Clin Genet 2014 Printed in Singapore. All rights reserved

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: 10.1111/cge.12547

Original Article

Resolving clinical diagnoses for syndromic cleft lip and/or palate phenotypes using whole-exome sequencing Pengelly R.J., Upstill-Goddard R., Arias L., Martinez J., Gibson J., Knut M., Collins A.L., Ennis S., Collins A., Briceno I. Resolving clinical diagnoses for syndromic cleft lip and/or palate phenotypes using whole-exome sequencing. Clin Genet 2014. © John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2014 Individuals from three families ascertained in Bogota, Colombia, showing syndromic phenotypes, including cleft lip and/or palate, were exome-sequenced. In each case, sequencing revealed the underlying causal variation confirming or establishing diagnoses. The findings include very rare and novel variants providing insights into genotype and phenotype relationships. These include the molecular diagnosis of an individual with Nager syndrome and a family exhibiting an atypical incontinentia pigmenti phenotype with a missense mutation in IKBKG. IKBKG mutations are typically associated with preterm male death, but this variant is associated with survival for 8–15 days. The third family exhibits unusual phenotypic features and the proband received a provisional diagnosis of Pierre Robin sequence (PRS). Affected individuals share a novel deleterious mutation in IRF6. Mutations in IRF6 cause Van der Woude and popliteal pterygium syndrome and contribute to nonsyndromic cleft lip phenotypes but have not previously been associated with a PRS phenotype. Exome sequencing followed by in silico screening to identify candidate causal variant(s), and functional assay in some cases offers a powerful route to establishing molecular diagnoses. This approach is invaluable for conditions showing phenotypic and/or genetic heterogeneity including cleft lip and/or palate phenotypes where many underlying causal genes have not been identified. Conflict of interest

The authors declare no conflicts of interest.

R.J. Pengellya,† , R. Upstill-Goddarda,† , L. Ariasb,† , J. Martinezb,† , J. Gibsonc , M. Knuta , A.L. Collinsd , S. Ennisa , A. Collinsa and I. Bricenob a Genetic

Epidemiology and Genomic Informatics, Faculty of Medicine, University of Southampton, Southampton, UK, b Department of Biomedical Sciences, Medical School, Universidad de La Sabana, Bogota, Colombia, c Centre for Biological Sciences, Faculty of Natural & Environmental Sciences, University of Southampton, Southampton, UK, and d Department of Clinical Genetics, Southampton General Hospital, Southampton, UK † These

authors equally contributed.

Key words: cleft lip and palate – exome sequencing – incontinentia pigmenti – Nager syndrome – Pierre Robin sequence – syndromic disease Corresponding author: Prof Andrew Collins, Genetic Epidemiology and Genomic Informatics, Faculty of Medicine, University of Southampton, Duthie Building (808), Tremona Road, Southampton, SO166YD, UK. Tel.: +44(0)2381206939; fax: +44(0)2380794264; e-mail: [email protected] Received 15 October 2014, revised and accepted for publication 26 November 2014

Cleft lip and/or palate (CLP) is a phenotypic feature of at least 275 genetic syndromes that arise through single-gene mutations, chromosomal abnormalities or teratogens (1). The syndromic designation refers to the

presence of additional physical or cognitive abnormalities, along with CLP. About 75% of CLP syndromes have a known genetic cause and include many arising through Mendelian inheritance at single genetic loci.

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Pengelly et al. An important example is Van der Woude syndrome (VWS), and its allelic disorder popliteal pterygium syndrome (PPS), which is caused by mutations in the IRF6 gene (2). VWS is the most common cause of syndromic clefting accounting for 2% of CLP cases. This gene functions as a transcriptional activator and shows high allelic heterogeneity as 100s of mutations in IRF6 have been reported to cause these disorders. The DNA-binding domains of IRF6 are particularly enriched for causal mutations, but mutations are also found extensively throughout the protein-binding domain (3). Furthermore, IRF6 mutations have also been linked to nonsyndromic forms of CLP (4). Because high allelic heterogeneity underlies many syndromes, targeted next-generation sequencing (NGS) of individual genes or panels of genes provides a route to establish molecular diagnoses that inform clinical management. A number of standard gene sequencing panels have been developed and provide particularly cost-effective routes to exploiting these technologies in a clinical setting. In contrast to standard panels, ‘custom’ gene panels are more expensive when used for a few samples but less so for larger sample sizes (5). However, developing optimal gene panels to screen samples representing genetically and phenotypically heterogeneous diseases or syndromes can be difficult particularly where there are ambiguous genotype–phenotype correlations. A cost-effective alternative strategy is to employ whole-exome sequencing (WES) which identifies the majority of coding variants in a DNA sample. For syndromic conditions, where underlying mutations are most likely to show Mendelian patterns of inheritance, exome sequencing, which screens up to 3% of the genome, is a powerful strategy to identify causal variation. As part of a study into the genetic basis of CLP phenotypes in Colombia, the utility of the WES strategy for CLP syndromes in families with particularly rare and/or atypical clinical phenotypes is considered here. We describe the results from the exome sequencing of six individuals from three families. Results confirm that genetic variants underlying CLP phenotypes in these Colombian families comprise both known and novel variants and establish new variant: phenotypic relationships. Materials and methods Patients

Families were ascertained at Operation Smile, Bogota, Colombia. All affected individuals or their parents gave written informed consent for the study. Ethical permission was obtained from the Research Ethics Committee at the Universidad de La Sabana, Bogota. The main clinical findings are summarized in Table 1, and the pedigrees in Figs. 1 and 2. Exome sequencing

