Next-generation sequencing-based gene panel tests for the detection of rare variants and hypomorphic alleles associated with primary open-angle glaucoma

Elena Milla; Javier Laguna; Mª Socorro Alforja; Beatriz Pascual; María José Gamundi; Emma Borràs; Imma Hernán; María Jesús Muniesa; Marta Pazos; Susana Duch; Miguel Carballo; Meritxell Jodar; on behalf of the EMEIGG group

doi:10.1371/journal.pone.0282133

. 2024 Jan 19;19(1):e0282133. doi: 10.1371/journal.pone.0282133

Next-generation sequencing-based gene panel tests for the detection of rare variants and hypomorphic alleles associated with primary open-angle glaucoma

Elena Milla ^1,^2,^‡,^*, Javier Laguna ^3,^‡, Mª Socorro Alforja ¹, Beatriz Pascual ⁴, María José Gamundi ⁴, Emma Borràs ⁴, Imma Hernán ⁴, María Jesús Muniesa ¹, Marta Pazos ¹, Susana Duch ², Miguel Carballo ⁴, Meritxell Jodar ^3,⁵; on behalf of the EMEIGG group^¶

Editor: Alvaro Galli⁶

PMCID: PMC10798505 PMID: 38241218

Abstract

Primary open-angle glaucoma (POAG) is a complex disease with a strong hereditably component. Several genetic variants have recently been associated with POAG, partially due to technological improvements such as next-generation sequencing (NGS). The aim of this study was to genetically analyze patients with POAG to determine the contribution of rare variants and hypomorphic alleles associated with glaucoma as a future method of diagnosis and early treatment. Seventy-two genes potentially associated with adult glaucoma were studied in 61 patients with POAG. Additionally, we sequenced the coding sequence of CYP1B1 gene in 13 independent patients to deep analyze the potential association of hypomorphic CYP1B1 alleles in the pathogenesis of POAG. We detected nine rare variants in 16% of POAG patients studied by NGS. Those rare variants are located in CYP1B1, SIX6, CARD10, MFN1, OPTC, OPTN, and WDR36 glaucoma-related genes. Hypomorphic variants in CYP1B1 and SIX6 genes have been identified in 8% of the total POAG patient assessed. Our findings suggest that NGS could be a valuable tool to clarify the impact of genetic component on adult glaucoma. However, in order to demonstrate the contribution of these rare variants and hypomorphic alleles to glaucoma, segregation and functional studies would be necessary. The identification of new variants and hypomorphic alleles in glaucoma patients will help to configure the genetic identity of these patients, in order to make an early and precise molecular diagnosis.

Introduction

It is estimated that almost 112 million people worldwide will have glaucoma in 2040, which will continue to be the second leading cause of blindness worldwide [1]. It is known that half of all glaucoma cases go undiagnosed, and therefore efficient screening methods for detecting glaucoma are essential. Genetic studies have enormously progressed in recent years with the advent of the Human Genome Project [2]. Increasingly, molecular genetics is seen as a diagnostic tool in daily clinical practice. Molecular genetic analysis could be used to detect the disease in its early stage (pre-symptomatic relatives), to perform phenotypic-genotypic correlations, to personalize treatment, to refine the prognosis of the disease and to offer genetic counselling [3].

Current next-generation sequencing (NGS) allows millions of DNA sequences to be produced in a single reaction, avoiding the limitations of the Sanger method of gene-to-gene and exon-to-exon sequencing [4]. In this way, through NGS, a large amount of data is obtained and a large number of variants will be observed in each individual. However, the classification of the gene variants is the challenge posed in current genetic molecular diagnosis.

Through linkage analysis, 23 loci and four genes (MYOC/TIGR, CYP1B1, OPTN, and WDR36) had already been associated with glaucoma in the past years. Early-onset glaucoma (<40 years) is more likely to be inherited according to a classic Mendelian pattern involving single genes, mainly CYP1B1 in primary congenital glaucoma (PCG) and MYOC in juvenile open-angle glaucoma (JOAG), whereas glaucoma in adults tends to be more complex due to its multifactorial inheritance [5, 6]. Family grouping is a known risk factor for glaucoma in the adult, with a risk 1–10 times greater than the observed in general population among the first-degree relatives of an affected individual [7, 8]. Therefore, the surveillance of these individuals is indicated for early detection and treatment [9, 10].

However, these disease-causing genes mentioned above account for <10% of primary open angle glaucoma (POAG) cases in the general population [11]. Therefore, in the past decade, efforts have been made to elucidate the genetic causes of adult glaucoma. The application of advanced genetic technology has increased the list of candidate genes [6]. Recent Big Data studies using genome-wide association study (GWAS) have identified over 100 loci related with oxidative stress, DNA repair mechanisms, mitochondrial DNA genes, sex hormones, and phenotypic traits associated with glaucoma [12]. Quantitative endophenotypic agents that act as risk factors (pachymetry, axial length, anterior chamber depth…) and glaucomatous risk stratification have been identified [13, 14]. Therefore, genetic screening appears increasingly promising not only for PCG and JOAG diagnosis, but also for POAG.

In 2006, our research group created the Spanish Multicentre Glaucoma Group (Estudio Multicéntrico Español de Investigación Genética del Glaucoma, EMEIGG), including 18 Eye Hospitals throughout Spain. We performed an initial study to identify pathogenic variants in the MYOC and CYP1B1 genes, which were the only fully-described glaucoma-causing genes at that time [15]. The aim of this study is to test a custom gene panel that gathered recently described candidate genes associated with POAG using NGS and to evaluate the impact of the implementation of NGS as a future method for diagnosis and early treatment of adult glaucoma. And, additionally, based on the NGS results, we set out to evaluate the impact of hypomorphic alleles of CYP1B1 in a new cohort of patients with POAG.

Materials and methods

Patients

A total of 74 patients signed written informed consent to participate in this study and were asked to fill in a questionnaire including personal, biographic, demographic, family, and clinical data. This study was carried out in accordance with the Declaration of Helsinki and approved by the Research Ethics Committee of the Hospital Clínic of Barcelona.

All participating ophthalmologists from the EMEIGG completed a standardized questionnaire to homogenize the data collection. A full history was taken, including systemic and ophthalmologic disease, family members affected by glaucoma (number and type of relative) and family history of consanguinity. POAG was diagnosed in the presence of compatible perimetric lesions correlated with typical glaucomatous changes of the optic nerve in patients over 40 years and absence of a cause for secondary glaucoma diagnosis. Patients affected with low-tension glaucoma (LTG) which is a form of POAG in which there is a glaucomatous optic neuropathy in the presence of intraocular pressures (IOP) lower than 20 mmHg were also included in the study. Patients with early-onset glaucomas who were diagnosed with PCG or JOAG were excluded.

Each referring ophthalmologist graded the disease stage, based on the ophthalmologic exams, the aspect of the optic disc, and visual field-testing results, and classified each case as initial, moderate, or severe glaucoma according to the Hodapp-Parrish-Anderson grading scale, which refers to visual field mean defect (MD) (initial if MD < −6 dB, moderate if MD between −6 and −12 dB, and advanced if MD > −12 dB). Data about the number of eye surgeries and current ocular hypotensive medication were recorded. The presence of thin pachymetry was also annotated as being an endophenotypic trait related to glaucoma conversion and severity.

Study of glaucoma-related genes using NGS

Sixty-one patients (all unrelated, except two siblings) with POAG (or LTG) (Table 1) were included in the NGS study, with a mean age at diagnosis of 51 ± 12 years (range: 18–76 years). Although the study included adult glaucoma cases, four patients were younger than 40 years at the time of diagnosis (18–27 years), but did not display the typical features of JOAG, and were considered as early-onset POAG.

Table 1. Clinical data of patients studied by NGS and Sanger sequencing.

			Patients NGS N = 61	Patients Sanger N = 13
Gender	Female		33	8
	Male		28	5
Diagnosis	POAG	Initial	9	2
		Moderate	14	8
		Severe	31	3
	LTG	Severe	3	-
	Early-onset POAG	Moderate	1	-
		Severe	3	-
Family history	Yes		50	11
	No		11	2
Surgery	Yes		38	6
	No		23	7
Ocular hypotensive medication	Yes		15	1
	No		46	12

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LTG: Low-Tension Glaucoma; POAG: Primary Open-Angle Glaucoma.

