Homozygous mutation in CEP19, a gene mutated in ...

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JMG Online First, published on November 10, 2017 as 10.1136/jmedgenet-2017-104758 Phenotypes

Original Article

Homozygous mutation in CEP19, a gene mutated in morbid obesity, in Bardet-Biedl syndrome with predominant postaxial polydactyly Esra Yıldız Bölükbaşı,1 Sara Mumtaz,2 Muhammad Afzal,2 Ute Woehlbier,3 Sajid Malik,2 Aslıhan Tolun1 ►► Additional material is published online only. To view please visit the journal online (http://d​ x.​doi.o​ rg/​10.​1136/​ jmedgenet-​2017-​104758). 1

Department of Molecular Biology and Genetics, Boğaziçi University, Istanbul, Turkey 2 Human Genetics Program, Department of Animal Sciences, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan 3 Center for Integrative Biology, Faculty of Science, Universidad Mayor, Santiago, Chile Correspondence to Dr Sajid Malik, Human Genetics Program, Department of Animal Sciences, Faculty of Biological Sciences, Quaid-iAzam University, Islamabad, Pakistan; ​malik@​qau.e​ du.​pk and Professor Aslıhan Tolun, Department of Molecular Biology and Genetics, Boğaziçi University, Istanbul, Turkey-34342; ​tolun@​boun.​ edu.t​ r Received 27 April 2017 Revised 16 October 2017 Accepted 29 October 2017

Abstract Background  Bardet-Biedl syndrome (BBS) is a ciliopathy with extensive phenotypic variability and genetic heterogeneity. We aimed to discover the gene mutated in a consanguineous kindred with multiple cases of a BBS phenotype. Methods  SNP genotype data were used for linkage analysis and exome sequencing to identify mutations. Modelling and in silico analysis were performed to predict mutation severity. Results  Patients had postaxial polydactyly plus variable other clinical features including rod-cone dystrophy, obesity, intellectual disability, renal malformation, developmental delay, dental anomalies, speech disorder and enlarged fatty liver. The 4.57 Mb disease locus harboured homozygous, truncating CEP19 c.194_195insA (p.Tyr65*) mutation. We also found glioma-associated oncogene homolog 1(GLI1) c.820G>C (p.Gly274Arg) in the homozygous state in most patients. In silico modelling strongly suggests that it is damaging. Also, different combinations of four possible modifier alleles in BBS-related genes were detected. Two are known modifier alleles for BBS, splicing variant CCDC28B c.330C>T and missense MKKS/BBS6 p.Ile339Val, and the others are C8ORF37/BBS21 p.Ala178Val and TMEM67/BBS14 modifier p.Asp799Asp. Some patients carry all those five known/possible modifier alleles. Such variants are highly significantly more abundant in our patients than in a control group. Conclusion  CEP19 encodes a centrosomal and ciliary protein, as all BBS genes do. Another truncating mutation p.Arg82* has been reported as responsible for morbid obesity in a family; however, in the family we present, not all homozygotes are obese, although some are severely obese. The variant in GLI1, encoding a transcription factor that localises to the primary cilium and nucleus and is a mediator of the sonic hedgehog pathway, possibly exacerbates disease severity when in the homozygous state.

Introduction To cite: Yıldız Bölükbaşı E, Mumtaz S, Afzal M, et al. J Med Genet Published Online First: [please include Day Month Year]. doi:10.1136/ jmedgenet-2017-104758

Bardet-Biedl syndrome (BBS; MIM 209900) is a rare ciliopathy resulting from dysfunction of primary cilia and exhibits extensive clinical variability. The primary features are retinal dystrophy, obesity, postaxial polydactyly, urogenital abnormalities, renal anomalies and intellectual disability (ID).1 2 Additionally, a variety of secondary features are associated with the syndrome including enlarged

