M indicates mutation; WT, wild-type.
Color fundus photographs (top left), fundus autofluorescence (FAF) imaging (top right), and optical coherence tomography (OCT) (bottom) for patients 3 (A), 4 (B), and 5 (C) (right eye [RE]), patient 8 (left eye [LE]) (D), and patient 9 (both eyes) (E). Patient 3, patient 4, and patient 5: midperipheral retinal pigment epithelium (RPE) atrophy with bone spicule hyperpigmentation and reduced autofluorescence in the midperiphery with a ring of increased macula autofluorescence corresponding to preserved inner segment ellipsoid (ISe) band on OCT. In patient 3, an epiretinal membrane was also present on OCT; in patient 4, reduced perifoveal dots of autofluorescence were also present; and in patient 5, vitreomacular traction with retinal cysts of the inner nuclear layer were also present on FAF imaging and OCT. Patient 8: midperipheral RPE atrophy and pigmentary change as before but with a large ring of increased autofluorescence outside of the macula on FAF imaging, with reduced anterior autofluorescence, and preserved retinal layers on OCT imaging. Patient 9: color fundus imaging from 2011 demonstrates narrowing of the vessels only; FAF imaging was normal in 2011, but in 2016, rings of increased autofluorescence were present in both eyes, as well as a partially preserved ISe band in 2011 with reduction in size demonstrated in 2016.
Full-field ERGs and pattern ERGs (PERGs) in patients 4 (age, late 30s), 5 (age, early 50s), 7 (age, early 40s), and 8 (age, late 30s), as well as traces from a representative individual without retinitis pigmentosa for comparison. The ERGs showed a high degree of interocular symmetry and are shown for 1 eye only; responses are consistent with rod-cone dystrophy. The PERG results were normal in 1 eye of patient 4 (mildly subnormal in the other eye) and showed reduction indicating symmetrical mild to severe macular dysfunction in the other patients. DA indicates dark adapted; LA, lighted adapted; and LF, large field.
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Hull S, Attanasio M, Arno G, et al. Clinical Characterization of CNGB1-Related Autosomal Recessive Retinitis Pigmentosa. JAMA Ophthalmol. 2017;135(2):137–144. doi:10.1001/jamaophthalmol.2016.5213
What can a detailed clinical and molecular genetic study of patients with CNGB1-related retinitis pigmentosa reveal about the disease presentation and progression?
This case series of 10 patients with retinitis pigmentosa identified childhood onset of nyctalopia with preserved visual acuity and central photoreceptors into adulthood.
The findings of this case series suggest that retinitis pigmentosa due to variants in CNGB1 is slowly progressive with a long potential treatment window.
There are limited published data on the phenotype of retinitis pigmentosa (RP) related to CNGB1 variants. These data are needed both for prognostic counseling of patients and for understanding potential treatment windows.
To describe the detailed clinical and molecular genetic findings in a series of patients with RP with likely pathogenic variants in CNGB1.
Design, Setting, and Participants
In this case series, 10 patients from 9 families underwent full ophthalmologic examination. Molecular investigations included whole-exome analysis in 6 patients. The study was conducted from April 17, 2013, to March 3, 2016, with final follow-up completed on March 2, 2016, and data were analyzed from October 27, 2014, to March 29, 2016.
Main Outcomes and Measures
Results of ophthalmologic examination and molecular genetic analysis of CNGB1.
In this case series, 7 women and 3 men from 9 families with a mean (SD) age of 47.4 (13.2) years identified as having CNGB1 variants were included in this study; there was a mean (SD) follow-up length of 3.7 (2.8) years. The first clinical presentation was with nyctalopia in childhood with visual field loss documented later at a mean (SD) age of 33.2 (8.0) years. All patients had preserved best-corrected visual acuity into adulthood, with a mean of 0.1 logMAR (Snellen equivalent, 20/25) in each eye (logMAR range, 0.0 to 0.3 [Snellen 20/20 to 20/40] in the right eye and −0.1 to 0.3 [Snellen 20/16 to 20/40] in the left eye). Fundus examination revealed midperipheral retinal pigment epithelial atrophy and intraretinal pigment migration. Optical coherence tomography of the macula demonstrated complete preservation of the inner segment ellipsoid band in 1 patient, with variable lateral extent in the other patients corresponding to the diameter of a paracentral ring of increased fundus autofluorescence. Electrophysiologic testing in 6 patients confirmed a rod-cone dystrophy phenotype. Molecular investigations identified a previously reported missense variant (p.[N986I]) and 7 variants not previously reported in disease including 4 nonsense (p.[(Q88*], p.[Q222*], p.[Q318*], and p.[R729*]), 2 frameshift (p.[A1048fs*13], p.[L849Afs*3]), and a splice site variant (c.761 + 2T>A).
