies were terminated, and whole-exome sequencing, followed by Sanger sequencing, was performed on the affected fetuses. Pre-implantation genetic testing for monogenic diseases was performed to avoid the recurrence. The compound heterozygous variants of c.712 + 2T > A and c.813C > G p.Tyr271* in the JAM3 gene (NM_032801.4) were identified in the proband and its affected brother, which were predicted to be pathogenic. The variant of c.813C > G p.Tyr271* but not c.712 + 2T > A was identified in the fourth fetus, implying a good prognosis. Our findings expanded the spectrum of the pathogenic mutations in the JAM3 gene and revealed an important application of fetal whole-exome sequencing in idiopathic fetal intracranial hemorrhage.

Case Report: Prenatal diagnosis of fetal tetrasomy 9p initially identified by non-invasive prenatal testing.

Tetrasomy 9p is a rare syndrome characterized by fetal growth restriction, Dandy-Walker malformation, cardiac anomalies, and facial abnormalities and is discovered by ultrasound during the prenatal examination. Herein, we report a fetus of tetrasomy 9p without obvious phenotypic manifestations during the first trimester that was identified by non-invasive prenatal testing (NIPT). NIPT revealed that the gain of 9p24.3-9p11 that was approximately 46.36 Mb in size. Karyotyping of amniocytes indicated an additional marker in all metaphase. Chromosome microarray and fluorescence in situ hybridization on uncultured amniocytes revealed tetrasomic of 9p24.3q13, and that the supernumerary chromosome is a dicentric isochromosome consisted of two copies of the 9p arm. Taken together, it was indicated that the fetal karyotype was 47,XY,+idic (9) (q13), and that multiple techniques are crucial to the prenatal diagnosis.

Case Report: Novel splicing mutations in RFX5 causing MHC class II deficiency.

Mutations of the Regulatory Factor X5 (RFX5) have been associated with the autosomal recessive major histocompatibility class II (MHC-II) deficiency, which is a severe immunodeficiency characterized by constitutive and interferon-gamma induced MHC II expression disorder and leads to the absence of cellular and humoral T-cell response to antigen challenge. The compound heterozygous splicing mutations of RFX5: c.353 + 6T>G (maternally inherited) and c.757 + 1G>A (paternally inherited) were identified in an infant diagnosed severe immunodeficiency. The mutation c.757 + 1G>A was classified as likely pathogenic while c.353 + 6T>G was classified as the variant of uncertain significance according to American College of Medical Genetics and Genomics (ACMG). To investigate the pathogenicity of RFX5: c.353 + 6T>G, reverse transcription PCR (RT-PCR) was conducted with the mother's peripheral blood. An insertion of 191-bp intronic sequence (intron 6) was found in the transcripts, and this resulted in a frameshift and premature truncation of the protein, especially reduced the DNA-binding domain (DBD) of the RFX5 protein. Our data expanded the spectrum of pathogenic mutations in MHC-II deficiency and put new insights into the genetic counseling, prenatal diagnosis and preimplantation genetic testing (PGT) for the disease.

Case Report: De novo DDX3X mutation caused intellectual disability in a female with skewed X-chromosome inactivation on the mutant allele.

Skewed XCI plays an important role in the phenotypic heterogeneities of many X-linked disorders, even involving in diseases caused by XCI-escaping genes. DDX3X-related intellectual disability is more common in females and less common in males, who usually inherit from unaffected heterozygous mothers. As an X inactivation (XCI) escaping gene, the role of skewed XCI in the phenotype of DDX3X mutant female is unknown. Here we reported a DDX3X: c.694_711dup18 de novo heterozygous mutation in a female with intellectual disability on the maternal X chromosome on the basis of SNPs detected by PCR-sanger sequencing. AR assay revealed that the maternal mutant X chromosome was extremely inactivated in the proband. Using RNA sequencing and whole-exome sequencing, we quantified allelic read counts and allele-specific expression, and confirmed that the mutant X chromosome was inactive. Further, we verified that the mutant DDX3X allele had a lower expression level by RNA sequencing and RT-PCR, and the normal and mutated DDX3X expression accounted for respectively 70% and 30% of total. In conclusion, we found a symptomatic female with extreme skewing XCI in the DDX3X mutant allele. It was discovered that XCI in the mutant allele was insufficient to reverse the phenotype of DDX3X-related neurodevelopmental disorder. It contributed to a better understanding of the role of skewed XCI in phenotypic differences, which can aid in the genetic counseling and prenatal diagnosis of disorders in females with DDX3X defects.

