Sensorineural deafness and male infertility: a contiguous gene deletion syndrome

Abstract

Syndromic hearing loss that results from contiguous gene deletions is uncommon.Three families with a novel syndrome characterised by deafness and infertility are described. Linkage was established by completing a genome-wide scan and candidate genes in the linked region were screened by direct sequencing. The deleted region is about 100 kb long and involves four genes (KIAA0377, CKMT1B, STRC and CATSPER2), each of which has a telomeric duplicate. This genomic architecture underlies the mechanism by which these deletions occur. CATSPER2 and STRC are expressed in the sperm and inner ear, respectively, consistent with the phenotype in persons homozygous for this deletion. A deletion of this region has been reported in one other family segregating male infertility and sensorineural deafness. We have identified three families segregating an autosomal recessive contiguous gene deletion syndrome characterised by deafness and sperm dysmotility. This new syndrome is caused by the deletion of contiguous genes at 15q15.3.

BACKGROUND

In developed countries, genetic deafness is estimated to affect one in 2000 newborns.¹ Syndromic and non-syndromic subclassifications are recognised, the former being differentiated from the latter by the presence of additional traits that segregate with the deafness phenotype. The most common types of syndromic hearing loss include Waardenburg syndrome and branchio-oto-renal syndrome, both of which are autosomal dominant, and Pendred syndrome which is recessive.² Syndromic hearing loss can also result from contiguous gene deletions, although these are uncommon.³ Examples include infantile hyperinsulinism associated with enteropathy, deafness and renal tubulopathy caused by a contiguous gene deletion located on chromosome 11p. We have identified three families segregating a new autosomal recessive contiguous gene deletion syndrome characterised by deafness and sperm dysmotility.

CASE PRESENTATION

Clinical findings in families D_SM, L705 and L1014 were consistent with a diagnosis of autosomal recessive non-syndromic deafness (figs 1 and 2). In each family, affected individuals had prelingual auditory impairment with normal vestibular function as evidenced by age-appropriate developmental motor milestones and physical tests of balance. No deaf person had evidence of syndromic features. However, based on the genotypic data, sperm motility was assessed in a consenting male in family D_SM. Although sperm counts were normal (78×10⁶/ml) with normal volume (∼4 ml) and colour, malformed sperm (>88%) were observed (mainly thin heads, micro- and irregular acrosomes). About 30% of sperm had short, coiled flagella. Less than 5% of sperm had full swimming capacities after liquidation. The reduction in sperm motility and viability was consistent with the diagnosis of asthenoteratozoospermia. These findings are suggestive of male infertility and are supported by the fact that the three affected males in family D_SM (II2, II4 and II5) were unable to father children without intracytoplasmic sperm injection.

Figure 1. Pedigree of family D_SM (A) and family L705 (B).

Haplotypes for markers on 15q15.3 are given. Regions shared by all affected individuals are shaded. Audiograms show moderate-to-severe hearing impairment at all frequencies. Normal auditory thresholds are above 25 dB (□, male; ⌃, female; ▪, affected male; diagonal line, deceased; X, left ear; O, right ear).

Figure 2. Pedigree of family L1014.

Audiograms show moderate-to-severe hearing impairment at all frequencies. Normal auditory thresholds are above 25 dB (□, male; ⌃, female; ▪, affected male; diagonal line, deceased; X, left ear; O, right ear).

INVESTIGATIONS

Family D_SM, family L705 and family L1014 were ascertained through a genetics clinic at The Welfare Science and Rehabilitation University in Iran. Informed consent was obtained from all participants. A sample (10 ml) of whole blood was obtained as a DNA source.

Linkage was established by completing a genome-wide scan using 400 fluorescence dye-labelled microsatellite markers with an average spacing of 10 cM across the 22 autosomes and chromosome X (Prism Linkage Mapping Set, version 2.5; Applied Biosystems, Foster City, CA). Candidate genes in the linked region were screened by direct sequencing.