Thirty-nine genes potentially associated with POAG were studied (Table 2). After a thorough search in OMIM, Orphanet and Human Gene Mutation Database (HGMD), we selected the classic known glaucoma-causing genes, genes associated with elevated IOP or POAG risk, anterior segment dysgenesis, glaucoma, glaucoma-related endophenotypic traits, optic nerve pathology or augmented susceptibility and retinal vessels anomalies and, finally, syndromic glaucoma-causing genes. Besides, additional genes have been included because a potential association with glaucoma has been suggested in some scientific literature studies [16–25].

Table 2. List of genes included in the custom glaucoma panel designed.

ADRB1	ERCC2	OCLM
ADRB2	GALC	OLFM2
AGTR2	GAS7	OPA1
ATOH7	GSTM1	OPTC
BCAS3	HK2	OPTN
BMP4	LMX1B	PAX6
CARD10	MFN1	PON1
CAV1	MFN2	SIX1
CAV2	MTHFR	SIX6
CDC7	MYOC	TGFBR3
CDKN2B	NCK2	TMCO1
COL8A2	NOS3	WDR36
CYP1B1	NTF4	XRCC1

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Libraries were generated through gene capture by hybridization with the SeqCap EZ system (NimbleGene, custom panel “cl1_GM1”) and subsequent massive parallel sequencing was carried out with a HiSeq™ 2000 platform sequencer (Illumina) at Sistemas Genómicos, S.L. Sequence reads were aligned with the reference genome GRCh38/hg38. Alignment was made using the BWA (Burrows-Wheeler Aligner) tool and scripts designed by Sistemas Genómicos, S.L. Genetic variants in coding and flanking sequences of genes with frequencies <1% were annotated. Annotated sequencing data was investigated using the filtering system for nonsense, missense, synonym and intronic variants located up to +10 bp in the flanking sequences. All exons and intronic regions up to +10 bp from the exons showed coverages greater than 20x. UTR regions were not analyzed. SNV annotation was made using ANNOVAR [26].

Sanger sequencing of CYP1B1 in a new cohort of POAG patients

After observing the results of the NGS study, we included 13 additional patients for the study of the CYP1B1 gene using Sanger sequencing. These 13 patients were not studied by NGS. The coding sequence of the CYP1B1 gene was directly sequenced, following the same selection criteria as before (Table 1). The mean age at diagnosis was 55 ± 10 years (range: 40–79 years).

Specific oligonucleotides were designed to amplify the two coding exons of CYP1B1 (NM_000104.4; S1 Table).

Interpretation and classification of variants

In silico prediction algorithms included in Varsome (https://varsome.com/) were used to classify variants. The in silico tools used were: CADD, Polyphen2, DEOGEN2, MutPred, FATHMM-XF, Mutation assessor, MVP, PROVEAN, EIGEN, LRT, SIFT, BLOSUM, DANN, LIST-S2, M-CAP, MutationTaster and PrimateAI. ClinVar database was also consulted to check the classification reported by other subscribers.

General population frequencies and the highest frequencies in a population were obtained from gnomAD v3.1.2. Final variant interpretation was performed according to the American College of Medical Genetics and Genomics (ACMG) and the Association for Molecular Pathology (AMP) recommendations [27]. In this way, we considered rare variants classified/described as pathogenic, likely pathogenic or variant of uncertain significance (VUS) with low frequencies in the general population (<1%), as well as variants that have been described as hypomorphic variants.

Results

Rare genetic variants detected in POAG patients by NGS

We detected nine rare or hypomorphic variants (Table 3) in 10 patients (16%) by NGS (Table 4). These variants were found in seven of the glaucoma-related genes. All variants were detected in heterozygosity and classified as VUS. The allelic frequency of these variants in general population was very low (<1%) (Table 3). Most patients described with variants showed a severe POAG phenotype (70%, Table 4). Additionally, all patients with a rare genetic variant had a family history of glaucoma and most of them had required surgery or maximal ocular medication (Table 4). The presence of thin pachymetry readings was detected in two patients (patient 1 and patient 8).

Table 3. Description and classification of variants detected in this study.

All variants were detected in heterozygosity.

Gene	Transcript	Variant (cDNA)	Variant (protein)	ACMG criteria	ClinVar classification (number of times)	ACMG classification	AF in general population	Highest AF and population
CYP1B1	NM_000104.4	c.241T>A	p.Y81N	PS3, PP3, BS1, BS2	VUS (2); LB (1); B (2)	VUS (Hypomorphic)	0.35%	1.36% European (Finnish)
CARD10	NM_014550.4	c.1307C>T	p.T436M	PM2, BP4	Not reported	VUS	0.01%	0.03% Ashkenazi Jewish
CARD10	NM_014550.4	c.2952C>A	p.C984X	PM2, BP4	Not reported	VUS	Not found	Not found
MFN1	NM_033540.3	c.1601G>A	p.R534Q	PM2, PP3	Not reported	VUS	Not found	Not found
MFN1	NM_033540.3	c.2101G>T	p.E701X	PM2	Not reported	VUS	Not found	Not found
OPTC	NM_014359.4	c.893G>A	p.R298H	PM2	Not reported	VUS	0.03%	0.21% Latino/Admixed American
OPTN	NM_001008212.2	c.1552C>T	p.Q518X	PM2	Not reported	VUS	Not found	Not found
SIX6	NM_007374.3	c.635C>T	p.T212M	PM2, PP3	VUS (2)	VUS (Hypomorphic)	0.02%	0.03% European (non-Finnish)
WDR36	NM_139281.3	c.892G>A	p.E298N	PM2, PP3	Not reported	VUS	0.01%	0.11% Ashkenazi Jewish
CYP1B1 ^¶	NM_000104.4	c.83C>G	p.S28W	PM2, PP5, PS1	Not reported	VUS (Hypomorphic)	0.004%	0.12% Ashkenazi Jewish

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^¶Variant detected only in the study by Sanger sequencing. ACMG: American College of Medical Genetics; AF: allelic frequency; B: benign; LB: likely benign; NGS: next-generation sequencing; VUS: variant of uncertain significance.

Table 4. Clinical characteristics of patients and variants detected in this study.

All variants were detected in heterozygosity.

Patient	Gender	Age at diagnosis	Diagnosis	Features	Family history of glaucoma	Consanguinity	Surgery	Ocular hypotensive medication	Gene	Transcript	Variant (protein)
Next-generation sequencing
Patient 1	Female	55	POAG	Initial glaucoma, with PDS and myopia	Yes (mother, sister)	No	No	No	CYP1B1	NM_000104.4	p.Y81N
Patient 2	Male	76	POAG	Severe glaucoma	No	No	Yes	Yes	CYP1B1	NM_000104.4	p.Y81N
Patient 3	Female	55	POAG	Moderate glaucoma	Yes (daughter)	No	Yes	No	SIX6	NM_007374.3	p.T212M
Patient 4^¶	Female	42	POAG	Severe glaucoma, with retinal venous and arterial occlusions	Yes (brother)	No	Yes	No	CARD10	NM_014550.4	p.T436M
Patient 5^¶	Male	30	POAG	Severe glaucoma, with retinal venous and arterial occlusions	Yes (sister)	No	Yes	No	CARD10	NM_014550.4	p.T436M
Patient 6	Female	65	POAG	Severe glaucoma	Yes (son, 2 siblings)	Yes	No	Yes	CARD10	NM_014550.4	p.C984X
									MFN1	NM_033540.3	p.R534Q
Patient 7	Female	65	POAG	Moderate glaucoma	Yes (4 siblings, nephew)	No	No	Yes	OPTC	NM_014359.4	p.R298H
Patient 8	Male	25	Early-onset POAG	Severe glaucoma	Yes (father, brother)	No	Yes	No	OPTN	NM_001008212.2	p.Q518X
Patient 9	Male	60	LTG	Severe glaucoma	Yes (daughter)	No	Yes	No	WDR36	NM_139281.3	p.E298N
Patient 10	Female	68	POAG	Severe glaucoma	Yes (father, brother)	No	Yes	No	MFN1	NM_033540.3	p.E701X
Sanger sequencing of CYP1B1
Patient 11	Male	58	POAG	Moderate glaucoma	No	No	Yes	No	CYP1B1	NM_000104.4	p.Y81N
Patient 12	Female	60	POAG	Initial glaucoma	Yes (maternal grandmother)	No	No	No	CYP1B1	NM_000104.4	p.Y81N
Patient 13	Male	60	POAG	Moderate glaucoma and myopia	Yes (mother, 2 siblings)	No	No	No	CYP1B1	NM_000104.4	p.S28W

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^¶Siblings. LTG: low-tension glaucoma; NGS: next-generation sequencing; PDS: pigment dispersion syndrome; POAG: primary open-angle glaucoma.

