Novel Compound Heterozygous BBS2 and Homozygous MKKS Variants Detected in Chinese Families with Bardet–Biedl Syndrome

Background Bardet–Biedl syndrome (BBS) is a rare multisystem developmental disorder. In this study, we report the genetic causes and clinical manifestations in two Chinese families with BBS. Materials and Methods Two families were recruited in this study. Family A was a four-generation family with four affected and 15 unaffected members participating in the study, and family B was a consanguineous family with one affected and three unaffected members participating. Whole exome sequencing was performed in the two families, followed by a multistep bioinformatics analysis. Sanger sequencing was used to verify the variants and to perform a segregation analysis. Comprehensive ocular and systemic examinations were also conducted. Results Novel compound heterozygous variants c.235T > G (p.T79P) and c.534 + 1G > T were detected in the BBS2 gene in family A, and known homozygous variant c.748G > A (p.G250R) was detected in the MKKS gene in family B. Both families presented with retinitis pigmentosa; however, except for polydactyly, all other systemic manifestations were different. All of the affected family members in family A were overweight with a high body mass index (range from 26.5 to 41.9) and high blood pressure. Family A also presented with a delay in the onset of secondary sex characteristics and genital anomalies, while other systemic abnormalities were absent in family B. Conclusions This study presents one family with two novel BBS2 variants, expanding the variant spectrum of BBS, and one family with a known homozygous MKKS variant. The different phenotypes seen between the families with BBS2 and MKKS variants will contribute to the literature and our overall understanding of BBS.

In this study, we report on two Chinese families with BBS, including their gene variants and clinical manifestations.

Patients and Methods
Two families were recruited from the Zhongshan Ophthalmic Center (Guangzhou, China). Nineteen participants, including four affected and 15 unaffected, were included from four-generation family A. Four participants, including one affected and three unaffected, were recruited from consanguineous family B. Written informed consent was obtained from each participating individual or a guardian prior to the study. is study was approved by the Institutional Review Board of the Zhongshan Ophthalmic Center. Genomic DNA was prepared from venous leukocytes for all 23 family members using a previously described method [29]. e diagnosis for BBS was made according to the criteria for an improved diagnosis of BBS [30]. All research adhered to the tenets of the Declaration of Helsinki.
Whole exome sequencing (WES) was performed in six family members in family A: III : 4, III : 5, IV : 1, IV : 5, IV : 8, and IV : 10 and one family member in family B: IV : 2. e WES was performed using the Illumina MiSeq platform (Illumina, Madison, WI, USA) and average sequencing depth was set to 100-fold. Strand NGS software version 2.0 (Strand Scientific Intelligence Inc., LA, USA) was used to set the sequencing reads to University of California Santa Cruz hg19. e Human Gene Mutation Database (HGMD; http:// www.hgmd.cf.ac.uk/ac/index.php), e Exome Aggregation Consortium (ExAC; http://exac.broadinstitute.org/), and dbSNP (https://www.ncbi.nlm.nih.gov/snp/) were taken into account to determine the frequency of the variants. Online algorithms (SIFT, PolyPhen-2, Mutation Taster, and PROVEAN) were used to estimate the pathogenicity of the mutated genes, and the Human Splicing Finder (HSF; http:// umd.be/HSF3/) was used to study pre-mRNA splicing sites [31]. Sanger sequencing was used to verify the identified variants, and a segregation analysis was performed in the available family members.
Detailed clinical data was collected, including age, gender, height, weight, blood pressure, and heart rate. Visual acuity was measured with a Snellen visual chart. e eyes were examined with a hand-held slit lamp. Fundus photography was taken with a hand-held nonmydriatic digital fundus camera (Optomed Oy, Oulu, Finland). e hearing test was performed using a hand-held device (Interacoustics, Middelfart, Denmark). Ultrasound (GE Healthcare, Chicago, USA) was performed on the heart, kidney, and genitals. Full field electroretinogram (RETIport, Roland Consult, Brandenburg, Germany) was performed in the probands of families A and B.

Genetic Information.
WES was performed in six family members (III : 4, III : 5, IV : 1, IV : 5, IV : 8, and IV : 10) of family A. Bioinformatics analysis revealed novel compound heterozygous variants c.235T > G (p.T79P) and c.534 + 1G > T in the BBS2 gene in family A. Sanger sequencing was performed in all of the available family members, which confirmed that all the affected members had the compound heterozygous variants and the unaffected members had no more than one of the two variants (Figure 1). Variant c.235T > G (p.T79P) was transmitted from the father (III : 4), and variant c.534 + 1G > T was transmitted from the mother (III : 5). Variant c.235T > G causes a substitution of residual 79 in BBS2 from threonine ( r) to proline (Pro). r79 is a conserved residue among vertebrates (Figure 2(a)). Hydrophilic r changes to hydrophobic Pro, causing the protein structure to change ( Figure 2(b)). e c.534 + 1G > T variant was predicted to change the splice site by HSF. WES was performed in IV:2 of family B, and a known homozygous variant, the c.748G > A (p.G250R) variant in the MKKS gene, was identified [32], which was segregated with BBS in this family and was passed to IV : 2 by each parent. However, the MKKS variant was absent in unaffected IV : 1 ( Figure 3).

