A novel LOXHD1 variant in a Chinese couple with hearing loss.

Introduction

Hearing loss (HL) is the most frequent sensory deficit in humans, with a prevalence of around 1/1000 in newborns.[1,2] Approximately 50% to 60% of hearing loss cases are caused by genetic factors.[3] The genetic mode of HL inheritance can be autosomal recessive, autosomal dominant, mitochondrial, or X/Y-linked. To date, 121 genes have been reported to be associated with hearing loss (http://hereditaryhearingloss.org/): 45 are autosomal dominant genes,71 are autosomal recessive, and 5 are X-linked. However, most of these genes have only been reported in one or a few families.[4] Epidemiological studies showed that variants in GJB2, SLC26A4, and 12S rRNA genes are highly correlated with hereditary HL.[2] The most frequent genetic cause of HL is variants in GJB2, and most of these cases occur with non-progressive HL. Variants in SLC26A4, CDH23, and MYO3A were also shown to be associated with naturally occurring progressive HL.[5,6]

The genetic diagnosis of HL is very important because the findings can be used to aid treatment decisions, and provide prognostic information and genetic counseling for the patient’s family.[7] Here, we describe a young couple with HL in whom the husband carried compound heterozygous variants of GJB2, and the wife had an extremely rare form of deafness and compound heterozygous variants of LOXHD1. We provided genetic counseling for this couple and followed them up during their pregnancy.

Patients and methods

Study population

We recruited a young Chinese couple (husband: 27 years old; wife: 25 years old) with congenital HL and 100 healthy controls (aged 25–30 years) from Gansu Provincial Maternal and Child Health Care Hospital. The couple had been married for 6 months and requested pre-pregnancy genetic counseling. The study was in accordance with the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of Gansu Provincial Maternal and Child Health Care Hospital. Written informed consent was obtained from all participants.

Sample collection and genomic DNA preparation

Blood samples (2–3 mL) were collected from the probands and their parents and control individuals. Genomic DNA was extracted using a Tiangen DNA extraction kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions and quantified spectrophotometrically.

Targeted next-generation sequencing (NGS) and Sanger sequencing

First, the coding exon and flanking sequences of GJB2 were screened by PCR and direct sequencing using primers and conditions described in Table 1. If no GJB2 variant was found, targeted capture of candidate disease genes (n = 165, Table 2) was performed using a GenCap custom enrichment kit (MyGenostics, Beijing, China). Briefly, 1 μg of DNA library was mixed with BL buffer and a GenCap hypercholesterolemia probe (MyGenostics) and heated in a PCR cycler at 95°C for 7 minutes then 65°C for 2 minutes. A total of 23 μL HY buffer (pre-warmed to 65°C; MyGenostics) was added and the mixture was incubated at 65°C for 22 hours for hybridization. MyOne beads (50 μL; Thermo Fisher Scientific Inc., Rockford, IL, USA) were washed three times in 500 μL binding buffer (1×) and re-suspended in 80 μL binding buffer (1×). Next, 64 μL binding buffer (2×) was added and the mixture was transferred into a tube containing 80 μL MyOne beads, and spun for 1 hour on a rotator. The beads were then washed once with WB1 buffer at room temperature for 15 minutes and three times with WB3 buffer at 65°C for 15 minutes. Elution buffer was used to elute the bound DNA, which was amplified as follows: 98°C for 30 seconds then 15 cycles of 98°C for 25 seconds, 65°C for 30 seconds, and 72°C for 30 seconds, followed by 72°C for 5 minutes. PCR products were purified using SPRI beads (Beckman Coulter Inc., Brea, CA, USA) following the manufacturer’s protocol. Enrichment libraries were sequenced on an Illumina HiSeq 2000 sequencer (Illumina, San Diego, CA, USA) for 100-bp paired reads.

Table 1.

Primers and PCR conditions for GJB2 and LOXHD1.

Primer name	Sequence (5′–3′)	Product size (bp)	Amplification reaction conditions
GJB2-F	CATGCTTGCTTACCCAGACTCA	873	95°C for 5 minutes, then 20 cycles of 94°C for 30 seconds, 62°C for 45 seconds, and 72°C for 1 minute95°C for 5 minutes, then 15 cycles of 94°C for 30 seconds, 58°C for 45 seconds, and 72°C for 1 minute.
GJB2-R	TAGCGACTGAGCCTTGACAGC
GJB2-S1	TGGGTTTTGATCTCCTCGATG	Sequencing primers
GJB2-S2	GCCTACCGGAGACATGAGAAG
LOXHD1-E14F	GGTAGTAGGGCTGGGTCTTCC	355
LOXHD1-E14R	AGTTGCCTAACCCATCAGCTC
LOXHD1-E19F	CACCAACTCCACGACAAGTTC	594
LOXHD1-E19R	GAGGTGGTGGAAGGATCTGAG

GJB2-S1 and GJB2-S2 are the sequencing primers for GJB2.