The only variant detected in two unrelated patients was the variant p.Y81N in the CYP1B1 gene (Table 4). Although different in silico algorithms and functional studies predict a possible pathogenicity of this variant, it has been identified in 528 individuals from genomes and exomes available in gnomAD, even in three of them in a homozygosis state. Nevertheless, the comparison of residues between organisms showed marked conservation of Y81 in different organisms (Fig 1) and its association with POAG has been reported several times [15, 28–31], being described as a hypomorphic variant in some cases [32–35]. Therefore, this variant was classified as a VUS.

Fig 1 — Comparison of the evolutionary conservation of p.S28W (left) and p.Y81N (right) in the *CYP1B1* gene in the reference genome of some organisms.

Although familial studies of VUS are not recommended in clinical practice [36], the segregation study of the p.Y81N variant in the CYP1B1 gene was performed in both families for research purposes (Fig 2). The variant segregated with the disease in the family of patient 1. The patient’s sister, who was also diagnosed with POAG, carried the p.Y81N variant in CYP1B1 in heterozygosity. The variant was also detected in the patient’s daughter, who had not signs of glaucoma, but has ocular hypertension. The family of patient 2 was smaller and not informative. The segregation study showed that the patient’s daughter, who had no signs of glaucoma or ocular hypertension, did not have the variant. His wife also had glaucoma, but she also underwent NGS testing, and no variant was detected.

Fig 2 — Pedigrees of patients in whom the p.Y81N variant in the *CYP1B1* gene was detected in the NGS study: patient 1 (left) and patient 2 (right). Arrows indicate the index case. Black symbols indicate glaucoma phenotypes, and carriers of the variant are indicated by black dots in the symbols. wt: wild-type allele.

We also observed two siblings with the variant p.T436M in the CARD10 gene. Both siblings (a 45-year-old female and a 43-year-old male) had a very aggressive POAG phenotype (Fig 3), with very high IOP and pigment dispersion syndrome (pigment dots scattered around the corneal endothelium and pigmented trabecular meshword), although other characteristic features of classic pigmentary glaucoma (typical Krukenberg spindle and iris atrophy) were missing. Both also developed retinal venous and arterial occlusions. Aside from the siblings, we detected another variant in the CARD10 gene (p.C984X) in a 65-year-old female, along with a second variant in the MFN1 gene (p.R534Q). This patient had a severe POAG phenotype, and was the only patient in the study with a family history of consanguinity (grandparents were first degree cousins). Regarding the MFN1 gene, we also detected another variant (p.E701X) in a 68-year-old patient with severe glaucoma. Both variants in the CARD10 gene and in the MFN1 gene have not been reported in ClinVar and most of them do not appear in gnomAD, except for the variant p.T436M in the CARD10 gene that was identified in 12 individuals in heterozygosity. All of them have one or two deleterious predictors and should therefore be classified as VUS.

Fig 3 — A: Optomap image of ocular fundus of the patient 4 showing macular whitening and a myriad of splinter retinal hemorrhages and venous tortuosity secondary to central retinal vein occlusion with cilioretinal artery occlusion in the right eye. B: Funduscopic image of the left eye of the patient 4 showing severe optic disk cupping in the context of advanced glaucoma with pigment dispersion syndrome. C: Optomap image of the right eye fundus of the patient 5 showing mild macular whitening with scant peripheral hemorrhages one month after non-ischemic central retinal vein occlusion associated with cilioretinal artery occlusion in the right eye. D: Retinography of the patient 5 (up) showing severe right optic disk cupping causing advanced visual field constriction seen on perimetry (below).

The other genetic variants were identified in OPTC (p.p.R298H), OPTN (p.Q518X), SIX6 (p.T212M), and WDR36 (p.E298N). As shown in Table 3, all these variants have very low allele frequencies in general population and most of them have not been described in ClinVar. It is interesting to note that one of these variants, p.T212M in the SIX6 gene, has also been reported in the literature as hypomorphic variant [37].

Analysis of hypomorphic CYP1B1 variants

It has been reported that CYP1B1 may play a role in the pathogenesis of POAG in a significant proportion of cases [30, 31, 34]. For this purpose, we sequenced the coding region of this gene in a new cohort of POAG patients. Heterozygous missense variants were detected in three POAG patients (23%, Table 4). None of these patients had severe glaucoma. The aforementioned variant p.Y81N in the CYP1B1 gene was also detected in two patients of these three patients (Table 4).

The variant c.83C>G (p.S28W) in the CYP1B1 gene was identified in a 66-year-old male patient, diagnosed with myopia magna and moderate glaucoma at the age of 60 (Fig 4). His mother and two siblings were also affected by glaucoma. He was taking ocular hypotensive medication, but his IOP remained very high despite maximal treatment. Finally, surgery was necessary in both eyes. To the best of our knowledge, this variant has never been reported in ClinVar and has been detected at a very low frequency in the general population (0.004%). According to the ACMG/AMP recommendations, the variant was classified as VUS following criteria: PM2 (absent from controls in Exome Sequencing Project, 1000 Genomes Project, or Exome Aggregation Consortium) and PP5 (reputable source recently reports variant as pathogenic, but not functional experimental evidence). Similarly to the variant p.Y81N, the comparison of residues between different organisms showed a marked conservation of S28 in mammals and other organisms (Fig 1).

Fig 4 — This figure shows the structural and functional damage of the right (RE) and left eye (LE). The optical coherence tomography shows severe peripapillary retinal nerve fiber layer thinning that correlates with the visual field findings that depicts a marked inferior arcuate scotoma for the RE and tubular island of vision for the LE.

Discussion

According to Lander and Schork [38], the genetic study of adult glaucoma is complex since its transmission mechanisms are sometimes unclear and have variable penetrance and late onset. Adult glaucoma is a multifactorial genetic disease whose outcome is also influenced by a number of environmental factors, many of them unknown. In addition, there are several ethnic and geographic disparities between populations, which further complicate the genetic study of glaucoma [39]. Thanks to recent technological improvements, such as NGS, the list of candidate genes possibly associated with glaucoma has increased considerably since the initial Sanger direct sequencing studies, when only four genes showed a strong association with glaucoma [6].

In this study, we applied NGS to study a cohort of patients with POAG. Hypomorphic alleles and rare variants that were not previously described in the literature or with a very low population frequency have been detected in 10 out of 65 patients assessed. The variant p.Y81N in CYP1B1 gene was the most frequent variant without considering related patients by NGS (two patients). Given the intimate relationship between CYP1B1 and glaucoma and the results observed in the first group, we sequenced the CYP1B1 gene in a different cohort of patients, detecting the variant p.Y81N in two more POAG patients. Thus, the variant p.Y81N was detected in four unrelated patients (two patients from the NGS study and two patients from the Sanger study).