Ocular Findings.
In family A, IV : 10 was the proband whose poor vision, night blindness, and seeking of medical help brought the entire family to the hospital. After an examination of all of the family members, it was found that all four of those affected by BBS had night blindness, poor visual acuity, and horizontal nystagmus. No light perception was detected in IV : 1 and IV : 2, and finger counting at 1.5 meters for the right eye and at 2.0 meters for the left eye was detected in IV : 3 (because of poor vision and illiteracy, she could not read the visual acuity chart). e electroretinogram of IV : 10 showed undistinguishable photopic and scotopic responses. e ocular findings of this family are summarized in Table 1.
e fundus examination showed vascular attenuation as well as disk pallor ( Figure 4). Considering the symptoms and ocular examinations, a diagnosis of retinitis pigmentosa was given to this family.
In family B, the proband IV : 2 had decreased vision with night blindness in both eyes since early childhood. Her visual acuity was finger counting at 5 centimeters for the right eye and hand movement at 20 centimeters with horizontal nystagmus. e fundus examination revealed vascular attenuation as well as disc pallor and retinal pigment epithelium changes ( Figure 4). e electroretinogram showed dramatically reduced photopic and scotopic responses, confirming the diagnosis of retinitis pigmentosa.

Systemic Clinical Data.
All of the detailed clinical data are summarized in Table 1. In family A, all the affected participants were overweight with a body mass index (BMI) ranging from 26.5 to 41.9, while the BMI ranged from 15.8 to 16.5 in the unaffected siblings. e blood pressure of III : 1, III : 2, and III : 3 was 132/90 mmHg, 135/95 mmHg, and 184/ 114 mmHg, respectively ( Table 1). All of those affected had polydactyly (six fingers and six toes bilaterally) but were without speech delay and dental anomalies (Figure 4). e extra fingers were surgically removed at birth ( Figure 5). IV : 1, IV : 2, and IV : 3 had delays in the onset of secondary sex characteristics and menarche, and genital anomalies were detected by B scan in IV : 1, IV : 2, and IV : 3 with small-sized ovaries and uteruses. IV : 10 had micropenis detected at birth and small-volume testes detected by B scan. Meanwhile, IV : 1, IV : 2, and IV : 3 had poor coordination, IV : 1 and IV : 3 had hyposmia, and IV : 3 had a hearing defect (Table 1). In family B, the proband had polydactyly with a blood pressure of 113/63 mmHg and BMI of 23.9. A blood test revealed normal glucose. B scans of the cardiovascular, urinary, and reproductive systems showed normal function. However, B scan showed the gallbladder polyps and thyroid nodules, and blood test detected high total cholesterol, high low-density lipoprotein cholesterol, high apolipoprotein B, and high uric acid in the proband of family B.

Discussion
In this study, we report one novel compound heterozygous variant in the BBS2 gene in a four-generation Chinese family and a known homozygous variant in the MKKS gene as well as on the comprehensive ocular manifestations and systemic features.
e primary features of BBS include rod-cone dystrophy, polydactyly, obesity, genital anomalies, renal anomalies, and         [34]. Within the same family, the most common variation was in limb distribution [34]. However, retinitis pigmentosa was detected constantly in all affected subjects with variants in BBS genes [34,35]. In a previous study of a patient with the c.748G > A (p.G250R) variant in the MKKS gene, the patient presented with obesity and mental delay [32]; however, the phenotype of the patient in this study with the same MKKS variant is different. Further studies are needed to illustrate the variabilities in phenotypes even in patients with the same variant in the MKKS gene. Referred to the diagnostic criteria of BBS, clinical diagnosis is made by the presence of either four major features or three major features and two minor features [36]. Family A met four major features (rod-cone dystrophy, polydactyly, obesity, and genital anomalies); however, family B only met two major features (rod-cone dystrophy and polydactyly). Nonetheless, the proband of family B presented with the gastrointestinal and endocrine/metabolic abnormalities, which were considered as minor features of BBS [35]. Forsyth and Gunay-Aygan pointed out the limitation of the clinical criteria that many of these clinical features emerge throughout infancy, childhood, and young adulthood, and for the individuals who are considered, the diagnosis of BBS, periodically review is needed [35]. Phenotypic spectrum of disease due to genetic variation should be taken into account, rather than just the clinical diagnosis itself.
Several studies have reported on the triallelic inheritance [30,37] of BBS genes, where a patient with a homozygous R315W variant of the BBS2 gene was also homozygous by descent for the BBS4 locus [37], and a patient with a homozygous D104A variant of the BBS2 gene also had a homozygous R632P variant in the BBS1 gene [37]. e third variant of the triallelic inheritance was considered to have a modifying effect that causes the phenotypic diversity [38]. However, in family A of the current study, WES was performed and only compound heterozygous variants was detected in the BBS2 gene, and no variants were detected in other BBS-associated genes except for single nucleotide polymorphism with high frequency.
is study presented one family with two novel BBS2 variants, expanding the variant spectrum of BBS, and one family with a known homozygous MKKS variant. e phenotypic similarity in family A with the BBS2 variant, and the phenotypic difference between family B and the family in the previous study with the same MKKS variant, will contribute to improved understanding of BBS.
Data Availability e data that support the findings of this study are available from the corresponding author (Xiaoyan Ding) upon reasonable request. Disclosure e authors alone are responsible for the content and writing of this article.

Conflicts of Interest
e authors report no conflicts of interest.