Conclusions and Relevance
The data from this study suggest that visual acuity and foveal structure in patients with RP are preserved into adult life such that a lengthy window of opportunity should exist for intervention with novel therapies.
Retinitis pigmentosa (RP) is an inherited disorder characterized by a progressive retinal dystrophy with primarily rod photoreceptor dysfunction at presentation. The disorder is highly heterogeneous and affects approximately 1 in 4000 individuals worldwide.1-4 Clinically, RP is characterized by night blindness, progressive constriction of peripheral visual fields, and, ultimately, in most patients, reduced visual acuity. The fundus typically shows midperipheral, intraretinal pigment migration associated with retinal pigment epithelium (RPE) atrophy, attenuated retinal vessels, and pallor of the optic nerve head.4-6
Approximately 15% to 20% of RP cases are autosomal dominant; 15% are autosomal recessive; 7% are X-linked; 43% are sporadic or simplex cases, most of which are likely to be autosomal recessive; and 15% are unknown.6,7 Rarely, RP may be caused by mutations in mitochondrial DNA.4 Genes associated with RP encode proteins that have a key role in retinal structure and function, including phototransduction or the visual cycle. Some genes are ubiquitously expressed but have a phenotype confined to the eye.6,8,9
Variants in genes encoding the 2 rod cyclic nucleotide-gated (CNG) channel subunits have been associated with autosomal recessive RP.8,10 The CNG channels are nonselective cation channels localized to the plasma membrane of rod and cone photoreceptors that translate light-mediated changes of second-messenger cyclic guanosine monophosphate into voltage signals.11,12 The CNG channels in rods form heterotetramers consisting of 3 α-subunits (CNGA1) and 1 β-subunit (CNGB1), whereas the cone channel is formed by 2 α-subunits (CNGA3) and 2 β-subunits (CNGB3).13,14 Variants in CNGB1 are an uncommon cause of RP, accounting for approximately 4% of autosomal recessive RP cases; there are limited reports describing the associated phenotypes.10,15-20 The present report describes the detailed clinical features of 10 affected patients harboring likely pathogenic variants in CNGB1.
Ten patients from 9 families were ascertained from the inherited retinal clinics of Moorfields Eye Hospital and Great Ormond Street Hospital for Children. The study was conducted from April 17, 2013, to March 3, 2016, with final follow-up completed on March 2, 2016. Data were analyzed from October 27, 2014, to March 29, 2016.
Informed written consent and peripheral blood samples were obtained for genetic analysis from all participants according to the protocols approved by the Research Management Committees of Moorfields Eye Hospital and Great Ormond Street Hospital for Children, in agreement with the Declaration of Helsinki.21 There was no financial compensation.
An accurate family history of each patient was recorded, and all underwent a complete ophthalmic examination, which included best-corrected visual acuity, slitlamp biomicroscopy of the anterior segment, and dilated fundus examination. Retinal fundus photographs were obtained by conventional 35° fundus color photographs (Topcon Great Britain Ltd) and in 1 patient by ultra-widefield (up to 200°) confocal laser scanning ophthalmoscopy (Optos plc). Fundus autofluorescence (FAF) imaging (30° and 55°) was performed with a confocal scanning laser ophthalmoscope (HRA+OCT Spectralis; Heidelberg Engineering Ltd). An optically pumped solid-state laser (488 nm) was used for excitation, and a 500-nm barrier filter was used to modulate the reflected light. Spectral-domain optical coherence tomography (OCT) was performed (HRA+OCT Spectralis). Optical coherence tomography imaging was acquired by a broadband 870-nm superluminescent diode that scanned the retina at 40 000 A-scans per second with an optical depth resolution of 7 μm. In particular, the central subfield thickness and integrity of the inner segment ellipsoid (ISe) band of the photoreceptors were assessed in the maculae of both eyes of all patients. Central subfield thickness was measured using an automated viewing module (Spectralis, version 22.214.171.124; Heidelberg Engineering Ltd) with slices visually inspected for segmentation accuracy.