fatty liver, type 2 diabetes, speech disorder, dental anomalies, developmental delay, brachydactyly and syndactyly. A generally recognised diagnostic criterion is the presence of at least either four primary features or three primary and two secondary features.1 Besides the substantial interand intra-familial variability in the types of clinical features,1 3 variable expressivity in the clinical features and reduced penetrance of the causal mutation have been reported.4 5 BBS is notable also for genetic heterogeneity. Only approximately 80% of all patients with BBS studied to date have mutations in known BBS genes, all of which encode proteins involved in the structure, assembly and/or function of the primary cilium.1 6 Twenty-three BBS-related genes are known, and biallelic mutations in 21 (BBS1-21) of them cause BBS (for reviews, see refs 2 3). Heterozygous alleles in one of those, ARL6/BBS3 and in the remaining two BBS genes, that is, CCDC28B and TMEM67, have been reported as modifier alleles that in the heterozygous state could increase the severity of BBS caused by a homozygous mutation in another BBS gene.7–9 Besides the genetic heterogeneity and modifier alleles, genetics of BBS is also complicated by the observation in some individuals that a homozygous mutation can cause BBS only when occurring together with a heterozygous mutation in another BBS gene. The hypothesis that BBS is a digenic, triallelic disease has gained support,3 5 10–12 but there has been opposition also.13–16 We present a Pakistani family with highly variable BBS phenotype. We mapped the disease locus and identified a novel homozygous truncating mutation in 19-kd Centrosomal Protein (CEP19) showing that the CEP19 mutation is responsible for the BBS in the family. Recently, in a large Arab family, another homozygous truncating CEP19 mutation was reported to cause morbid obesity (body mass index (BMI) >40) and azoo/oligospermia, and Cep19 knockout mice were similarly morbidly obese and azoospermic.17 In contrast, the majority of the BBS members of the family we present are not even obese, although some are severely obese. The large size of the consanguineous family allowed us to search for possible contributing mutations in known BBS-related genes. A very rare homozygous missense mutation is detected in glioma-associated oncogene homologue (GLI1),

Yıldız Bölükbaşı E, et al. J Med Genet 2017;0:1–9. doi:10.1136/jmedgenet-2017-104758

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Phenotypes

Figure 1  Pedigree of the kindred. A horizontal line above the symbol indicates that the individual was physically examined. *Available for all genetic studies; +Available for mutation analysis; #Exome sequenced individuals. encoding a transcriptional factor activated by the sonic hedgehog (SHH) signal transduction cascade, which has vital functions during brain development (reviewed in ref 18 19) and is functionally related to the primary cilium.20–22 The protein localises to the distal tip of the primary cilium in addition to the nucleus,22 23 as reviewed in ref24. Impairment of its function is expected to affect the downstream targets of the pathway.18 Furthermore, GLI1 is suggested to interact with GLI3 (STRING tool),25 deficit of which can cause postaxial polydactyly types 1A and B or preaxial polydactyly type IV. Also, a link between postaxial polydactyly observed in patients with BBS and SHH signalling has been reported.26

Methods

The study was conducted in accordance with the Declaration of Helsinki and national guidelines.

Family and clinical evaluation

The highly consanguineous kindred is from Southern Punjab, Pakistan. The five-generation pedigree constructed showed that all parents originated from the same village and almost all marriages were consanguineous, suggestive of autosomal recessive inheritance (figure 1). Initial physical and medical examination of the three male and five female patients and their seven unaffected relatives was carried out with the help of physicians at Nishtar Hospital in Multan. Later, five patients (501, 503, 505, 508 and 509) were subjected to chorioretinal evaluation and ultrasonography of vital organs including kidneys, spleen and liver, whereas three (505, 508 and 509) of those also underwent ECG evaluations at the District Headquarters Hospital in DG Khan. Brain MRI was available for one patient (503).

Genetic studies

DNA extracted from peripheral blood samples of seven affected and seven unaffected relatives were available for the genetic study (see figure 1). SNP genotype data were generated using Illumina Human OmniExpress-24 Bead Chip. Multipoint logarithm of odds (LOD) score calculations were performed assuming a disease frequency of 0.0001 and using marker 2

