Bardet–Biedl syndrome (BBS) is a genetically heterogeneous autosomal recessive ciliopathy initially reported by Georges Bardet and Arthur Biedl. The primary clinical features of BBS include retinal degeneration/dystrophy (RD), obesity, polydactyly, hypogonadism and genital abnormalities, intelligence disability, renal abnormalities, and behavioral dysfunction. Some secondary clinical features have also been reported (Khan et al., 2016).

The incidence of BBS varies widely across the population, ranging from nearly 1 in 125,000–160,000 in North America and Europe (Ajmal et al., 2013) and 1 in 156,000 in North Africa (M'Hamdi et al., 2011) to a high morbidity such as 1 in 17,000 among the Bedouins in Kuwait (Farag and Teebi, 1989), 1 in 13,000 in Newfoundland (Brinckman et al., 2013), and 1 in every 3,700 individuals in the Faroe Islands (Ajmal et al., 2013; Hjortshoj et al., 2009). This phenomenon is caused by the founder effect in these particular populations (Fattahi et al., 2014). In addition, the high rate of consanguinity and the geographic isolation also play a vital role (Farag and Teebi, 1989; Moore et al., 2005).

Thus far, 21 BBS genes located on different chromosome have been identified (M'Hamdi et al., 2014), the majority of which are involved in the formation and proper functioning of a multi-subunit complex (BBSome). The stable complex participates in signaling receptor trafficking to cilia, which is required for proper cilia functions (Khan et al., 2016). These identified BBS genes can explain approximately 80% of the clinical examined affected. BBS1 is the most common gene accounting for about 23% of the cases in Europe and North America (Forsythe and Beales, 2013). The typical inheritance pattern of this syndrome is autosomal recessive. However, it has been hypothesized that a more intricate and provocative mode called “triallelic inheritance” or “digenic trait” played an important role in 10% of the BBS families (Katsanis, 2004; Forsythe and Beales, 2013).

In this study, we reported a couple who suffered from three continuous pregnancies with fetal polydactyly, renal abnormalities, and cerebral ventriculomegaly. We sequenced the whole exome of the couple, and only an unclassified missense mutation BBS1: c.1339G > A was found both in male and female samples. Further functional analysis clarified the pathogenicity, pathogenesis, and classification of this mutation, which expanded the mutation spectrum. Meanwhile, it enhanced our understanding that the aberrant splicing effect caused by silence or missense mutations falling in the last nucleotide of exon cannot be ignored.

Comparing with prokaryotes, eukaryotes exhibit a much higher degree of variety and complexity, which greatly attributes to the function of alternative splicing. Up until now, 127 splice variants of BBS1 have been included in the human genome mutation database (HGMD^® Professional 2021.2), most of which located in the intron region near the exon boundary, except for two silence mutation c.1110G > A, p. (Pro370 = ), c.1695G > A, p. (Lys565 = ) and one missense mutation c.479G > A, p. (Arg160Gln). Missense mutations in important domains alter amino acid sequences, which may contribute to the changes of protein features. It is challenging to define the pathogenicity of this novel missense mutation. Therefore, we still do not fully understand some of the missense mutations detected by WES. In this study, we employed WES to explore the genetic cause of a suspicious BBS carrier couple and a missense variant BBS1: c.1339G > A was detected, which was first reported by Taylor's study (Taylor et al., 2017) in 85 children with inherited retinal disease. Although the identified missense mutation has been included in the ClinVar database and human genome mutation database (HGMD^® Professional), its classification was still disputable because available evidence (PM2 and PP4) only support the classification of VUS. To the author’s knowledge, it was reported in Chinese population for the first time and was the original study about its function. With transcript analysis, we proved this missense mutation created a new 5′ splice site that led to extra 115 bp originating from intron 13 into cDNA, which generated a predicted premature termination codon (PTC) in BBS1 protein (NP_078925). According to the recommendations for interpreting the loss of function PVS1 ACMG/AMP variant criterion (Abou et al., 2018), the aforementioned results of the in vitro experiment provide the PVS1 evidence for this mutation. However, there were still some limitations of this study. RNA has tissue specific expression patterns. We attempt to see whether other tissue or cell type from the mutation carrier has the same consequence. Oral mucosa cells from saliva were considered as the applicable materials. Bernoulli experiment for three times failed, and cells from other tissue were difficult to acquire unless invasive operation.