Full-field electroretinography (ERG) (6 patients) and pattern ERG (PERG) (5 patients) were performed to incorporate the International Society for Clinical Electrophysiology of Vision Standards.22,23 The ERGs were recorded under dark-adapted conditions to flash strengths of 0.01 and 10.0 candelas/s/m−2 (cd/s/m−2) and light-adapted ERGs to a flash strength of 3.0 cd/s/m−2 (30 Hz and 2 Hz). An additional larger-field PERG (30° × 24°) was recorded in 2 patients as previously described.24
Genomic DNA was isolated from peripheral blood lymphocytes (Puregene kit; Gentra Puregene Blood Extraction Kit; QIAGEN). Whole-exome sequencing was performed on patients 1 to 5 and patient 9 as part of the National Institute for Health Research BioResource–funded Specialist Pathology: Evaluating Exomes in Diagnostics study (Cambridge Biomedical Centre). As part of this study, more than 600 unrelated patients from Moorfields Eye Hospital and Great Ormond Street Hospital with a range of inherited retinal diseases underwent whole-exome sequencing or whole-genome sequencing, with patients 1 to 5 and patient 9 all from the exome cohort. Exome enrichment was performed (EZ 64 Mb Human Exome Library, version 3.0; Roche NimbleGen, Inc). The libraries were sequenced (HiSeq 2000; Illumina). Reads were aligned to the GRCh37 reference genome (NovoAlign, version 2.08.03; Novocraft Technologies Sdn Bhd). Duplicate reads were marked (Picard tools MarkDuplicates; Mark Institute). Calling was performed using the haplotype caller module of Genome Analysis Toolkit, version 3.3-0 (GATK) (https://www.broadinstitute.org/gatk), creating genome Variant Call Formatted (GVCF) files for each sample. The individual GVCF files for the exomes discussed in this study, in combination with approximately 3000 clinical exomes (University College London Exomes Consortium), were combined into merged VCF files for each chromosome containing, on average, 100 samples each. The final variant calling was performed using the GATK GenotypeGVCFs module jointly for all samples (cases and controls). Variant quality scores were then recalibrated separately according to GATK best practices for indels and single-nucleotide polymorphisms. Resulting variants were annotated using ANNOVAR (http://www.openbioinformatics.org) based on Ensembl gene and transcript definitions. Candidate variants were filtered based on function (nonsynonymous, presumed loss of function or splicing, defined as intronic sites within 5 base pairs of an exon-intron junction) and minor allele frequency (<0.5% minor allele frequency in our internal control group, as well as the National Heart, Lung, and Blood Institute GO Exome Sequencing Project data set (EVS; http://evs.gs.washington.edu/EVS/).
Next-generation sequencing of the coding regions of 105 genes for patients 7 and 8 and, more recently, for 176 retinal genes for patient 10 was performed at the Manchester Centre for Genomic Medicine (Manchester, England) with enrichment (SureSelect Target Enrichment Kit; Agilent Technologies Inc) and then run on a sequencer (SOLiD 4; Life Technologies).25 More than 500 unrelated patients with a range of inherited retinal dystrophies recruited from Moorfields Eye Hospital have undergone this molecular investigation.
Confirmatory bidirectional Sanger sequencing of CNGB1 was performed in all probands and available family members. Amplification of DNA was performed using specifically designed primers by polymerase chain reaction, and the resulting fragments were sequenced using standard protocols.
Variant nomenclature was assigned in accordance with GenBank Accession number NM_001297 .4, with nucleotide position 1 corresponding to the A of the ATG initiation codon. Variants were identified as novel if not previously reported in the literature and if absent from dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/), EVS, and the Exome Aggregation Consortium database (ExAC; http://exac.broadinstitute.org) containing 61 486 exomes, all accessed on March 21, 2016. Where relevant, potential splice site disruption was assessed using Splice Site Prediction by Neural Network (http://www.fruitfly.org/seq_tools/splice.html).