spacing of 0.01 Mb and sets of 30. Multipoint linkage analysis in autosomal recessive model with full penetrance was performed separately for the two branches of the family because the pedigree exceeded the capacity of software Allegro. Unaffected sibs were not included in order to not overlook regions possibly harbouring candidate variants with reduced penetrance. A new linkage analysis (fine mapping) was performed for the regions yielding LOD scores >3 using all markers and in sets of 10, and identity by descent (IBD) for the haplotypes in the regions with new scores >3 was investigated by genotype comparison on MS Excel and/or haplotype construction. Final linkage analysis at the identified gene locus included the genotype data of all participants. Linkage analysis in a dominant model (80% penetrance) was performed similarly but for branch A only, as the whole pedigree exceeded the capacity of Allegro. For the two regions yielding the highest LOD score of 1.6, linkage analysis was performed also for branch B. We searched for genomic deletions or duplications in BBS-related genes or in any other part of the genome, shared by at least some of the patients using cnvPartition (V.3.2.0) CNV Analysis Plug-in for Genome Studio. Hg19 build GRC37 map was used throughout the study. Exome sequencing was performed for one patient in each branch of the family (patients 503 and 509) using the Agilent SureSelect Target Enrichment System and the Illumina HiSeq2000 platform. The reads were mapped to the reference genome using Burrows-Wheeler alignment (0.5.9). Variant calling was performed with Sequence Alignment/Map tools (0.1.14) and Annotate Variation to annotate variants. In addition, the aligned reads in the linked region were scrutinised by Integrative Genomics Viewer for any variants that possibly escaped detection. Bedtools was employed to ascertain that each of the target exons was sufficiently covered in at least one of the sequenced exomes. For a recessive model, variants with frequencies 0.6, and for a dominant model 0.25, respectively, were filtered. Splicing variants and exonic variants deduced to affect protein function were evaluated. In the search for modifier alleles, we considered both homozygous and heterozygous variants possibly damaging to protein with population frequencies 2 in branch A plus >0 in branch B (table 2). The most striking is the very rare missense GLI1 c.820G>C (p.Gly274Arg) (NM_005269) at 12q13.3. Four of the seven patients tested were homozygous for it, and two others plus three unaffected relatives were heterozygous (figure 3). Computational algorithms SIFT, PolyPhen-2 and Mutation Taster predicted the variant to be deleterious, possibly damaging and disease causing, respectively. It is reported in only ExAC Latino and non-Finnish European samples, with frequencies of 0.00025 (three alleles in 11572) and 0.000015 (one allele in 66280), respectively. The substituted glycine residue at position 274 as well as the neighbouring 103 residues of the 1106 amino acid proteins are completely conserved across mammals. Conservation of the amino acids altered by the mutations we detected are presented in online supplementary figure S5. We searched for possibly damaging variants with frequencies T (formerly c.430C>T; p.Phe110Phe; rs41263993) (NM_024296) and missense MKKS/ BBS6 c.1015A>G (p.Ile339Val; rs137853909) (NM_018848). Two other variants are also in BBS-related genes. C8ORF37 c.533C>T (p.Ala178Val; rs375314973) (NM_177965) is predicted as deleterious, possibly damaging and disease causing, respectively. Residue Ala178 is completely conserved across species. Adjacent Arg177 has been reported as altered in BBS and isolated recessive retinal dystrophy.33 The other is synonymous TMEM67 (BBS 14, modifier) c.2397T>C (p.Asp799Asp) (NM_153704), which was predicted as disease causing (donor marginally increased) by Mutation Taster; SIFT and PolyPhen-2 are not applicable for synonymous changes. Father 403 homozygous for this variant did not have an obvious phenotype and died at the age of 53 years of infection of a wound. These four variants are present in different combinations in patients and unaffected relatives (figure 3). Patients are either heterozygous or non-carriers, except that patient 502 is homozygous for the CCDC28B allele. 3

4

Overweight (25.6)





N/A

 Obesity (BMI)†

 Intellectual disability

 Renal anomaly

 Urogenital abnormality



F

+

+

+

–/–



N/A

++

Sparse hair; – enuresis; enlarged head; frontal bossing

+



–/–





++

+

+ –

+



+

N/A



++, #



N/A



++

++

B/A Over weight (27.0)

Parenchymal disease

16

F

507

B/–

+

25

F

508



+

N/A

–/–



N/A











N/A

N/A

N/A





+



–/–



+











+

N/A

Parenchymal disease

+

– ++ (23.0; 74th (37.5) centile)

B/–

+, optic nerve N/A atrophy

– (20.0)