Prenatal diagnosis of trisomy 8 mosaicism, initially identified by cffDNA screening.

BACKGROUND: So called cell-free fetal DNA (cffDNA) in the maternal plasma, which is derived from placenta, is widely used to screen fetal aneuploidies, including trisomy 21, 18, 13 and sex chromosomes. Here we reported a case of trisomy 8 mosaicism (T8M), which was initially identified via cffDNA screening in noninvasive prenatal testing (NIPT). METHODS: A 35-year-old woman received cffDNA screening at 17th week of gestation. Amniocentesis was performed subsequently, and karyotyping, single-nucleotide polymorphism array (SNP-array) and BACs-on-Beads™ (BoBs™) were used to determine fetal chromosome content. Interphase fluorescence in situ hybridization (FISH) was applied to determine the copy number of chromosome 8. RESULTS: An enhanced risk for fetal trisomy 8 was identified by cffDNA screening in the studied pregnant woman. After amniocentesis trisomy 8 was found in 1 of 73 metaphases. SNP-array on DNA derived from cultured amniocytes and neonatal cord blood cells suggested the presence of T8M. Interphase FISH on native neonatal cord blood cells confirmed T8M with a percentage of 10%. The Bobs™ fluorescence data also suggested that 8q23-8q24 was amplified. CONCLUSIONS: The current study shows that NIPT is suited to provide hints on rare autosomal trisomies, which have to be further validated and confirmed by other approaches.

Case report: FOXP1 syndrome caused by a de novo splicing variant (c.1652+5 G>A) of the FOXP1 gene.

FOXP1 syndrome is a rare neurodevelopmental disorder characterized by global developmental delay, intellectual disability, and language delay, with or without autistic features. Several splicing variants have been reported for this condition, but most of them lack functional evidence, and the actual effects of the sequence changes are still unknown. In this study, a de novo splicing variant (c.1652 + 5 G>A) of the FOXP1 gene was identified in a patient with global developmental delay, mild intellectual disability, speech delay, and autistic features. Assessed by TA-cloning, the variant promoted the skipping of exon 18 and a premature stop codon (p.Asn511*), resulting in a predicted truncated protein. This variant, that is lacking the forkhead-box DNA-binding domain and nuclear localization signal 2, may disrupt the protein function and thus cause FOXP1 syndrome-related symptoms. Our study extends the phenotypic and allelic spectra of the FOXP1 syndrome.

Case report: Prenatal diagnosis of Kagami-Ogata syndrome in a Chinese family.

The aim of this work was to explore the genetic cause of the proband (Ⅲ2) presenting with polyhydramnios and gastroschisis. Copy number variation sequencing (CNV-seq), methylation-specific multiplex PCR (MS-PCR), and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) were used to characterize the genetic etiology. CNV-seq revealed a deletion of 732.26 kb at 14q32.2q32.31 in the proband (Ⅲ2) and its mother (Ⅱ2). MS-PCR showed the maternal allele was missing in the proband, while paternal allele was missing in its mother. MS-MLPA showed deletion of the DLK1, MEG3, MIR380, and RTL1 genes of both the proband and its mother. MEG3 imprinting gene methylation increased in the proband, while decreased in its mother. It was indicated that a maternally transmitted deletion was responsible for Kagami-Ogata syndrome in the proband (Ⅲ2), and the de novo paternal deletion resulted in Temple syndrome in the mother (Ⅱ2). Prenatal diagnosis was provided at 17+3 weeks of pregnancy on the mother's fourth pregnancy (Ⅲ4). Fortunately, the karyotype and single-nucleotide polymorphism array (SNP array) results were normal. The current investigation provided the detection methods for imprinted gene diseases, expanded the phenotype spectrum of the disease, and obtained the insight into the diagnosis, prenatal diagnosis, and genetic counseling of the disease.