CYP1B1 is an enzyme involved in drug, fatty acid and steroid metabolism located in the endoplasmic reticulum membrane, peripheral membrane protein and microsome membrane. CYP1B1 participates in the metabolism of an as-yet-unknown biologically-active molecule that participates in eye development. Genetic variants in this gene had always been associated with PCG, an autosomal recessive inherited trait [40, 41]. However, in recent years it has been observed that CYP1B1 also could play an important role in the development of adult glaucoma [30, 31]. Hypomorphic alleles pose a challenge in the interpretation of genomic variants [42]. A hypomorphic allele results in a partial loss of the normal (wild-type) gene function, often characterized by reduced expression of the gene product (protein or RNA), although this reduction does not reach a 100% reduction in normal gene function. In the literature we found few articles about hypomorphic alleles in POAG, and most of them focus on genetic variants in CYP1B1 [31–33, 43, 44] and SIX6 [37]. In the current study, hypomorphic variants in CYP1B1 and SIX6 genes have been identified in 8% of the total POAG patient assessed. As commented before, the genetic variant p.Y81N has been identified in heterozygous state in four unrelated patients. This genetic variant has been reported at a low frequency (0.35%) in the general population and has been described as a heterozygous hypomorphic variant for POAG [32–35], with a significantly reduced enzymatic activity and reduced protein stability (18–40% of the wild-type activity) [32]. In one of the families, the segregation study revealed the presence of a variant in an individual who had not yet shown signs of glaucoma, but who had ocular hypertension. These results suggest that it might be interesting to propose familial studies in patients with CYP1B1 hypomorphic alleles to identify asymptomatic carriers of the variant for close ophthalmological follow-up.Besides, we also identified for the first time the variant c.83C>G (p.S28W) in the CYP1B1 gene in a patient with a moderate glaucoma and myopia. Interestingly, a different genetic variant but at the same position (c.83C>T) which produces the same amino acid change as the variant found in our study (p.S28W) has been described as a hypomorphic variant and reported in patients with POAG [45] and PCG [46].

Both variants identified in our study in the CYP1B1 gene caused amino acid changes and affected conserved residues located in structural domains, having the potential to modify enzyme activity by incorrect insertion of the protein into the endoplasmic reticulum membrane (p.S28W) and modification of substrate binding (p.Y81N) [45].

Additionally, we detected the variant p.T212M in the SIX6 gene in a patient with a moderate glaucoma. This variant was also described as a hypomorphic variant. SIX6 is a transcription factor with a known function in the retinal progenitor cell proliferation during the eye development [47]. In vivo analyses in zebrafish have been useful to study the effect of this variant in the eye. Modified animal models with this variant have been found to have a smaller eye size, hypothesizing that the presence of hypomorphic alleles in SIX6 could reduce the number of retinal ganglion cells and increase the risk of glaucoma [37].

Regarding the rare variants detected in other genes, we have observed variants in CARD10, MFN1, OPTC, OPTN and WDR36. Most of them have not yet been associated with glaucoma. Additional studies, including functional and segregation analyses, are imperative to confirm whether these genes and variants are indeed associated with the disease. Interestingly, we found two siblings with the variant p.T436M in the CARD10 gene. This variant has not been reported in the general population, but in silico tools predict a benign impact. Nevertheless, the variant is still considered a VUS. CARD10 encodes for a caspase recruitment domain-containing protein, which is a signaling protein in the regulation of the NF-κB (nuclear factor kappa B) pathway. Since NF-κB is involved in the regulation of cellular apoptosis, it is likely that there is a relationship between CARD10 and cell apoptosis, especially retinal ganglion cell apoptosis, producing higher optic nerve susceptibility to IOP elevations and POAG [48, 49]. In fact, Zhou et al. [18] observed a higher frequency of variants in this gene (4.28%) in patients with severe POAG compared to the control population (0.27%).

Besides, we found one patient with severe glaucoma and two VUS in CARD10 and MFN1. In these cases, the use of polygenic risk scores may be of interest. These scores have been reported in recent years as promising for stratifying individual risk and prognosis of POAG [50, 51], although further studies are still needed.

The association of POAG with some of the rare variants identified in this study should be treated with caution. For example, we detected the nonsense variant p.Q518X in the OPTN gene in one patient with POAG. Recent studies have reported that the missense variant p.E50K is the only known variant in the OPTN gene associated with glaucoma, whereas loss-of-function variants in this gene are associated with amyotrophic lateral sclerosis (ALS) [52]. However, there are cases where ALS and glaucoma coexist, as seen in one of the patients reported by Maruyama et al. [53]. Therefore, variants in the OPTN gene may contribute to some additional risk of glaucoma in certain patient populations.

Similarly, contradictory results are reported for the association of pathogenic variants in the WDR36 gene with glaucoma. Whereas several studies have indicated that genetic variants in WDR36 gene are contributing risk factors for glaucoma progression and severity [54–58], recent clinical trials and meta-analyses have suggested a lack of effect [59–61]. However, it is known that this gene has a connection to glaucoma susceptibility and even to retinal homeostasis [62, 63]. Monemi et al. demonstrated WDR36 gene expression in the lens, iris, ciliary muscles, ciliary body, trabecular meshwork, retina, and optic nerve by RT-PCR with four pathogenic variants (p.N355S, p.A449T, p.R529Q and p.D658G) associated with adult-onset POAG with implications for both high- and low-pressure glaucoma [64]. In our study, we detected the variant p.E298N in the WDR36 gene in a patient with severe LTG, whose phenotype was very similar to another reported LTG patient with a variant (p.N355S) in the WDR36 gene [58].

Some of the patients carrying rare variants (patient 1 and patient 8) presented with thin pachymetry, which is a known endophenotypic trait that increases the degree of severity of the glaucoma cases, whether this trait has been directly linked to the variant described or is a consequence of other genetic/epigenetic influences still has to be elucidated. Further studies are warranted to explore this relationship more comprehensively.

Certainly, addressing the limitations of our study is crucial. While NGS is a powerful tool for detecting genetic variations, it may have limitations in detecting copy number variations (CNV), including deletions or duplications. We acknowledge that our study focused primarily on single nucleotide variations and small indels, and we did not specifically investigate CNV. This limitation is important to consider, especially in cases where CNV could be contributing to the disease [65]. Future research efforts could explore complementary techniques or arrays designed for CNV detection to provide a more comprehensive genetic assessment of glaucoma-related variations.

Our findings suggest that NGS could be a valuable tool for the genetic assessment of glaucoma. However, to irrefutably show the contribution of these rare variants and hypomorphic alleles to glaucoma, additional studies, including functional evidence, will be necessary. The progressive identification of new rare variants and hypomorphic alleles in patients clinically diagnosed with glaucoma will help to configure the genetic identity of these patients, in order to make an early and precise molecular diagnosis. And as a clinical application of these findings, the presence of hypomorphic alleles in asymptomatic relatives of our glaucoma patients acts, in our opinion, as a red flag that suggests close monitoring of these patients and early treatment decision in case of glaucoma suspicion.

Supporting information

S1 Table. Primers designed to amplify coding region of the CYP1B1 gene.

(DOCX)

Click here for additional data file.^{(17KB, docx)}

Acknowledgments

We would like to thank the rest of members of the EMEIGG group (Estudio Multicéntrico Español de Investigación Genética del Glaucoma): Esperanza Gutiérrez and Marta Montero (Hospital Doce de Octubre, Madrid); Cristina Vendrell (Hospital de Viladecans, Barcelona); José Abreu (Hospital Universitario de Canarias); Carmen Cabarga (Hospital Ramón y Cajal, Madrid); Soledad Jiménez (Hospital Universitario Puerta del Mar, Cádiz); Miguel Ángel Almela (Hospital Lluis Alcanyís, Xàtiva); Jordi Loscos (Hospital Universitari Germans Trias i Pujol, Badalona); Carlos Martínez Bello (Hospital Dos de Maig, Barcelona); Sergio Torregrosa (Hospital Punta de Europa, Cádiz); Rosa Martínez (Hospital Infanta Leonor, Madrid); Tiburcio Ibáñez (Hospital San Agustín, Linares); Lourdes Iglesias (Hospital Universitario La Princesa, Madrid); Pere Viñallonga (Institut Oftalmològic, Menorca); Lluís Soler (Hospital de Manresa, Barcelona); and Carmen Carrasco (Hospital de Alcorcón, Madrid).

Data Availability

The minimal dataset is contained within the paper and its Supporting Information file.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0282133.r001

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PONE-D-23-03532Next-generation sequencing-based gene panel tests for the detection of rare variants and hypomorphic alleles associated with primary open-angle glaucomaPLOS ONE

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Reviewer #1: The approach taken is interesting and there is useful data here, but there is much work to do before this is of a standard required for publication in a peer reviewed journal. My criticisms fall into two areas.