Table 2.

Genes in the hearing loss panel.

Nuclear genes associated with hereditary HL
ACTG1	ADGRV1	ALX3	BSND	CABP2	CCDC50	CDH23
CEACAM16	CHD7	CIB2	CLDN14	CLPP	CLRN1	COCH
COL11A1	COL11A2	COL1A1	COL1A2	COL2A1	COL4A3	COL4A4
COL4A5	COL4A6	COL9A1	COL9A2	CRYM	DFNB59	DIABLO
DIAPH1	DIAPH3	DSPP	ECM1	EDN3	EDNRB	ELMOD3
ESPN	ESRRB	EYA1	EYA4	FGF3	FGF8	FGFR1
FGFR3	FLNA	FOXI1	FREM1	FXN	GATA3	GIPC3
GJB1	GJB2	GJB3	GJB6	GLYAT	GPSM2	GRHL2
GRXCR1	GSDME	HARS	HARS2	HGF	HMX1	HOXA2
HSD17B4	IL13	ILDR1	KARS	KCNE1	KCNJ10	KCNQ1
KCNQ4	KITLG	KRT9	LAMA3	LARS2	LHFPL5	LOXHD1
LRTOMT	MARVELD2	MIR96	MITF	MPZ	MSRB3	MYH14
MYH9	MYO15A	MYO1A	MYO1E	MYO3A	MYO6	MYO7A
NDP	NDRG1	NEFL	NELL2	NF2	OPA1	OTOA
OTOF	OTOG	OTOGL	P2RX2	PABPN1	PAX3	PCDH15
PCDH9	PDZD7	PMP22	PNPT1	POLR1C	POLR1D	POU3F4
POU4F3	PROK2	PROKR2	PRPS1	PTPN11	PTPRQ	PTPRR
RDX	RPGR	SALL1	SALL4	SEC23A	SEMA3E	SERPINB6
SIX1	SIX5	SLC17A8	SLC19A2	SLC26A4	SLC26A5	SMAD4
SMPX	SNAI2	SOX10	STRC	TBC1D24	TCIRG1	TCOF1
TECTA	TIMM8A	TJP2	TMC1	TMEM126A	TMIE	TMPRSS3
TMPRSS4	TNC	TPRN	TRIOBP	TRMU	TSPEAR	TYR
USH1C	USH1G	USH2A	WFS1	WHRN
Mitochondrial gene
MT-RNR1	RNR-TL1	MT-CO1	RNR-TS1	MT-TK	RNR-TE

After sequencing, high-quality reads were retrieved by filtering out adaptors, low-quality reads, and short sequences (<40 bp). Data quality control standards were: 10× > 95%, depth = 200 ± 30. The SOAPaligner program (SOAP v2.21) was used to align clean read sequences to the human reference genome (UCSC Genome Browser hg19). After removing duplicates with Picard software (v1.119), single nucleotide polymorphisms (SNPs) were identified using SOAPsnp v1.03. Subsequently, reads were realigned to the reference genome using the Burrows–Wheeler alignment program (0.7.12-r1044), and insertions or deletions (InDels) were detected by the HaplotypeCaller of GATK software (https://software.broadinstitute.org/gatk/, GATK-3.5) and filtered by VariantFiltration of GATK software. We annotated the identified SNPs and InDels using the Exome-assistant program. Short read alignment and candidate SNP and InDel validation were performed using MagicViewer.

We performed Sanger sequencing for all identified variants in the probands and their parents. PCR primers for Sanger sequencing were designed by Primer 3.0 software (http://bioinfo.ut.ee/primer3-0.4.0/). Primers and PCR conditions for GJB2 and LOXHD1 are shown in Table 1. DNA sequencing was performed on an ABI 3500DX Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

Bioinformatics analysis

If a novel variant was found that was not reported in the Human Gene Variant Database (http://www.hgmd.cf.ac.uk/) or ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/), we used PolyPhen2 (http://genetics.bwh.harvard.edu/pph2) and PROVEAN (http://provean.jcvi.org/index.php) tools to predict its possible functional role. To exclude the possibility that the variant was a polymorphism, we also performed direct sequencing in 100 healthy controls.