The series consisted of 10 patients (7 women). Eight were white British individuals, 1 was Bangladeshi (patient 4), and 1 was Afghani (patient 9). The clinical findings are summarized in the Table, with family pedigrees and identified variants shown in Figure 1. Mean (SD) age at last review was 47.4 (13.2) years (range, 15-65 years) with a mean follow-up of 3.7 (2.8) years (range, 0-11 years). The initial symptom in all patients was nyctalopia, with onset from infancy to 14 years. No patient reported photophobia. A fine nystagmus was observed in patient 9. Symptomatic peripheral visual field loss occurred later at a mean age of 33.2 (8.0) years (range, 13-40 years), although it was detectable on formal kinetic perimetry in patient 9 at 12 years. Six of 10 patients developed visually significant posterior subcapsular lens opacities in both eyes during follow-up, with subsequent cataract surgery.
Mean best-corrected visual acuity was 0.1 logMAR (20/25 Snellen) in the right eye (range, 0.0 to 0.3; Snellen 20/20 to 20/40) and 0.1 logMAR in the left eye (range, −0.1 to 0.3; Snellen 20/16 to 20/40). Myopic refractive errors were present in 3 of the patients for whom data were available. Confrontation visual field testing demonstrated variable peripheral field loss in all participants. There was sparing of the central 20° to 30° in 7 patients, sparing of the central 10° to 20° in 1 patient, and sparing of the central 5° to 10° in 2 patients, with documented slow progression during follow-up.
Fundus examination of all participants but patient 9 revealed arteriolar attenuation, optic disc pallor, RPE atrophy, and midperipheral intraretinal pigment migration. For patient 9, the youngest participant, narrow retinal vessels and midperipheral RPE mottling were the only observable abnormalities at 18 years (Figure 2). Perifoveal RPE atrophy was additionally present in patients 1, 4, and 10.
Fundus autofluorescence imaging showed a loss of autofluorescence in the midperiphery, with macular or perimacular rings of increased autofluorescence in all patients. Patient 9, with initial preserved autofluorescence, developed a macular ring of increased autofluorescence over a 5-year follow-up period (Figure 2). Three patients (patients 1, 4, and 10) had an additional patchy perifoveal ring of reduced autofluorescence corresponding to their perifoveal RPE atrophy.
One patient (patient 8) with a large paracentral ring of FAF had complete preservation of the ISe band evident on OCT (Figure 2). In other participants, the lateral extent of the ISe band corresponded to the diameter of the ring of increased signal on FAF, with the most severe loss of the ISe band in patients 1 and 10. In addition, both eyes of patients 3 and 6 and the left eye of patient 7 had an epiretinal membrane; patient 5 had bilateral vitreomacular traction associated with macular edema, and patient 10 had a small, left lamellar macular hole without apparent vitreomacular traction. Interval OCT imaging during a 5-year period in patient 9 demonstrated a marked reduction in the diameter of the ISe band (Figure 2). Mean central subfield thickness, excluding patient 5 (macular edema) and patient 9 (information unavailable), was 302.3 (35.0) μm in the right eye and 295.3 (33.0) μm in the left eye compared with a mean normative value of 270.2 (22.5) μm.26 Excluding patients with all concurrent macular abnormality resulted in similar values for the central subfield thickness for patients 1, 2, 4, 7 (right eye), 8, and 10 (right eye) of 297.7 (37.1) μm in the right eye and 291.8 (42.8) μm in the left eye.
Full-field ERG and PERG were performed in 6 patients at the mean age of 40 (13.2) years. Full-field ERG performed in patient 9 at 13 years showed profoundly attenuated rod-specific responses (dark-adapted, 0.01) with subnormal and delayed cone responses. In the other 5 patients, rod-specific responses (dark-adapted, 0.01) were undetectable bilaterally; the brighter flash dark-adapted ERGs (dark-adapted, 3.0 and 10.0) showed markedly reduced or undetectable function from both eyes (Figure 3). Light-adapted 30-Hz flicker ERGs and single-flash cone ERG b-waves were delayed and subnormal in most patients, subnormal without delay in patient 8, and with only a residual single flash cone ERG detectable in patient 7. The PERG P50 responses in 5 patients ranged from undetectable to normal (Table, Figure 3). Patients 7 and 8 underwent large-field PERG testing, with lack of enlargement of the response for patient 7 indicating marked paracentral retinal dysfunction and the expected enlargement relative to the standard PERG for patient 8 indicating relative preservation of paracentral macular function (Figure 3).