A/A

+, retinitis pigmentosa early features

22

M

25

505

503

Overriding sixth toe, left; hyperphagy; insomnia; low hairline, hypertensive

+

+

2/3 toes syndactyly, right/–



N/A

+

+

+





+

N/A



+

++ (36.3)

A/MA

+, early features

24

M

509



N/A

N/A

–/–



N/A











N/A

N/A

N/A



+ (33.6)

–/A

N/A

18

F

511 F

Goitre





–/–



N/A











N/A

N/A

N/A



– (20.6)



N/A

52







–/–



N/A











N/A

N/A

N/A



+ (30.4)



N/A

61

M

402

Unaffected relatives 401







–/–



N/A











N/A

N/A





– (23.5)



N/A

50

M

403







–/–



N/A











N/A

N/A

N/A



− (23.5)



N/A

44

F

404







–/–



N/A











N/A

N/A

N/A



Over weight (28.8)



N/A

22

F

504







–/–



N/A











N/A

N/A

N/A



Over weight (25.5)



N/A

18

F

506







–/–



N/A











N/A

N/A

N/A



– (22.5)



N/A

20

M

510







–/–



N/A











N/A

N/A

N/A



– (24.5; 85th centile)



N/A

16

F

512



6/7

3/7

1/8, 0/9

0/8

1/3

3/8

3/8

1/8

2/8

1/8

3/5

N/A

2/5

4/8

3/8

6/8, 6/8

4/7

Concordance in patients investigated

*Age at the time of last examination. †For ages 20 years and over, normal BMI is 18.5–24.9, overweight 25–30, obese >30 and morbidly obese >40. BMI for ages below 20 was calculated using https://nccd.cdc.gov/dnpabmi/Calculator.aspx?CalculatorType=Metric as recommended by http://www.who.int/growthref/ who2007_bmi_for_age/en/. #, walks with support; +, feature present; ++, severe phenotype; –, feature absent; A, bilateral postaxial type A; B, bilateral postaxial type B; bilateral MA, mesoaxial; BMI, body mass index; F, female; M, male; N/A, phenotype not ascertained.

Retroverted uterus



N/A

 Diverse

+

–/–





 Shortness of breath +

 Exotropia of right eye

Others

–/–

 Syndactyly/ brachydactyly

N/A





 Diabetes

 Ataxia/poor coordination







 Dental anomalies

 Developmental delay









 Speech disorder



N/A

N/A

N/A



 Aggressive behaviour





 Enlarged fatty liver

 Physical disability; neuromotor problem

Secondary features

–/A

B/A

 Polydactyly in hands/feet

Over weight (26.3)

N/A

 Rod-cone dystrophy –

Primary features

Clinical findings

M

F

28

 Sex

 Age (years)

25

502

Patients

501

Features

Table 1  Characteristics of the family members

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Phenotypes

Yıldız Bölükbaşı E, et al. J Med Genet 2017;0:1–9. doi:10.1136/jmedgenet-2017-104758

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Phenotypes

Figure 3  Genotypes of family members for the six variants that we considered relevant to the disease. *Available for genetic studies; +Available for mutation analysis; #Exome sequenced individuals.