Firstly a general comment. The paper as written confuses the search for potentially Mendelian or near Mendelian alleles (the latter would be what I think they mean when they say hypomorphic alleles) with a search for association. If this is considered a search for alleles of large effect size then it is valid, with the proviso, as they rightly say, that these results are tentative and would be given more credence if they were replicated in other screens, as well as confirmed by segregation in families or functional tests. However, the use of chi-squared tests suddenly make it feel like an association screen. I am no statistical geneticist, but even I know that those stats are meaningless. They have performed thousands of tests - arguably every nucleotide they sequenced is a test, or at least every variant from reference they examined. If you do a thousand tests then your p value needs to be below 0.05/1000 before it is statistically significant. So this distinction needs to be discussed and made crystal clear, and those chi-squared tests need to be removed.

Secondly there is much information missing or in need of clarification.

The cohort they study is not well described. The section in mat/mets only talks about the 65 patients, not the 34, so reading that I get the impression the 34 are a subset of the 65, but on lines 97 and 296 these are clearly referred to as a separate cohort. Also it seems bizarre that they would weaken a genetic study by making their cohort so diverse - surely there are plenty of POAG patients across 18 Spanish centres without introducing heterogeneity by including NTG and OHT? All patients are said to be unrelated - was any attempt made to recruit affected relatives where possible, as this would allow segregation studies? In the introduction the authors rightly suggest genetic effects may be enriched in patients with early onset or strong family history - was any attempt made to recruit particularly among those groups? Also, if there is no overlap between the 65 patients and the possibly additional 34, the differences between them deserve some note. The second group seem milder, with more OHT and less surgery.

The process by which candidate genes were selected for screening is also poorly described. The authors simply say they looked at Pubmed and chose these 72 genes. Why these and not others, what criteria were used to select candidates? Also when was the selection made, because Pubmed is a rapidly changing and developing resource that may well give a very different set of candidates if they look again now. There are the obvious ones in their list but there are a few for which I was unaware of a link. I did also wonder why a couple of big hits from GWAS were not included - TCMO1, FOXC1, GAS7 and the CDKs are in this list, but not AFAP1 or ABCA1? I'm not asking for a one by one justification, just some objective criteria.

There are also gaps in the technical data. How many nucleotides did exons of the 72 genes amount to, how much flanking DNA was included, were 5' and 3' sequences included in the sequencing, how much intronic, 5' and 3' sequence was analysed, what kind of HiSeq was used, what depth of coverage was obtained?

Finally there was very little said about what was found other than the variants cherry-picked for presentation. I think we can safely presume that the nine variants presented were not the only things they found. There needs to be a brief summary statement of everything identified, then a list of everything that passed certain minimal criteria that need to be clarified - below 1% in the population (clear), in the coding sequence and +/-10bp (not clear), non-synonymous (not clear) then presumably predicted VUS, likely pathogenic or pathogenic (again not clear) - should be attached in a supplementary file. Lastly they need some further explanation of why only these nine were chosen for further examination.

In addition I have a few mostly minor corrections.

On line 68 and below "Through linkage analysis, 23 loci and four genes.... (ref 5)"; this is clumsy, they seem to be saying at a certain time there were only these findings then later there were more, but the "later" they quote is reference 7 that was actually published in 2016, a year before reference 5?

Line 130 - they need to decide whether they prefer NTG or LTG and stick with one. As far as I know these are not two different diseases but different names for one.

Legend to table 3 needs to state that (I assume) all variants were heterozygous.

Were mutations found in NGS rechecked by Sanger? Perhaps not essential with very modern NGS machines but if the data were older it might be reassuring, and also allows segregation if other family members are available.

Figure 1 is described in the text as homology "modelling". To me modelling means something different, this is just comparing evolutionary conservation of these residues, and since it only compares across mammals it isn't very conclusive - are these residues conserved in birds, reptiles, amphibians or fish? If not it would be useful to know.

Figure 2d is too small and fuzzy to derive anything from it.

Line 284 - "Adult glaucoma is a multifactorial genetic disease upon which a series of environmental factors must hatch" - clumsy - I suggest "Adult glaucoma is a multifactorial genetic disease the outcome of which is also influenced by a series of environmental factors" - or similar.

Also in the discussion there is much talk of hypomorphic alleles - a clear definition of what the authors mean by that would be useful.

Reviewer #2: The authors analysed a cohort of 65 patients with primary open-angle glaucoma (POAG) using a custom gene panel of 72 genes associated with the disease, to test the use of NGS as a diagnostics method. They reported 9 variants in 7 genes in 15% of patients. However some of the genes included in the panel have been reviewed and showed to have no association to POAG and none of the reported variant presented evidence to support a pathogenic impact. Additionally, the cohort is quite small to identify variants that contribute to <10% of the disease, especially with no variants reported in the main contributor to POAG (e.g. MYOC). Therefore, the evidence presented in this study does not support NGS for genetic diagnosis of glaucoma and does not support the conclusions.

Although variants in Mendelian genes account for around 5% of POAG cases, this is mainly driven by MYOC. It is surprising that no variants were reported in MYOC in this study. Have the authors identified any variants in the gene in their cohort?

Some of the genes included in the panel such as WDR36 and NTF4 have been reclassified by initiatives conducting evidence-based reviews (e.g. ClinGen & PanelApp) as having no association with POAG. Similarly, some rare variants in genes such as EFEMP1 and TEK have been associated with JOAG/POAG in a monogenic manner but have not been included in the list. The list of genes associated with POAG needs to be revised for proper interpretation of results in the context of using NGS as a diagnostic strategy.

Specifically, for WDR36 the initial variants reported in the literature were found to be too common in the general population, a meta-analysis showed no association in case-controls, the gene is ubiquitously expressed therefore expression in eye tissues does not constitute supporting evidence and no functional evidence inc. animal models reported a glaucoma phenotype. However, none of this is presented in the discussion, which only highlights evidence for an association to POAG. This gene has now been reclassified as having no association and should not be included in a gene panel for POAG assessing NGS for diagnostics.

Most of the genes included in the panel have been associated with POAG or its endophenotypes in genome-wide association studies, however rare variants have not yet been associated with the disease for most genes. The rare variants identified in these genes need to be discussed in this context, highlighting that additional studies, including functional evidence, would be needed before these addition of these genes to gene panels is justified.

A definition of hypomorphic variants is missing. In general, hypomorphic alleles refer to variants that can be common, do not cause disease on their own but functional evidence supports an impact. It is unclear from the paper which of the variants identified are rare vs hypomorphic, with evidence to support classification.

Please specify what in silico tools were used for variant impact prediction.

In addition to the above comments, the limitations need to discuss the detection (or lack of) of CNVs using NGS and its impact on detecting deletions or duplications associated with glaucoma (e.g. TBK1 duplications are known to cause POAG).

Other comments:

Some references are missing in the text while others reference incorrect papers. line 55 reference 1 does not report on the rate of undiagnosed glaucoma. line 69 reference 5 does not report on the loci and genes associated with POAG. line 86 ref 10 reports on optic disc parameters but no references are provided for the endophenotypes listed (AL, ACD, CCT). line 303 ref 27 and 28 did not report the first association of CYP1B1 to PCG. There are no references provided for the prevalence of glaucoma (line 53), the familial risk (line 75), the gene contribution to POAG (line 78), the 100 loci reported for POAG (line 84).

Table 3:

- Please note that the use of the ACMG PP5 and BP6 criteria has been discontinued (PMID 29543229) and should be removed for CYP1B1 Y81N.

- PVS1 only applies when LoF is a known disease mechanism. Therefore it does not apply to OPTN variants which cause POAG through a gain of function mechanism since LoF variants such as nonsense are not expected to cause glaucoma. Therefore, there is no evidence to support a role of the nonsense OPTN variant reported with POAG. ClinGen recently reviewed the association of OPTN to POAG and concluded that the only variant showing an association is E50K (https://search.clinicalgenome.org/kb/gene-validity/CGGV:assertion_1d8edc80-413c-4c76-bf06-89198175ac65-2022-05-10T170000.000Z)

- All in silico tools (CADD, SIFT, PP2, REVEL, Mutation Taster) predict a benign impact for CARD10 T436M which should then meet BP4. Please revise and acknowledge in the discussion.