Results

Variant analysis

PCR and direct Sanger sequencing identified c.235delC (rs80338943)/c.299-300delAT (rs111033204) compound heterozygous variants of GJB2 in the husband. c.235delC was inherited from his mother and c.299-300delAT was inherited from his father (Figure 1).

Figure 1.

Results of Sanger sequencing. Compound heterozygous variants were detected in the proband.

c.235delC (rs80338943, left)/c.299-300delAT (rs111033204, right) of GJB2. The father carried the heterozygous variant c.299-300delAT, while the mother and fetus carried the heterozygous variant c.235delC.

No GJB2 variants were identified in the wife, so targeted NGS was used to search for potential pathogenic variants. She was shown to carry c.1828G>A (p.Glu610Lys, rs535637788)/c.2825-2827delAGA compound heterozygous variants of LOXHD1, with c.2825-2827delAGA inherited from her mother and c.1828G>A from her father (Figure 2). Her hearing loss is an extremely rare form known as DFNB77 (OMIM: 613079). Variant c.2825-2827delAGA has previously been reported to be associated with DFNB77,[7] but variant c.1828G>A (p.E610K) was only reported in a Mexican-American individual in the 1000 Genomes database. It has not been reported to be associated with DFNB77. PCR and direct Sanger sequencing did not identify this variant in any of our 100 healthy controls.

Figure 2.

Results of Sanger sequencing. Compound heterozygous variants were detected in the proband.

c.1828G>A(p.Glu610Lys, rs535637788, left)/c.2825-2827delAGA (right) of LOXHD1. The father and fetus carried the heterozygous variant c.1828G>A, while the mother carried the heterozygous variant c.2825-2827delAGA.

Bioinformatics analysis score

PolyPhen2 and PROVEAN tools were used to evaluate the possible functional role of variant c.1828G>A. PolyPhen2 gave a score of 1, suggesting that the site might be a damaging variation. The PROVEAN score was –3.203, and the site was considered “deleterious”.

Pregnancy outcome

Following molecular analysis, we provided genetic counseling to the young couple with HL. We explained that their children were unlikely to have HL because they both carried different genetic variants. During their pregnancy, they underwent regular prenatal checkups which detected the presence of the c.235delC GJB2 variant and the c.1828G>A LOXHD1 variant in the fetus (Figures 1 and 2). The baby was born in June 2018, and both ears passed the hearing screening test.

Discussion

We identified causative variants of HL in both individuals of a young Chinese couple. The variants of the husband were common and the c.2825-2827delAGA LOXHD1 variant of the wife was previously associated with DFNB77; however, the c.1828G>A LOXHD1 variant of the wife has only been reported in a Mexican-American individual in the 1000 Genomes database, and not in the HGMD database or elsewhere. PolyPhen2 and PROVEAN tools suggested that it is a likely pathogenic variant.

LOXHD1 is located on chromosome 18q12-q21 and contains at least 43 exons.[8] It encodes lipoxygenase homology domain 1-containing protein 1 which has 15 PLAT domains[4] that are involved in targeting proteins to the plasma membrane and mediating protein interactions.[9-12] Mouse studies showed that the Loxhd1 product is localized to the stereocilia of sensory hair cells, and that Loxhd1 variants can induce deafness with defects in the stereocilia followed by hair cell degeneration.[13] This indicates that LOXHD1 plays an important role in maintaining normal hair cell function.

Although DFNB77 has previously been associated with LOXHD1 variants, it is a highly heterogeneous disease both phenotypically and genetically. More than 23 probands with DFNB77 have been reported worldwide on PubMed, and 37 different disease-causing variants have been identified (Table 3).[5,7-21] Most of these probands come from Asia, suggesting that it has a high incidence of DFNB77. They show different auditory characteristics and audiometric phenotypes, varying from mild to profound and from stable to progressive sensorineural HL.[5,7-21] Animal studies revealed that homozygous missense variants of Loxhd1 induced profound deafness while homozygous nonsense variants caused progressive HL.[13] However, Wesdorp et al.[4] found that the type of variant (nonsense or missense) did not associate with HL severity, and that the combination of a nonsense and missense variant could cause different audiometric phenotypes. Such research is limited, so correlations between LOXHD1 variants and phenotypic characteristics of HL remain unclear.

Table 3.

Known LOXHD1 variants causative of hearing loss.

