The co-occurrence of rare non-ocular phenotypes in patients with inherited retinal degenerations

Patients and clinical evaluation

Twenty-five patients from 15 families were included in this study. Patients were ascertained by the inherited retinal degeneration clinic at Schneider Children’s Medical Center of Israel (SCMCI) and by the Laboratory of Visual Genetics at the Technion. The tenets of the Declaration of Helsinki were followed, the study adhered to the ARVO statement on human subjects, the study was approved by institutional review boards in participating Medical Centers, and written informed consent was obtained from all participants. Ophthalmic examination included best-corrected visual acuity (BCVA), biomicroscopic examination, cycloplegic refraction, retinal imaging (fundus photos, optical coherence tomography (OCT), and fundus autofluorescence), and electroretinography (ERG) testing in most patients. In addition, patients and their families were evaluated by clinical geneticists before and after the molecular diagnosis was made, and were advised about the relevant genetic testing needed, as well as the patient’s prognosis, further needed medical evaluation, and future family planning, accordingly.

Genetic analyses

Venous blood samples were obtained using K3EDTA vacuette tubes (Greiner Bio-One, Kremsmunster, Austria) and kept at 4 ºC for up to 48 h. Genomic DNA was extracted from venous blood samples using a high salt solution according to a standard protocol [9]. Homozygosity mapping in family 1 was performed with the Infinium Human Linkage-12 Genotyping BeadChip Kit (Illumina, Inc., San Diego, CA), which is capable of genotyping 6,090 highly informative single nucleotide polymorphisms (SNPs) with an average genetic distance of 0.58 cM across the human genome. Homozygous regions were calculated using HomozygosityMapper [10]. Chromosomal microarray analysis (CMA) in family 2 was performed using the CytoScan^TM 750K Chip Kit (PE Applied Biosystems, Foster City, CA).

Whole exome sequencing (WES) in families 2, 4, 5, 10, and 13 was performed using SureSelectXT Human All Exon V5 (Agilent Technologies, Santa Clara, CA) and HiSeq2500 or HiSeq4000 (Illumina, San Diego, CA). For family 12, a Roche NimbleGen Exome Enrichment Kit (Roche NimbleGen, Inc., Pleasanton, CA) was used for target enrichment.

Testing for specific mutations and verification of mutations identified with WES were performed by PCR amplification with specifically designed primers. PCR was performed on 50 ng of genomic DNA in a 25 µl reaction volume in the presence of 5X Readymix (LAROVA GmbH, Teltow, Germany) and 10 pmol of each forward and reverse primers. This was followed by direct sequencing with the BigDye Terminator Cycle Sequencing Kit on an ABI 3130xl Genetic Analyzer (PE Applied Biosystems). The primer sequences used for PCR amplification and Sanger sequencing are listed in Appendix 1. Information regarding the genes in which mutations were found is provided in Appendix 2.

Family 1

Family 1 is a consanguineous family of Moroccan Jewish origin. Patient 1–1 has RP and congenital profound neurosensory HL. This phenotypic combination led to an initial diagnosis of type 1 Usher syndrome (USH1). However, her sister (patient 1–2) had HL, without RP (Figure 1). Homozygosity mapping performed on individual 1–1 revealed many large homozygous regions. None of these regions included a known USH causative gene. However, two regions harbored known causative genes for AR nonsyndromic RP and for AR nonsyndromic HL: A 24 Mb interval located on chromosome 2 (61–85 Mb) included the FAM161A gene, and a 58 Mb interval located on chromosome 9 (31–89Mb) included the TMC1 gene. Sequencing of all coding exons of these two genes in patient 1–1 revealed homozygous mutations in both: FAM161A (NM_001201543; OMIM: 613596) harbored the c.1355_1356del mutation, which is one of the most common RP causative mutations in the Israeli Jewish population [11]; TMC1 (NM_138691; OMIM: 606706) harbored the p.Ser647Pro founder mutation, which is prevalent among Moroccan Jews [12] (Table 1). Testing of additional family members revealed that both parents were heterozygotes for both mutations, while patient 1–2 was homozygous for the TMC1 mutation, but heterozygote for the FAM161A mutation (Figure 1).