- Please report the highest AF in a population instead of the general population as some of these variants are more common in some populations than others, which should be used when classifying variants.

- Add the ACMG classification for each variant based on evaluation in Table 3.

- Move nucleotide nomenclature to Table 3 instead of Table 4.

- CYP1B1 S28W is missing from Table 3.

Table 4:

- OPTN R298H in Patient 8 should be Q518*.

The prevalence of glaucoma in 2040 reported by Tham et al. is 112 million, not 120 million.

line 127 the range of age at diagnosis is listed as 25-76y but line 129 reports a patient diagnosed at age 18y.

line 267 CYP1B1 S28W is in gnomAD, please correct.

Reviewer #3: The authors have screened two different cohorts of POAG patients for disease causative mutations by using two different techniques; NGS panel for 72 genes associated with POAG and Sanger sequencing for Cyp1b1, a frequently mutated gene in patients with glaucoma of different types. The main aim is the applicability of custom NGS panel for POAG. The study is well planned and methods are up to date. The results are as per expectations but the interpretations need some more work to reach a definite conclusion. Following are some points for improvement of the draft

1. For first cohort of 65 patients, subjected to NGS, 53 patients have family history. It is important to check segregation of the alleles in other family members, to confirm its association with the disease.

2. The most common gene was CYP1B1 as per results. The non-penetrance of various CYP1B1 alleles is reported and its diagnosis in a proband need segregation studies as well to establish its pathogenic role.

3. Why CARD10 gene was not selected for sanger sequencing as it is second common gene as per NGS results

4. Authors should discuss the findings in the light of already reported data of the glaucoma genes in Spanish patients

Reviewer #4: I would like to make several recommendations which the authors may find useful to improve their paper:

• As the authors pointed out several times the importance of genetic diagnosis of glaucoma, I would recommend classifying detected variants (e.g. in table 3, not only ClinVar data). Additionally, in the same table, the ACMG criteria are listed in the column “Predicted effect in silico”. It is not clear are those criteria taken from Varsome database or the authors selected those by themselves (which I would recommend).

• Lines 77-78. Missing references.

• Lines 191-192. What variants were found correlated?

• The variant c.83C>G (p.Ser28Trp) in the CYP1B1 gene was not presented in tables 3 and 4.

• Lines 255-260. It is misleading how many variants were detected in CYP1B1 (2 in the tables, here one more, 4 in discussion...)

• Lines 307-309. Four patients with variants in CYP1B1?

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Reviewer #2: No

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Reviewer #4: Yes: Milena Jankovic

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PLoS One. 2024 Jan 19;19(1):e0282133. doi: 10.1371/journal.pone.0282133.r002

Author response to Decision Letter 0

2 Nov 2023

Thank you for this comment. In our study, we are searching for rare variants described as pathogenic, likely pathogenic or VUS with low frequencies (<1%) in the general population, as well as variants that have been described as hypomorphic potentially associated with adult glaucoma. We agree with the reviewer's opinion, and the chi-squared test can be confusing. We thought it was a way to statistically demonstrate that the allele frequencies observed in our study are higher than those observed in the general population. We do not intend to conduct an association study because we would need a larger number of patients. For this reason, we have removed the column with the frequencies of our study in the Table 3 and the chi-squared test from the manuscript and Table 3.

Additionally, as reviewer suggested, we have added the familial segregation of some variants (see figure 2).

Secondly there is much information missing or in need of clarification. The cohort they study is not well described. The section in mat/mets only talks about the 65 patients, not the 34, so reading that I get the impression the 34 are a subset of the 65, but on lines 97 and 296 these are clearly referred to as a separate cohort.

Thank you for this comment. As mentioned at the end of the comment, these are two different cohorts. In the "Materials and Methods" section, the first three subsections address the patients included in the study. In the first subsection ("Patients"), we refer to all patients included in both cohorts. In the second subsection ("Study of glaucoma-related genes using NGS"), we only refer to the patients who underwent NGS study. In the third subsection ("Sanger sequencing of CYP1B1 in a new cohort of POAG patients"), we are discussing the patients in whom only the CYP1B1 gene was studied by Sanger sequencing. We have rewritten certain parts of the text in these subsections to make it clearer.

Also it seems bizarre that they would weaken a genetic study by making their cohort so diverse - surely there are plenty of POAG patients across 18 Spanish centres without introducing heterogeneity by including NTG and OHT?

We excluded patients with OHT alone to ensure that only patients with glaucoma were included, making the groups more homogeneous and avoiding a predominance of patients with OHT in the Sanger group. This exclusion did not affect the results as no variants were found in patients with OHT alone.

All patients are said to be unrelated - was any attempt made to recruit affected relatives where possible, as this would allow segregation studies?

All detected variants were classified as VUS, and therefore, it is not recommended to perform familial segregation analysis for VUS variants in clinical practice (Pollard et al., Nat. Rev. Genet. 2019). However, given the potential role of CYP1B1 hypomorphic variants in adult glaucoma as described by others in the literature and the availability of samples from relatives, we performed the segregation study of the p.Y81N variant in the relatives of two index samples. We have added this information to the manuscript and a new figure with the pedigrees (Figure 2).

In the introduction the authors rightly suggest genetic effects may be enriched in patients with early onset or strong family history - was any attempt made to recruit particularly among those groups?

In the current study, our objective was the study of patients with primary open-angle glaucoma. This is the most common form of glaucoma in the population and typically manifests after the age of 40. Early-onset glaucoma, such as congenital and juvenile glaucoma, were not included in the study as they do not fall within the scope of the research. This point is not clear in the manuscript, so we have added the following clarification: “Patients with early-onset glaucoma who were diagnosed with PCG or JOAG were excluded”.

Also, if there is no overlap between the 65 patients and the possibly additional 34, the differences between them deserve some note. The second group seem milder, with more OHT and less surgery.

As mentioned above, to ensure the homogeneity between the groups and to avoid over-representation of OHT patients in the cohort of patients studied by Sanger, we excluded patients with OHT alone. This exclusion did not affect the results, as no variants were found in patients with OHT alone.

Thank you very much for the comment, which is entirely appropriate, considering that genes associated with glaucoma are constantly being updated. The gene panel was designed based on a thorough search of the OMIM, Orphanet, and HGMD databases, complemented by an exhaustive search on PubMed. For this reason, we have included the references selected in this search. As the reviewer rightly points out, it's very likely that if we were compiling this list of genes today, we would include more genes. However, we think that the study provides valuable insights into the genes that were under investigation at the time and that these findings can serve as a foundation for future research. Nevertheless, some of the genes we reviewed have been excluded after a careful consideration.

All the exons of the genes included in the study and intronic regions up to +10 bp from the exon were analyzed, with coverage greater than 20x. UTR regions were not analyzed. In addition, our libraries were sequenced using the HiSeq™ 2000 System. We have included this information in the manuscript.

As we have mentioned above we have reported rare variants classified/described as pathogenic, likely pathogenic or VUS with low frequencies in the general population (<1%), as well as variants that have been described as hypomorphic variants associated with glaucoma in other studies. The variants classified as benign o likely benign that are not associated with glaucoma are not described due to they are not associated with the disease. All variants reported were found in coding regions, but as we mentioned in the 'Materials and Methods' section, we have also analysed intronic regions up to 10 bp with respect to the exons.

Besides, to clarify this point, we have included the ACMG classification in Table 3. The variants included in this table are the ones that passed these filters and are therefore candidates for description in the manuscript as they may have some association with adult glaucoma.

In addition I have a few mostly minor corrections. On line 68 and below "Through linkage analysis, 23 loci and four genes.... (ref 5)"; this is clumsy, they seem to be saying at a certain time there were only these findings then later there were more, but the "later" they quote is reference 7 that was actually published in 2016, a year before reference 5?

Thank you for the note. We have removed reference 5 to avoid any confusion.

Line 130 - they need to decide whether they prefer NTG or LTG and stick with one. As far as I know these are not two different diseases but different names for one.

We prefer the use of "low-tension glaucoma," so we have eliminated the use of "normal tension glaucoma" to avoid any confusions.

Legend to table 3 needs to state that (I assume) all variants were heterozygous.