Case Report: Prenatal Diagnosis of Postaxial Polydactyly With Bi-Allelic Variants in Smoothened (SMO).

Postaxial polydactyly is a common congenital malformation which involves complex genetic factors. This retrospective study analyzed the cytogenetic and molecular results of a Chinese fetus diagnosed with postaxial polydactyly of all four limbs. Fetal karyotyping and chromosomal microarray analysis (CMA) did not find any abnormality while trio whole-exome sequencing (trio-WES) identified bi-allelic variants in smoothened (SMO) and (NM_005631.5: c.1219C > G, NP_005622.1: p. Pro407Ala, and NM_005631.5: c.1619C > T, NP_005622.1: p. Ala540Val). Sanger sequencing validated these variants. The mutations are highly conserved across multiple species. In-depth bioinformatics analysis and familial co-segregation implied the compound heterozygous variants as the likely cause of postaxial polydactyly in this fetus. Our findings provided the basis for genetic counseling and will contribute to a better understanding of the complex genetic mechanism that underlies postaxial polydactyly.

A Novel Homozygous Missense Mutation of PIEZO1 Leading to Lymphatic Malformation-6 Identified in a Family With Three Adverse Pregnancy Outcomes due to Nonimmune Fetal Hydrops.

Lymphatic malformation-6 (LMPHM6) is a rarer form of nonimmune hydrops that often manifests as widespread lymphedema involving all segments of the body, namely, subcutaneous edema, intestinal/pulmonary lymphangiectasia, chylothoraces, and pleural/pericardial effusions. Here, we detected one rare and previously unobserved homozygous missense variant in PIEZO1 (c.5162C>G, p.Ser1721Trp) as a novel genetic cause of autosomal recessive LMPHM6, in a family with three adverse pregnancy outcomes due to nonimmune fetal hydrops. Although, the loss-of-function mutations such as those usually including nonsense, frameshift, splice site, and also fewer missense variants in PIEZO1 have been proved to lead to LMPHM6, among these, the biallelic homozygous mutations resulting in the loss of function of PIEZO1 have not been reported before. Here, we first strongly implicated impaired PIEZO1 function-associated LMPHM6 with a homozygous missense mutation in PIEZO1.

Case Report: Identification Pathogenic Abnormal Splicing of BBS1 Causing Bardet-Biedl Syndrome Type I (BBS1) due to Missense Mutation.

Conventionally, protein features affected by missense mutation was attributed to destroy an important domain with amino acid alternation, and it was difficult to clearly specify the pathogenicity of a novel missense mutation. Nevertheless, the associations between missense mutations and abnormal splicing are nowadays increasingly reported. Rarely, some missense mutations, locating at the non-canonical splicing sites, are observed to damage the splicing process. In this study, a couple has three adverse pregnancy history that the affected fetus presented typical polydactyly, renal abnormalities, and cerebral ventriculomegaly. To identify its genetic etiology, whole-exome sequencing (WES) was performed and a missense mutation c.1339G > A was identified, which was located at the non-canonical splicing sites of the BBS1 gene. Then, reverse transcription polymerase chain reaction was carried out and demonstrated extra 115bp originating from intron 13 cut into cDNA, which generated a predicted premature termination codon (PTC) in the BBS1 protein. Further expression analysis by using real-time reverse-transcribed PCR confirmed the occurrence of nonsense-mediated decay (NMD). Therefore, the pathogenicity of the missense mutation c.1339G > A was explicit and our study helped to extend the spectrum of pathogenic mutations in Bardet-Biedl syndrome type I.