Family 2

Patient 2–1 is a Jewish female, born to non-consanguineous parents of North African/Turkish origin. She was referred for ophthalmic evaluation at the age of 16 years, due to suboptimal visual acuity, even with optical correction. The first thing noticed were her unusual facial features, which included bilateral microtia with a hearing aid in the left ear, and profound micrognathia. The ophthalmic exam was positive for bilateral lipodermoid lesions on the temporal aspect of the bulbar conjunctiva. BCVA was 20/40 in oculus dexter (OD) and 20/50 in oculus sinister (OS). The fundus exam revealed optic disc pallor, macular atrophy, multiple bone spicules in retinal midperiphery, and vessel attenuation, leading to a diagnosis of RP. The combination of RP and facial features led us to consider a syndromic form of RP. However, CMA revealed a heterozygous 656 kb duplication on chromosome 14q22.3, which includes exons 1–3 of the OTX2 gene (NM_021728.3; OMIM: 600037; Appendix 3). OTX2 duplications are a known cause of AD hemifacial microsomia [13]. This mutation was likely inherited from the patient’s father, who had similar facial features, according to descriptions by the family; unfortunately, he was unavailable for clinical or genetic evaluation. In addition, WES revealed that she was compound heterozygous for two mutations in the RDH12 gene (NM_152443; OMIM: 608830), a known causative gene for arRP (Table 1). Both mutations in RDH12 were previously identified in Jewish patients of North African and Oriental descent with RP [14] (Figure 1).

Family 3

The index patient (patient 3–1) is a female, born to nonrelated Jewish parents of North African origin (Figure 1). Both parents were diagnosed with advanced RP. Their daughter was diagnosed with familial Mediterranean fever (FMF) and treated with colchicine. FMF, an AR disease caused by mutations in the MEFV gene (NM_000243.2; OMIM: 608107), is common in the Israeli population and predominantly affects North African Jews [15], with a carrier frequency of approximately 1:5 [16]. Patient 3–1 was found to be homozygous for the common MEFV founder mutation p.Met694Val. At the age of 10 years, during an episode of headaches and vomiting, she was referred to the eye clinic for a fundus examination to rule out papilledema and elevated intracranial pressure. Upon exam, BCVA was 20/20 in each eye, and the retinal midperiphery was noted to have mild hypopigmented lesions and few bone spicules. ERG showed no rod response, with severely decreased cone response, thus confirming a diagnosis of RP. Molecular testing confirmed she was homozygous for the p.Arg523* mutation in FAM161A, which is a prevalent RP causative mutation among Jews [11].

Family 4

A 2.5-year-old male, born to non-consanguineous Ashkenazi Jewish parents, came to the clinic due to progressive myopia and nyctalopia. His medical history was positive for diabetes insipidus (DI) and HL, thus raising the possibility of syndromic IRD. On his exam, BCVA was 20/30 in both eyes, and the fundus exam seemed normal. Family history revealed that his maternal grandfather had been diagnosed with RP (Figure 1). WES revealed that the patient harbors three mutations, each causing one of his diseases (DI, HL, and RP; Table 1). Two mutations are XL: a novel nonsense mutation, p.Glu758*, in RPGR (NM_001034853.1; OMIM: 312610; Appendix 3), and a novel nonsense mutation, p.Cys224*, in AVPR2 (NM_000054.5; OMIM: 300538; causing RP and DI, respectively). The third mutation is a large homozygous deletion on chromosome 15q15, involving the STRC (NM_153700.2; OMIM: 606440) and CATSPER2 (NM_172095.3; OMIM: 607249) genes. STRC mutations are a known cause of AR HL [17]. Deletions in STRC-CATSPER2 have been previously described in several patients with AR deafness-infertility syndrome (DIS) [18-20]. Due to the patient’s young age, his fertility was not assessed.