Correct, all variants were detected in heterozygosity. We have added this information in the tables and in the manuscript.

No, the mutations detected by NGS were not rechecked by Sanger sequencing. Sanger sequencing was not demmed necessary. The NGS equipment used is highly accurate, so there is no need for additional validation by Sanger sequencing. Besides, the results of quality controls and coverage were satisfactory. In our routine work, Sanger sequencing is only required to validate frameshift variants and in some cases where the variant appears in less than 30% of the reads. This was not the case for any of the variants reported in the manuscript.

Thank you for this note. Yes, these residues are conserved in species other than mammals. Regarding residue 28, we can see that it is conserved in a multitude of species, except for some bird and amphibian species. As for residue 81, we have observed its presence in all types of species assessed. Additional examples of birds, reptiles, amphibians and fish have been included in Figure 1. Furthermore, we have updated the figure caption to clarify that it is a comparison of residues in different species.

Figure 2d is too small and fuzzy to derive anything from it.

We have enlarged the image for better visibility.

Thank you, we have added this clarification to the text.

Also in the discussion there is much talk of hypomorphic alleles - a clear definition of what the authors mean by that would be useful.

Thank you, we have added a definition of hypomorphic alleles in the discussion.

As the reviewer points out, it is noteworthy that we did not find variants in the MYOC gene in our cohort of adult glaucoma patients. In our centres, we have detected patient with pathogenic variant in MYOC, but these typically involve younger patients, with diagnoses often made in their 20s or 30s. In these cases, the main diagnosis is juvenile open-angle glaucoma. In fact, it is considered that the MYOC gene is primarily associated with juvenile open-angle glaucoma. Patients with congenital or juvenile glaucoma were excluded from our cohorts, and this fact could explain why we did not identify any patient with MYOC variants. However, the MYOC gene was included in the panel.

We have data on younger cohorts of glaucoma patients in which we have found variants in MYOC (data not published yet). However, as mentioned earlier, the age of diagnosis was lower, and the glaucoma signs led to the diagnosis of juvenile open-angle glaucoma, which is not the focus of this article.

Regarding the gene panel, it was designed based on a thorough search of OMIM, Orphanet, and HGMD databases, complemented by an exhaustive search on PubMed. For this reason, we have added the references selected in this search. As the reviewer rightly points out, it's highly likely that if we were to compile this list of genes today, we would include more genes. However, we think that the study provides valuable insights into the genes that were under investigation at the time and that these findings can serve as a foundation for future research. Nevertheless, some of the genes we reviewed have been excluded after a careful examination.

We agree with the reviewer. EFEMP1 and TEK are genes associated with glaucoma. Regarding EFEMP1, it is a gene with a very recent association, which OMIM has not yet included. TEK is a better-known gene. However, both are associated with juvenile and congenital glaucoma, respectively, so we would not have expected to find variants in these genes. Nevertheless, they are genes associated with glaucoma and should have been included in the panel.

Regarding NTF4, the first study that linked NTF4 and glaucoma was conducted by Pasutto et al. (Am. J. Hum. Genet. 2009). The authors examined the postmortem retina of a patient with no history of eye disease and used in situ hybridization to confirm a specific NTF4 signal localized to the ganglion cell layer of the retina. However, a subsequent study revealed that NTF4 does not play a significant role in the pathogenesis of POAG (Liu et al., Am. J. Hum. Genet. 2010). Nevertheless, since then, we can find different studies with contradictory results and multiple reported cases of glaucoma patients with NTF4 variants (Jemmeih et al., Ophthalmic Epidemiology, 2022; Kumar et al., Gene, 2016; Vithana et al., Mol. Vis. 2010; Huang et al., Sci. Rep. 2018). Besides, if we search for the NTF4 gene in OMIM or Orphanet, it appears associated with POAG. Thus, a meta-analysis of different studies in glaucoma patients with population-based controls would be required to clarify the role of NTF4 in glaucoma. In our case, until this is resolved, we have decided to include the gene in the gene panel. In our cohort, we did not detect any variant in NTF4.

Regarding the inclusion of WDR36 in our panel, clinical studies have yielded contradictory results. Some of these studies indicate a lack of effect of WDR36 in certain populations (Kramer et al., Arch. Ophthalmol. 2006; Frezzotti et al., Br. J. Ophthalmol. 2011; Liu et al., Medicine. 2017). However, other research has demonstrated that WDR36 is a contributing risk factor for glaucoma progression and severity (Footz et al., Hum. Mol. Genet. 2009; Blanco-Marchite et al., Investig. Ophthalmol. Vis. Sci. 2011; Huang et al., Investig. Ophthalmol. Vis. Sci. 2014; Mookherjee et al., Mol. Vis. 2011; Meer et al., Genes 2021). In fact, there is a known genetic interaction between WDR36 and glaucoma susceptibility. It also plays a significant role in retinal homeostasis. Therefore, given the potential of the WDR36 gene as a causative gene for glaucoma, its investigation in these cases is important, although further studies are needed to fully understand its role in glaucoma. To clarify this point, we have added the relevant explanation about WDR36 in the discussion and had to include some additional references.

Indeed, the reviewer is correct. We have further discussed the variables, and, as expected, the variants are classified as VUS. Most of them have not yet been associated with glaucoma, thus emphasizing the need for additional studies to confirm these findings.

Thank you, we have added a definition of hypomorphic alleles in the discussion. Currently, as we mentioned in the discussion, there are only hypomorphic variants described in CYP1B1 and SIX6, and we have detected some of them in this study. Regarding rare variants, these will be those classified as pathogenic, likely pathogenic, or VUS and that are infrequent in the population, whose effects are not fully understood. We have also added this information to the manuscript.

Please specify what in silico tools were used for variant impact prediction.

The in silico tools used were: CADD, Polyphen2, DEOGEN2, MutPred, FATHMM-XF, Mutation assessor. MVP, PROVEAN, EIGEN, LRT, SIFT, BLOSUM, DANN, LIST-S2, M-CAP, MutationTaster and PrimateAI. We have added this information to the manuscript.

We thank you for this comment, and we have included this limitation in the study, and we have discussed it in the Discussion section.

Other comments:

The reference 1 was incorrectly placed in the text. We have now placed it in the correct location. With regard to reference 5, we have removed this reference.

Regarding the line 86, we have added the following reference: Asefa NG, Neustaeter A, Jansonius NM, Snieder H. Heritability of glaucoma and glaucoma-related endophenotypes: Systematic review and meta-analysis. Surv Ophthalmol. 2019;64(6):835-851.

Regarding the sentence with references 27 and 28, we did not intend to imply that these references report the first association of the CYP1B1 gene with PCG. Our intention was to emphasize that the CYP1B1 gene has always been associated with the genetics of primary congenital glaucoma, not with POAG. We have rewritten the sentence to clarify this point.

There are no references provided for the prevalence of glaucoma (line 53), the familial risk (line 75), the gene contribution to POAG (line 78), the 100 loci reported for POAG (line 84).

The reference for the sentence in line 53 is reference 1, which was incorrectly placed. We have now placed it in the correct location.

Regarding the other lines, as the reviewer recommends, we have added the following references:

• Line 75:

o Le et al. Risk factors associated with the incidence of open-angle glaucoma: the visual impairment project. Invest Ophthalmol Vis Sci. 2003:44(9):3783-9.

o Green et al. How significant is a family history of glaucoma? Experience from the Glaucoma Inheritance Study in Tasmania. Clin Exp Ophthalmol. 2007;35(9):793-9.

• Line 78: Rao et al. Complex genetic mechanisms in glaucoma: an overview. Indian J Ophthalmol. 2011;59(Suppl. 1):S31-42.

• Line 84: Han et al. Large-scale multitrait genome-wide association analyses identify hundreds of glaucoma risk loci. Nat Genet. 2023;55(7):1116–25.

Table 3:

- Please note that the use of the ACMG PP5 and BP6 criteria has been discontinued (PMID 29543229) and should be removed for CYP1B1 Y81N.