Figure 2  Pictures of autopods of patients. To assess whether the mutational load in BBS-related genes in our patients are heavier than in a control group, we screened 10 unrelated Pakistani exomes for exonic and splicing variants with frequencies T as a third BBS allele were reported to be more severely affected.8 As compared with control individuals a significantly higher fraction of the total 226 patients with BBS screened were found to carry this variant, and a strong overtransmission of the variant was observed. In contrast, the variant had not led to a more severe phenotype in 52 other patients.44 We did not find a genotype–phenotype correlation; homozygote 502 had a rather mild phenotype, as he had polydactyly only in the feet, did not have ID or developmental delay and was not obese. The variant resulted in only approximately 30% of wild-type mRNA molecules in an unaffected homozygote.8 ExAC reports its frequency in South Asians as 0.01075 (102 alleles in 9484). MKKS c.1015A>G (p.Ile339Val) was found in two unrelated patients with BBS in the heterozygous state but not in an affected sibling.45 46 Residue Ile339 is not highly conserved in mammals. Computational algorithms SIFT, PolyPhen-2 and Mutation Taster predicted the variant to be tolerated, benign and polymorphism, respectively, and ClinVar lists it as likely benign (see online supplementary table S4). ExAC reports its frequency as 0.006275 (41 alleles in 6534, three homozygotes) in Europeans and as 0.00604 (98 alleles in 16 224, two homozygotes) in South Asians. It might be a modifier allele in our patients as well, as the severe cases are among the heterozygotes for the allele and those with milder phenotypes are non-carriers. Missense C8ORF37 (BBS21) variant and synonymous TMEM67 variant are both predicted to be damaging by algorithms and have ExAC South Asian frequencies of 0.000548 (nine alleles in 16 422) and 0.02291 (378 alleles in 16 498), respectively. We were unable to make any prediction of a potential structural and/or functional impact of these alleles since the protein structures were not available. All of our patients carry at least one modifier/possible modifier allele, and the most severe of them (503) carries all five of those alleles, one in the homozygous state, but patient 501 with mild phenotype has the same genotype. It might well be that there are yet other modifier alleles in the family. Hence, our findings do not refute oligogenic inheritance (as no patient is without a modifier allele), but whether it supports modifier hypothesis is not clear, as a straightforward genotype–phenotype correlation was not observed. We attempted to assess whether the modifiers/possible modifiers we found might be circumstantial and evaluated 10 unrelated Pakistani exomes as controls. We searched for exonic and splicing variants similarly in the same genes and found that patients had a highly significantly more of such variants (PT was not affected, and another unaffected father was homozygous for BBS1 Met390Arg (as his BBS sibs) but not carrying the CCDC28B variant.8 We cannot speculate whether the GLI1 variant we identified is damaging in the homozygous state in humans, as we did not observe homozygosity in any unaffected individual. Of note, Gli1 knockout mice are viable and seem normal.47 We did not find any reported evidence for interaction between CEP19 and GLI1. Future studies could unravel whether the proteins of the three possible modifier genes we identified herein interact with CEP19 and/or one another and the variants contribute to the disease phenotype. The finding of a high number of modifier/possible modifier alleles in our patients supports cumulative mutational load hypothesis. A model of total mutational load of ciliary signalling was proposed to explain complex inheritance in BBS,8 and phenotypic variability was proposed to be due to a different mutational load in cilia-associated genes among siblings.48 In summary, we identified CEP19 p.Tyr65* as responsible for BBS in the family and GLI1 Gly274Arg as a possible modifier of severity, and the three variants in other BBS-related genes could be exacerbating the disease severity. We hope that our findings would facilitate the furthering of our understanding of the molecular processes underlying BBS. Including CEP19 in BBS screening panels could benefit families, considering that only approximately 80% of all patients with BBS studied to date have mutations in known BBS genes,1 6 and usually polydactyly is the only obvious feature at birth, and hence, the disease is diagnosed in late childhood. Acknowledgements  We would like to thank the family members for their cooperation. We would also like to thank Rashid Qureshi and Muhammad Sadiq for making the extended clinical evaluation possible. Contributors  AT and SajM were responsible for the concept and design of the study. EYB, SarM and UW generated and interpreted the data. SajM and MA contributed clinical data. AT, SajM, EYB and UW drafted the manuscript. All authors revised the manuscript. Funding  This research was supported by Boğaziçi University Research Fund (project 10860; to AT), the Scientific and Technological Research Council of Turkey (114Z829; to AT) and URF-QAU, Pakistan (to SajM). UW is supported by FONDECYT no. 1150743. Competing interests  None declared. Patient consent  Guardian consent obtained. Ethics approval  Informed consent was obtained from/for participants in accordance with the regulations of the Ethical Review Committee of Quaid-i-Azam University and Boğaziçi University Institutional Review Board for Research with Human Participants, both of which approved the study protocol. Provenance and peer review  Not commissioned; externally peer reviewed. 7

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References

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Homozygous mutation in CEP19, a gene mutated in morbid obesity, in Bardet-Biedl syndrome with predominant postaxial polydactyly Esra Yildiz Bölükbasi, Sara Mumtaz, Muhammad Afzal, Ute Woehlbier, Sajid Malik and Aslihan Tolun J Med Genet published online November 10, 2017

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