No.	Nucleotide or amino acid change	Variant type	Zygosity	Type of HL	Progressiveness	Population
1[5]	c.879 + 1G>A	Splice	Hom	Profound	Non-progressive	Japanese
2[5]	c.5869G>T	Nonsense	Het	Moderate-severe	Non-progressive	Japanese
3[5]	c.4480C>T	Nonsense	Het	Moderate-severe	Non-progressive	Japanese
4[7]	c.884C>T	Missense	Het	Moderate-severe	Progressive	–
5[7]	c.2825_2827delAGA	Frameshift	Het	Moderate-severe	Progressive	–
6[7]	c.2797C>T	Nonsense	Het	Profound	Non-progressive	–
7[7]	c.1730T>G	Frameshift	Het	Profound	Non-progressive	–
8[7]	c.2722G>A	Missense	Het	Profound	Non-progressive	–
9[7]	c.3015_3017delCTT	Frameshift	Het	Profound	Non-progressive	–
10[7]	c.766G>T	Nonsense	Het	Profound	Non-progressive	–
11[7]	c.3596T>C	Missense	Het	–	–	–
12[7]	c.2696G>C	Missense	Het	–	–	–
13[7]	c.4526G>A	Missense	Hom	Profound	Progressive	–
14[7]	c.4480C>T	Nonsense	Hom	Profound	Progressive	–
15[7]	c.3206G>A	Missense	Het	Moderate-severe	Non-progressive	–
16[7]	c.894T>G	Nonsense	Het	Moderate-severe	Non-progressive	–
17[7]	c.1501delG	Frameshift	Het	Profound	Progressive	–
18[7]	c.1193G>A	Missense	Het	Profound	Progressive	–
19[7]	c.1147C>T	Nonsense	Het	Profound	Progressive	–
20[8]	c.4714C>T	Nonsense	Hom	Profound	Non-progressive	Jewish
21[9]	c.5674G>T	Missense	Het	Moderate-severe	Non-progressive	Japanese
22[9]	c.4212 + 1G>A	Splice	Het	Moderate-severe	Non-progressive	Japanese
33[13]	c.2008C>T	Nonsense	Hom	Moderate-severe	Progressive	Iranian
24[14]	p.Gly398Glu	Missense	Het	Profound	Progressive	American
25[14]	p.Arg383X	Nonsense	Het	Profound	Progressive	American
26[15]	c.2863G>T	Nonsense	Hom	–	–	Turkey
27[15]	c.4480C>T	Nonsense	Hom	–	–	Turkey
28[16]	c.1588C>T	Nonsense	Hom	Profound	Progressive	Qatar
29[17]	c.71delT	Frameshift	Hom	–	–	Turkish
30[18]	c.3371G>A	Missense	Het	Profound	Non-progressive	Cameroonian
31[18]	c.3979T>A	Missense	Het	Profound	Non-progressive	Cameroonian
32[19]	c.1751C>T	Missense	Het	Moderate-severe	Progressive	Chinese
33[19]	c.5815G>A	Missense	Het	Moderate-severe	Progressive	Chinese
34[20]	p.A1406V	Missense	Het	–	–	–
35[20]	p.K148*	Nonsense	Het	–	–	–
36[21]	c.797 G > A	Missense	Het	–	–	Chinese
37*	c.1828G>A	Missense	Het	Profound	Non-progressive	Chinese

References shown as superscript numbers in first column; * This study

Hom, homozygous; het, heterozygous

LOXHD1 variants have not only been linked to HL but are also associated with late-onset Fuchs corneal dystrophy (FCD), a genetic disorder of the corneal endothelium.[22] A case–control study by Stehouwer et al.[23] reported a significant association between FCD and hearing disorders, but this should be investigated in larger sample sizes.[22] We believe that it is important to check for ophthalmology disorders in patients with HL caused by LOXHD1 variants; however, we found no FCD phenotype in the current proband with LOXHD1 variants.

In conclusion, we carried out molecular diagnosis in a young couple with congenital HL and identified different disease-causing variants in the two individuals. The husband had compound heterozygous variants of GJB2, while the wife had the extremely rare HL known as DFNB77 and compound heterozygous variants of LOXHD1. We followed up the pregnancy outcome of this couple, and report that both ears of their baby passed the hearing screening test. To the best of our knowledge, this is the third case reported in Chinese individuals and the first in the northwest of the country. PCR and direct Sanger sequencing cannot provide effective detection of diseases caused by such rare variants. However, with the development of molecular diagnostic technology, the cost of tests is decreasing and NGS will become a more effective way of providing accurate molecular diagnosis and genetic counseling for rare diseases.