Family 5

This is a consanguineous Muslim Arab family (parents are first cousins). The index patient (5–1) had liver transplantation during childhood due to cholestatic liver disease. In addition, he had progressive HL and RP, which presented as nyctalopia since childhood, with progressive visual fields loss and visual deterioration. On exam (at the age of 25 years), BCVA was counting finger (CF) in each eye. Full-field ERG (ff-ERG) was non-recordable for cone and rod responses. His brother (patient 5–2) is also affected by cholestatic liver disease and RP. Data regarding his hearing status were not available (Figure 1). WES of patient 5–1 identified a novel homozygous nonsense mutation, p.Glu785*, in the TJP2 gene (NM_004817; OMIM: 607709), which is known to cause AR cholestatic liver disease [21] and AD progressive HL [22] (Appendix 3). A mutation causing RP was not found.

Family 6

This is a consanguineous Israeli Muslim Arab family (parents are double cousins). Patient 6–1 was clinically diagnosed at the age of 2 years with chronic granulomatous disease (CGD), and underwent bone marrow transplantation. At the age of 7 years, he was referred for ophthalmic evaluation due to visual disturbances. On exam, BCVA was 20/80 OD and 20/50 OS. The fundus exam showed perivascular chorioretinal punched out lesions, and perivascular pigmentary changes. ERG was consistent with rod-cone disease, i.e., RP (Figure 1). The combination of CGD and RP raised the possibility of the XL contiguous gene deletion syndrome known as the McLeod phenotype [23]. However, genetic analysis revealed that in patient 6–1 CGD is inherited as an AR trait, caused by a homozygous nonsense mutation in the NCF2 gene (NM_001127651.2; OMIM: 608515; Table 1). This mutation, p.Arg66*, has been previously reported in patients with CGD [24,25]. The genetic cause of RP remains unknown.

Family 7

This is a consanguineous Muslim Arab family. The parents are healthy. Their older daughter (patient 7–1) was diagnosed at the age of 17 years with a bestrophinopathy, and the parents were advised to bring in her siblings for ophthalmic exams. One of the siblings (patient 7–2; age 14 years) was asymptomatic at presentation, with BCVA of 20/20 in each eye. However, on the fundus exam he was noted to have mild foveal RPE changes. OCT showed a small subfoveal accumulation. In addition, he had ichthyosis presenting with severe perioral and hand skin atrophy. Both siblings were found to have a previously reported homozygous missense mutation in the BEST1 gene (NM_004183.3; OMIM: 607854), p.Arg25Gln [26], thus confirming a diagnosis of ARB (Table 1). A causative mutation for ichthyosis was not found; however, intrafamilial marriage for several generations in this family, and the occurrence of additional affected individuals in previous generations, supports an AR inheritance mode (Figure 1).

Family 8

Family 8 is a non-consanguineous Jewish family of Yemenite origin. One of the daughters (patient 8–1) has severe RP, due to homozygosity for a known and common mutation (p.Gly1961Glu) in ABCA4 (NM_000350.2; OMIM: 601691). Her intelligence is normal. Her brother (patient 8–2) is intellectually disabled. He was referred to the eye clinic for vision evaluation at the age of 11 years, because his teachers suspected he had impaired vision. On exam, BCVA was 20/80 OD, 20/160 OS, with exotropia in the left eye. The fundus exam showed pallor of the optic disc, arterial narrowing, abnormal macular appearance with RPE changes, and no bone spicules. FF-ERG demonstrated a significant decrease in rod and cone responses, thus confirming a diagnosis of RP. Genetic analysis confirmed homozygosity for the p.Gly1961Glu mutation in ABCA4. The cause of the intellectual disability (ID) remains unknown.