Samples from six individuals with syndromic phenotypes from three families were exome-sequenced. DNA derived from peripheral blood was sequenced for five

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Table 1. Description of the probands Proband diagnosis/ N affected Family in family/N number exomes sequenced CLP1

CLP2

CLP3

Phenotype

Nager syndrome/1/1

Age at examination: 9 years; history of swallowing disorder because of retrognathia; bilateral dacryostenosis; micrognathia; atresia of the right external auditory canal; agenesis of first finger (bilateral); and normal external genitalia. Incontinentia Age at examination: 34 years; pigmenti/11/2 cleft lip palate on left side; nail hyperpigmentation; nail clubbing; and cutaneous syndactyly. Pierre Robin syndrome Age at examination: /5/3 18 months; history of respiratory failure because of micrognathia; and cleft palate.

individuals during 2012–2013 on an Illumina HiSeq 2000 sequencer at the Wellcome Trust Centre for Human Genomics using the Agilent SureSelect v5 capture kit encompassing 51 Mb of genome sequence. A sample from an additional individual (CLP2, II10, the half-uncle of the proband) was exome-sequenced at the Beijing Genomics Institute (BGI) during 2014. Paired-end exome sequence reads were aligned to the hg19 human reference genome using novoalignMPI (V2.08.02, Novocraft Technologies, Selangor, Malaysia). Picard (v1.34) and SAMtools (v0.01.18) were used to merge, sort and manipulate format aligned sequence files (Sam and Bam files) and create a ‘pileup’ of reads for each sample. Coverage statistics were calculated using BEDTools (v2.13.2) and are described in Table S1, Supporting Information. The mean read depths across the exomes are in the range 57–128. Sample provenance was ensured using an optimized panel of 24 SNPs (6). For filtering of single nucleotide polymorphism (SNP) variants and indel calls, we established a comprehensive list of genes previously implicated in any form of CLP phenotype including search terms related to the clinical diagnoses made for the patients. First, we queried the Human Gene Mutation Database (HGMD professional http://www.hgmd.org/), in July 2014, using the following search terms: cleft lip, cleft palate, cleft, syndactyly, brachydactyly, Pierre Robin, incontinentia pigmenti, Nager syndrome, hyperpigmentation, craniofacial, clubbing, dysmorphic, dysmorphia and micrognathia. This list comprised 363 genes. Additional genes were included after a corresponding interrogation of OMIM (http://omim.org/, accessed July 2014), and a small number of additional CLP-related genes from the review

III2

III3

III4

III5

III6

III7

III8

II2

I V1

female

III9

II3

I V2

P I I I 10

II4

I V3

I I I 11

II5

I2

I I I 12

II6

I I I 13

II7

I I I 14

II8

I I I 15

II9

I I 10

I3

III1

II2

II3

II4

I2

II5

III2

II6

II7

II8

II9

II10

I3

II11

I4

II12

I5

III3

II13

P III4

II14

Fig. 2. Pedigree of family CLP3. Phenotypes of affected individuals: II5 and III1 unilateral cleft lip and palate; III2 bilateral cleft lip and palate (exome-sequenced); II9 cleft palate (exome-sequenced); III4 (proband, exome-sequenced) cleft palate, micrognathia.

II1

I1

Fig. 1. Pedigree of family CLP2. II5, II6, II7 facial clefting, cause of death uncertain; II10 (half-uncle of proband) facial clefting, syndactyly, proximal thumbs, brachydactyly (exome-sequenced) ; III2, III3, III4, II5 (males) post-natal death at 8–15 days and facial clefting; III6 (female) post-natal death at 8 days and cleft lip and palate; IV1 pre-natal death and facial clefting; III10 (proband, exome-sequenced), unilateral (left side) cleft lip and palate, clubbing, nail hyperpigmentation, cutaneous syndactyly.

III1

II1

I1

Resolving clinical diagnoses for syndromic cleft lip and/or palate phenotypes

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Pengelly et al. were also included by Collins et al. (7). The complete list of 865 genes considered in variant filtering is given in Table S2. We filtered the lists of called variants to identify all novel non-synonymous (NS), stopgain, stoploss, splicing and indel variants in these genes as well as known rare variants with an allele frequency of less than 1% in the 1000 Genomes Project database (http://www.1000genomes.org/) (Table 2 and Table S3). More frequent variants were excluded from further consideration as unlikely causes of rare syndromic disease. For NS variants, we used the scaled predictive scores (8, 9) from dbNSFP v2 (10, 11) and only considered NS variants classed as deleterious or damaging by any of: PhyloP (larger positive scores represent conserved sites while negative scores indicate non-conserved sites) (12); SIFT (scores 0.95 considered damaging; 16) and GERP++ (scores range from 100) (19). All variants were also annotated with combined scores for deleteriousness: PHRED-scaled CADD (higher scores indicate that a variant is more likely to be deleterious) (20); Logit (the conditional probability that a variant is Mendelian disease-causing given prediction scores from 13 programs, including sift, PolyPhen2, LRT, MutationTaster, PhyloP, GERP++ and CADD, under a logistic regression model) (10, 11). We also produced a combined rank for variants with PhylopP, GERP++, CADD and Logit scores based on the summed ranks across all four scores (Table 2 and Table S3). We excluded variants found in homopolymer/repeat regions that can arise through miss-alignment between the sequenced reads and reference sequence. Any variants with read depth of