Furthermore, the symptoms of BBS were variable, which include six primary features [retinal degeneration, truncal (central) obesity, postaxial polydactyly, hypogenetelism, intelligence disability, and renal abnormalities] and some secondary features (include 12 kinds of features). These symptoms are not always present in all BBS patients. The disorder of cilia as BBS had a significant overlap of clinical features and genetic overlap within the BBS and other ciliopathies such as Alström and McKusick–Kauffman syndromes or Meckel Gruber syndromes. The clinical diagnosis of BBS requires that a patient displays either four primary features or a combination of three primaries and two secondary features, which can effectively differentiate this syndrome from other phenotype overlapped syndromes (Beales et al., 1999). As the previous study showed (Fu et al., 2018; Lord et al., 2019; Petrovski et al., 2019), the truth that for the same illness, the molecular diagnostic rate for likely pathogenic variants associated with malformed fetal phenotypes is slightly lower than the rates reported in larger scale clinical exome sequencing, studies on postnatal patients might be due to the fact that prenatal phenotypic evaluation is limited and genotype–phenotype assessments are extremely difficult. In this case, the ultrasound examination suggested polydactyl, lateral ventricle widening, and bilateral renal echo enhancement that were consistent with BBS. However, it still did not meet the diagnostic criteria because most symptoms listed before can only be detected postnatally even until childhood. Even so, the available evidence still supported the clinical diagnosis of BBS and matched with the evidence of PP4. Therefore, according to the ACMG/Association for Molecular Pathology (AMP) guideline (Richards et al., 2015), this mutation can be classified as “Pathogenic” (PVS∗1 + PM∗1 + PP∗1).

As is well-known, the incidence of BBS varies widely worldwide. The global average AF value of c.1339G > A was 0.00004609, while it was 0.0002506 in East Asia, which was significantly higher than other ethnic groups. In addition, the non-consanguinity couple in this pedigree carried the same mutation in BBS1. Ethnic specific variants had been reported for other autosomal recessive disease previously (Alanay et al., 2010; Cabral et al., 2012; Essawi et al., 2018). Therefore, we cannot rule out a founder effect of this mutation in Chinese population or a relatively isolated region subjectively. Furthermore, the AF and incidence of this BBS1 variant can be estimated within large scale exon data and confirmed whether it was a founder mutation or do haplotype analysis and mutation dating as follow-through (Zhu et al., 2021). The identification of ethnic specific founder mutation can help to determine screening strategy for disease prevention and facilitate genetic counseling. The BBS1: c.1339G > A was a defined pathogenic variation that should be included in expended carrier screening so that this severe condition can be prevented at an early stage.

Our study provides further evidence that WES facilitates genetic diagnosis of fetal structure abnormalities, allowing for more accurate detection of etiology. Performing WES on trios was benefit for detecting de novo or compound heterozygous variants, and reducing the percentage of VUS. In turn, de novo variants can be used to identify potential novel disease-causing genes. In this case, the samples of abnormal fetus were unavailable so that we had to obtain suspected variants from the couple. We found the causative mutation fortunately and identified the correlation between genotype and phenotype. However, the application of this indirect analysis was limited. Therefore, the proband’s sample is indispensable as the direct evidence for genetic etiology identification, which needs to be better implemented, especially in primary care institutions.

To our knowledge, it is the first study to determine the pathogenicity and pathogenesis of the missense mutation BBS1: c.1339G > A. Our work helped to expand the spectrum of pathogenic mutations in the BBS1 gene and the expanded carrier screening. At the same time, it is an alerting sign that the aberrant splicing effect caused by silence or missense mutations falling in the last nucleotide of exon cannot be ignored.