Family 9

Patient 9–1 was born to healthy non-related Jewish parents of Iraqi and Tunisian origin. She had HL and used hearing aids since 9 months of age. She was referred for ophthalmic evaluation at the age of 2 years due to her mother’s concern of nyctalopia. On exam, BCVA was fix and follow in each eye. The fundus exam revealed an abnormal macular reflex and bone spicules in the midperiphery. ERG showed no rod response and significantly attenuated cone response, thus leading to a diagnosis of RP. The combination of RP and HL led to an initial diagnosis of USH. However, further questioning revealed the existence of non-syndromic RP in several maternal relatives (Figure 1). The mode of segregation in the extended family appeared to be AD with reduced penetrance, and a heterozygous mutation in the PRPF31 (NM_015629; OMIM: 606419) gene (p.Glu370*) was previously detected in some of the affected individuals. Genetic testing of patient 9–1 confirmed the presence of the same heterozygous mutation (Appendix 3). The cause of the HL currently remains unknown.

Family 10

Family 10 is a non-consanguineous Jewish family of mixed origin. Patient 10–1 was seen at the age of 34 years. Medical history was positive for pacemaker insertion at the age of 25 years due to bradycardia. In addition, he had mild ID. He was referred to the ophthalmic clinic due to reduced vision. BCVA was CF OD, and 20/200 OS. FF-ERG demonstrated significantly reduced cone response and slightly reduced rod response, suggesting a cone-rod dystrophy (CRD; Figure 1). The combination of cardiac disease, cognitive impairment, and retinopathy led to a possible diagnosis of XL Danon disease [27]. The causative gene of Danon disease, LAMP2 (NM_002294.2; OMIM: 309060), was sequenced, but no mutation was found. WES analysis revealed that the patient was homozygous for a known missense mutation in the ABCA4 gene (p.Arg1161His), which confirmed a diagnosis of arCRD. The causes of cardiac disease and cognitive impairment remain unknown.

Family 11

This is a consanguineous Muslim Arab family. Patient 11–1 was diagnosed with early-onset RP (her ERG was non-recordable at the age of 18 years). In addition, she was diagnosed with primary ciliary dyskinesia (PCD). The combination of these two phenotypes initially raised a suspicion of a ciliopathy, affecting the lung and the retina. However, further questioning of the family revealed that three additional siblings were affected by early-onset RP, but without PCD (Figure 1). In addition, in RP-affected individuals from the same village, we previously identified a known mutation (c.389del) in GUCY2D (NM_000180; OMIM: 600179) [28]. Genetic testing confirmed homozygosity for this mutation in all four siblings. The cause of PCD in patient 11–1 remains unknown.

Family 12

This is a non-consanguineous Jewish family of Ashkenazi origin. Patient 12–1 was followed up in the ophthalmology clinic due to suboptimal BCVA even with refractive correction (20/40 OD and 20/30 OS). His mother reported he had difficulty in night vision. In addition, he had a single kidney and amelogenesis imperfecta. His family history was positive for nyctalopia in three maternal male relatives, and a single kidney in his mother’s grandfather and his brother (suggestive of an XL inheritance pattern for both traits; Figure 1). WES detected a novel single nucleotide variant located at position −9 of CACNA1F (NM_005183; OMIM: 300110) intron 36. This variant (c.4294–9G>A) is rare (not present in the Genome Aggregation Database, gnomAD), and was predicted to generate a novel acceptor splice site, 7 bp upstream of the original splice site of intron 36 (Human Splicing Finder). The use of this novel acceptor site will generate a 7-bp insertion, leading to a frameshift (Appendix 3). Mutations in CACNA1F are associated with XL congenital stationary night blindness (OMIM: 300071). The causes of the kidney and teeth phenotypes are currently unknown (Table 1).

Families 13, 14, and 15

These families were previously described in detail elsewhere. Patient 13–1 is homozygous for pathogenic mutations in two IRD-related genes (MYO7A (NM_000620; OMIM: 276903) and PDE6B (NM_000283; OMIM: 180072), causing syndromic and non-syndromic RP [29]. Patients 14–1 and 15–1 have a combination of non-syndromic IRD, caused by homozygosity for a recessive mutation in NRL (NM_006177.3; OMIM: 162080), and early-onset oculopharyngeal muscular dystrophy, caused by homozygosity for a dominant mutation in PABPN1 (NM_004643; OMIM: 602279) [30].