Likely pathogenic variants in CNGB1 were identified in all 9 probands and, after segregation analysis, in a further 3 affected family members, 1 of whom was also available for examination (patient 6). One previously reported variant, c.2957A>T p.(N986I) in exon 29, was identified in patients 2, 3, 5, 7, and 8 (Figure 1).16 Four novel variants were detected: 3 nonsense, c.262C>T p.(Q88*) in exon 3, c.664C>T p.(Q222*) in exon 10, c.2185C>T p.(R729*) in exon 22, and 1 splice site variant c.761 + 2T>A in intron 10 predicted to abolish the canonical splice donor site. These were all absent from the ExAC database. In addition, 3 variants were detected that had not been previously reported in an affected patient but were present at a very low allele frequency in the ExAC database: c.952C>T p.(Q318*) in exon 13 (1 of 120 768 alleles), c.3142_3143insGTGG p.(A1048fs*13) in exon 31 (2 of 120 522 alleles), and c.2544dupG p.(L849Afs*3) in exon 26 (4 of 120 644 alleles). For patient 3 and patients 5 and 6, further segregation to establish the phase was not possible from either the antecedents or children; however, the 2 variants found segregated with additional affected individuals in both families (Figure 1).
This report describes the findings in 10 patients (7 women and 3 men) from 9 families with a typical RP phenotype and likely pathogenic variants in CNGB1. Seven likely pathogenic variants not previously reported in an affected patient were identified.
There are limited published data on the CNGB1 retinal phenotype.10,15-20 To our knowledge, only 7 families have been identified with recessive RP due to CNGB1 comprising 3 missense variants, 3 splice site variants, and 1 frameshift variant. Of the 4 families with clinical details, there was a childhood onset of nyctalopia with a later development of peripheral visual field loss reported in 4 patients at 10, 20, and 30 (2 patients) years.10,15,17,19 Severe loss of visual acuity was present in 3 patients at 24, 57, and 67 years. There were fundus abnormalities typical of RP with midperipheral RPE atrophy and intraretinal bone-spicule pigmentation and variable macular atrophy. Two patients had undetectable rod responses on ERG and severely abnormal cone responses at 24 and 30 years, and 1 patient had undetectable ERG responses at 44 years; PERG was not performed. The patients in the present series had similar features: onset of nyctalopia was seen in childhood with symptomatic visual field loss occurring later, central visual acuity was preserved well into adult life, fundus abnormalities were consistent with RP, and electrophysiologic testing demonstrated a rod-cone dystrophy phenotype. Pattern ERGs showed variable degrees of central or paracentral macular involvement and could be relatively preserved in patients with ERG evidence of severe generalized photoreceptor dysfunction.
To our knowledge, this is the first report to describe retinal imaging other than fundus appearance; all patients demonstrated reduced midperipheral autofluorescence, with macular rings of increased autofluorescence corresponding to the size of the remaining ISe band. Abnormal parafoveal rings of increased FAF have been reported in approximately 59% of patients with RP.27 All patients in the present series demonstrated such abnormal rings; the largest FAF ring surrounded an area that included most of the vascular arcades in a patient (patient 8) with OCT evidence of preserved outer retina (Figure 2) and relatively preserved PERG (Figure 3). The diameter of smaller FAF rings corresponded with the lateral limit of the remaining OCT ISe band, consistent with previous studies of patients with RP that have shown spatial correspondence or correlation between these variables.28,29 Central subfield thickness was within normal limits. The findings of our study suggest that RP associated with variants in CNGB1 has a good prognosis for central vision despite the early onset of night blindness. The good visual prognosis is reflected by preserved central macular thickness and morphology of the ISe band of the photoreceptors even in adults.