Thank you for the notice. We have removed both criteria from the variant. Additionally, we have reviewed the classification of this variant, and the correct criteria are: PS3, PP3, BS1, and BS2. We have added the PS3 criterion because there is a study (Lopez-Garrido et al., Clin Genet. 2010) that conducts a functional analysis of the Y81N variant. The authors reported that the presence of six variants in the CYP1B1 gene (including the Y81N variant) reduces enzymatic activity, with activity ranging from 18% to 40% of the wild-type protein. Therefore, the variant is classified as a VUS, which has also been described as a hypomorphic variant.

Thank you for the comment, we agree with what the reviewer has stated. It is indeed a loss of function and should not be classified under the PVS1 criterion. We have removed it from the table, and the variant is still classified as a VUS. Loss of function in the OPTN gene is associated with amyotrophic lateral sclerosis (ALS), and variants predominantly described in association with glaucoma are missense mutations. Nevertheless, there are cases where ALS and glaucoma coexist, as seen in one of the patients reported by Mayurama et al. (Nature, 2010). Although recent studies have reported that E50K is the only variant known to be causative of glaucoma associated with the OPTN gene, the presence of variants in this gene may contribute to some added risk for glaucoma in certain patient populations, as indicated by Fox and Fingert (Progress in Retinal and Eye Research, 2023) in their latest review. For all these reasons, the variant found in our study should be considered a VUS.

We have included this information in the text, and we have also added the two references mentioned.

- All in silico tools (CADD, SIFT, PP2, REVEL, Mutation Taster) predict a benign impact for CARD10 T436M which should then meet BP4. Please revise and acknowledge in the discussion.

We have added the BP4 criterion in the table. Nevertheless, the variant continues to be classified as a VUS. We have added this information to the manuscript.

- Add the ACMG classification for each variant based on evaluation in Table 3.

We have added the highest AF and the ACMG classification for each variant to the Table 3.

- Move nucleotide nomenclature to Table 3 instead of Table 4.

We have made the change to move nucleotide nomenclature to Table 3 instead of Table 4.

- CYP1B1 S28W is missing from Table 3.

Originally, Table 3 only displayed the results of the NGS study. The p.S28W variant in CYP1B1 was found in the Sanger study. To clarify this point, we have decided to include all variants in Table 3.

Table 4:

- OPTN R298H in Patient 8 should be Q518*.

It is absolutely right; it was a mistake in the table preparation. We have already corrected it.

The prevalence of glaucoma in 2040 reported by Tham et al. is 112 million, not 120 million.

That's correct. The prevalence of glaucoma in 2040 reported by Tham et al. is 112 million, not 120 million. We have already made the change in the manuscript.

line 127 the range of age at diagnosis is listed as 25-76y but line 129 reports a patient diagnosed at age 18y.

Thank you for this observation. The age range for patients with POAG is indeed 18 to 76 years. It has now been corrected.

line 267 CYP1B1 S28W is in gnomAD, please correct.

We have already corrected this data. The variant has a very low population frequency (0.004%).

Following are some points for improvement of the draft

All detected variants were classified as VUS, and therefore, conducting studies on relatives of VUS variants whose association is not fully understood is not recommended in clinical practice. However, due to the role of CYP1B1 in glaucoma and the description of hypomorphic variants in the literature, we decided to perform the segregation study of the Y81N variant in the relatives of the patients detected in the NGS study. We have added this information to the manuscript and a new figure with the pedigrees. Relatives of the patients detected in the Sanger study were not available for the segregation study.

3. Why CARD10 gene was not selected for sanger sequencing as it is second common gene as per NGS results

The CARD10 gene was not selected for Sanger sequencing because, despite being the second most common gene according to NGS results, we decided to study CYP1B1 due to its well-documented nature. Its association with glaucoma is more established, and in scientific literature, we can find hypomorphic alleles described in relation to POAG. Furthermore, the cost of studying CARD10 by Sanger sequencing is 10 times more expensive than the cost of studying CYP1B1 (CARD10 has 20 exons, whereas CYP1B1 has only 2 exons). However, we are open to revisiting this decision based on the significance of the CARD10 gene in further analyses.

4. Authors should discuss the findings in the light of already reported data of the glaucoma genes in Spanish patients

To our knowledge, there are previous studies with Spanish populations affected by pseudoexfoliative glaucoma, primary congenital glaucoma, juvenile glaucoma and anterior segment dysgenesis. Regarding POAG, there is a study from 2007 that retrospectively investigated the contribution of myocilin (MYOC) and optineurin (OPTN) to a cohort of OHT and POAG Spanish patients. They found that a minority of adult-onset high-pressure POAG patients carried heterozygous disease-causing mutations in the MYOC gene and that OPTN was not involved in either OHT or POAG. (Lopez-Martinez F et al. Mol Vis. 2007 Jun 14;13:862-72.). The same authors published another article several years later and found heterozygous hypomorphic CYP1B1 mutations in Spanish POAG patients (López-Garrido MP et al. Clin Genet. 2010 Jan;77(1):70-8).

Reviewer #4: I would like to make several recommendations which the authors may find useful to improve their paper:

Thank you for the comment. We have clarified this in the table and added a column with the ACMG classification of the detected variants.

• Lines 77-78. Missing references.

We have added the following reference: Rao et al. Complex genetic mechanisms in glaucoma: an overview. Indian J Ophthalmol. 2011;59(Suppl. 1):S31-42.

• Lines 191-192. What variants were found correlated?

The presence of thin pachymetry was detected in two patients (patient 1 and patient 8). The variant p.Y81N in CYP1B1 was detected in patient 1, while the variant p.R298H in OPTC was detected in patient 8. As discussed, the presence of thin pachymetry readings is a recognized endophenotypic trait that amplifies the severity of glaucoma. Nevertheless, further studies are warranted to explore this relationship more comprehensively.

• The variant c.83C>G (p.Ser28Trp) in the CYP1B1 gene was not presented in tables 3 and 4.

Thank you for this comment. The variant has already been included in the tables.

• Lines 255-260. It is misleading how many variants were detected in CYP1B1 (2 in the tables, here one more, 4 in discussion...)

• Lines 307-309. Four patients with variants in CYP1B1?

The variant p.Y81N was detected in four patients (two in the NGS study and two in the Sanger study). Additionally, in the Sanger study, we found one patient with a different variant in the CYP1B1 gene. Therefore, as shown in Table 4, a total of five patients with variants in CYP1B1 were identified (four patients with p.Y81N and one patient with p.S28W).

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PLoS One. doi: 10.1371/journal.pone.0282133.r003

Decision Letter 1

Alvaro Galli

22 Nov 2023

Next-generation sequencing-based gene panel tests for the detection of rare variants and hypomorphic alleles associated with primary open-angle glaucoma

PONE-D-23-03532R1

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Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

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PLoS One. doi: 10.1371/journal.pone.0282133.r004

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Alvaro Galli

8 Jan 2024

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Supplementary Materials

S1 Table. Primers designed to amplify coding region of the CYP1B1 gene.

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Data Availability Statement

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PERMALINK

Next-generation sequencing-based gene panel tests for the detection of rare variants and hypomorphic alleles associated with primary open-angle glaucoma

Elena Milla

Javier Laguna

Mª Socorro Alforja

Beatriz Pascual

María José Gamundi

Emma Borràs

Imma Hernán

María Jesús Muniesa

Marta Pazos

Susana Duch

Miguel Carballo

Meritxell Jodar

Roles

Abstract

Introduction

Materials and methods

Patients

Study of glaucoma-related genes using NGS

Table 1. Clinical data of patients studied by NGS and Sanger sequencing.

Table 2. List of genes included in the custom glaucoma panel designed.

Sanger sequencing of CYP1B1 in a new cohort of POAG patients

Interpretation and classification of variants

Results

Rare genetic variants detected in POAG patients by NGS

Table 3. Description and classification of variants detected in this study.

Table 4. Clinical characteristics of patients and variants detected in this study.

Fig 1.

Fig 2.

Fig 3. Ophthalmologic studies in patients 4 and 5 (siblings) with the variant p.T436M in the CARD10 gene.

Analysis of hypomorphic CYP1B1 variants

Fig 4. Ancillary tests of patient 13.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Alvaro Galli

Roles

Author response to Decision Letter 0

Decision Letter 1

Alvaro Galli

Roles

Acceptance letter

Alvaro Galli

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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