A total of 8 different variants in CNGB1 were identified. The previously reported missense variant, p.(N986I), was detected in 5 patients, all British white individuals, suggesting it to be a common CNGB1 variant in this population.16 p.(N986I) is found in 133 of 120 752 alleles with no homozygotes in the ExAC database (minor allele frequency, 0.0011), including 84 of 66 728 non-Finnish European alleles (minor allele frequency, 0.0013). Patient 1, who was biallelic for nonsense variants, had a more severe phenotype compared with the other patients; the best-corrected visual acuity was 0.3 logMAR (Snellen 20/40) at 47 years, and the visual field was restricted to the central 10° in both eyes. This patient is predicted as being nullizygous for CNGB1 due to nonsense-mediated decay of the transcribed messenger RNA as are patients 4, 9, and 10, suggesting that there is no direct correlation between predicted nullizygous variants and phenotype severity.30 Of the previously reported patients with clinical details, 2 had splice site variants (c.413-1G>A and c.3444 + 1G>A) and visual loss at 24 and 67 years, respectively; 2 patients with missense variants had preserved central vision into at least their fourth decades.10,15,17,19 Both splice site variants have been shown in vitro to lead to aberrant splicing and premature termination codons.19,31 To our knowledge, there is no demonstrable genotype-phenotype correlation. Further functional work and larger numbers of patients may help to elucidate potential associations.
The slowly progressive RP phenotype in patients with CNGB1 variants is consistent with findings in prior canine and murine model studies.32-34 In Papillon dogs with a homozygous frameshift variant in CNGB1, there was marked reduction or absence of rod ERG responses with a partial preservation of cone ERGs.32 Optical coherence tomography imaging of the central macula when it was fully developed (at approximately 8 weeks) showed retinal layer thickness comparable to that of a normal control with a gradually progressive thinning of the outer nuclear layer with age, confirming a slow retinal degeneration. In Cngb1−/− mice, a progressive loss of rod photoreceptor function was noted with a later degeneration of cone photoreceptors.33 The degeneration was slow with loss of 20% to 30% of rods at 4 months, 30% to 50% at 6 months, and 80% to 90% at 1 year. Although the rods degenerated early, cone photoreceptors started to degenerate only at 6 months and were still present at 11 months.33,34
This report expands the phenotype of patients with RP due to variants in CNGB1 and describes 7 additional pathogenic variants. The phenotype, similar to that in previous reports in patients and animal models, indicates slow degeneration and therefore a lengthy window of opportunity for therapeutic intervention. The results from proof-of-concept gene therapy studies in a Cngb1 knockout mouse model lead to optimism that human RP associated with variants in CNGB1 may ultimately be treated successfully using a similar approach.35
Corresponding Author: Andrew R. Webster, MD (Res), FRCOphth, University College London Institute of Ophthalmology, 11-43 Bath St, London EC1V 9EL, England (firstname.lastname@example.org).
Accepted for Publication: November 1, 2016.
Published Online: January 5, 2017. doi:10.1001/jamaophthalmol.2016.5213
Author Contributions: Drs Hull and Attanasio contributed equally to this work, had full access to all of the data in this study, and take responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design: Hull, Attanasio, Michaelides, Raymond, Moore, Webster.
Acquisition, analysis, or interpretation of data: Hull, Attanasio, Arno, Carss, Robson, Thompson, Plagnol, Michaelides, Holder, Henderson, Raymond, Webster.
Drafting of the manuscript: Hull, Attanasio, Plagnol, Michaelides, Webster.
Critical revision of the manuscript for important intellectual content: Arno, Carss, Robson, Thompson, Michaelides, Holder, Henderson, Raymond, Moore, Webster.
Statistical analysis: Plagnol, Webster.
Obtained funding: Michaelides, Moore, Raymond, Webster.
Administrative, technical, or material support: Robson, Michaelides, Holder, Raymond.
Study supervision: Michaelides, Holder, Henderson, Raymond, Moore, Webster.
Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none were reported.
Funding/Support: This study was supported in part by the NIHR England, which supported the NIHR BioResource–Rare Diseases and Biomedical Research Centres at Cambridge University Hospitals (grant RG65966), Moorfields Eye Hospital, and the University College London Institute of Ophthalmology, and by the Foundation Fighting Blindness, Fight for Sight; Moorfields Eye Hospital Special Trustees and Rosetrees Trust; and Research to Prevent Blindness USA.
Role of the Funder/Sponsor: The funding organizations had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Additional Contributions: The NIHR BioResource–Rare Diseases Consortium performed the molecular investigations described in this report as part of the UK National Specialist Pathology: Evaluating Exomes in Diagnostics study.