In both families D_SM and L705, a genome-wide screen using short tandem repeat polymorphic (STRP) marker analysis identified a single region of homozygosity by descent in affected persons on 15q15.1–15.3. In family D_SM, we narrowed the breakpoints to a 2.5-kb interval flanked by two critical mismatches, SNM7 and SNM8 (fig 3A, table 1). The absence of additional mismatches in this interval precluded us from narrowing the breakpoint further. As we expected, amplification across the breakpoint region generated PCR products in all persons in family D_SM and in all controls. However by sequencing, all controls demonstrated the presence of nucleotide sequence mismatches SNM7a, SNM7b and SNM8a, SNM8b (fig 3B), indicating the absence of a deletion. Affected persons from family D_SM, in contrast, failed to demonstrate nucleotide sequence mismatches (missing SNM7a and SNM8b; fig 3B) consistent with the presence of a deletion. Total RNA isolated from blood in all four affected individuals in this family showed that only the nucleotide mismatch of CKMT1A was present (fig 3A); in controls, both mismatches of CKMT1A and CKMT1B were expressed. Based on these data, affected individuals in family D_SM are homozygous for a approximately 100-kb deletion with the proximal breakpoint at the 5′ flank of KIAA0377 (SNM8a-SNM7a) and the distal breakpoint at the 5′ flank of ΨKIAA0377 (SNM8b-SNM7b). Included in this deleted region are CATSPER2, STRC and CKMT1B (fig 3A). In family L705, we narrowed the breakpoints to the interval flanked by the two mismatches, SNM6 and SNM4 (fig 3A, table 1), which is telomeric to the breakpoint interval in family D_SM. All non-affected individuals in family L705 show mismatches at SNM6 and SNM4 by sequencing long-PCR amplicons across this region, while the three affected persons are missing the mismatch of SNM6b and the mismatch of SNM4a (fig 3B). The deletion size in this family is also approximately 100 kb, and extends proximally from CKMT1B (5′ – intron 7; SNM6a-SNM4a) to CKMT1A distally (5′ – intron 7; SNM6b-SNM4b). Functional copies of STRC and CATSPER2 are deleted (fig 3A). In family L1014, we observed the first loss of a single nucleotide mismatch at SNM5a and the last loss at SNM8b in all three patients but not in unaffected individuals (fig 3B) consistent with a approximately 90-kB deletion starting in the mid portion of CKMT1B and ending 5′ of ΨKIAA0377. The deletion includes CATSPER2 and STRC (fig 3A).

Figure 3. The genomic organisation of 15q15.3 includes a duplication with several single nucleotide mismatches, which were used to define the deletions in each family (A).

Electropherograms show representative non-deleted and deleted alleles (B).

Table 1. Single nucleotide mismatches position in non-pseudo and pseudo region.

SNM	Location on 15q	Nucleotide	Region
SNM4a	41676103	C	Centromeric
SNM4b	41775935	T	Telomeric
SNM5a	41672406	G	Centromeric
SNM5b	41772158	C	Telomeric
SNM6a	41670757	T	Centromeric
SNM6b	41770509	A	Telomeric
SNM7a	41665337	T	Centromeric
SNM7b	41765172	C	Telomeric
SNM8a	41662746	C	Centromeric
SNM8b	41762581	T	Telomeric

DISCUSSION

Rather than being random events, many rearrangements characterised by non-allelic homologous recombination reflect genome architecture.⁴ Abundant repeat elements, for example, lead to large deletions of MECP2 in approximately 25% of cases of classic Rett syndrome. In the region we describe on chromosome 15q15.3, another architectural feature, a large tandem repeat, is present that is prone to rearrangement. A duplicated four-gene array, which includes KIAA0377 (recently named by HUGO as HISPPD2A, histidine acid phosphatase domain containing 2A), CKMT1B, STRC and CATSPER2 and spans 83 kb, is separated by a 10 kb of intermediate unique sequence which contains a 6-kb LINE sequence.⁵ A comparison of the two duplicated portions shows a 30-kb region of high sequence homology (99.97% identical) from KIAA0377 to CKMT1B (30 kb centromeric) and from pseudo-KIAA0377 to CKMT1A (30 kb telomeric). Using site-specific nucleotide dosage mapping, we identified a 100-kb deletion in family D_SM that extended 5′ of centromeric KIAA0377 to 5′ of telomeric pseudo KIAA0377, deleting all of CKMT1B, STRC and CATSPER2 (fig 3A). Within the breakpoints lies a pair of imperfect inverted repeats (fig 4A) which are known to promote secondary structure formation. This finding suggests that this region is prone to deletions triggered concordantly by inverted repeat pairing and misalignment due to replication slippage between tandem sequences.

Figure 4. A schematic representation of the breakpoint regions in family D_SM (A) showing the predicted alignments of the two inverted repeats, IR1 and IR2, with 76% base pair matching as predicted by EMBOSS (B), and the resultant secondary DNA structure (C).

The disease phenotype associated with these deletions is characterised by deafness and infertility. The hearing loss phenotype is similar by audioprofiling to DFNB16-related hearing loss, suggesting that deletion of STRC is causally related to the deafness in DIS, while deletion of CATSPER2 appears to be responsible for the male infertility. The two other genes in the deletion interval, CKMT1 (creatine mitochondrial kinase 1) and KIAA0377, are both ubiquitously expressed. CKMT1 is present as two transcribed copies, CKMT1A and CKMT1B, which encode the isoenzyme of mitochondrial creatine kinase that is responsible for the transfer of high energy phosphate from mitochondria to creatine in tissues with large fluctuating energy demands. Another isoenzyme in this family is encoded by CKMT2, the expression of which is limited to sarcomeric tissues of heart and skeletal muscle.⁶ Deleting only two of four copies of CKMT1 does not give an obvious phenotype, presumably due to functional redundancy. KIAA0377 contains a single histidine acid phosphatase domain with unknown function. The large deletions in families L705 and L1014 do not affect the expression of KIAA0377. However in family D_SM, the 5′ portion of KIAA0377 (5′UTR to exon 3) is replaced by its counterpart in ΨKIAA0377. Over the replacement interval, KIAA0377 and ΨKIAA0377 differ by only one nucleotide (intron 2, SMN8), and hence the expression of KIAA0377 would be predicted to be unaltered. We confirmed this prediction by demonstrating KIAA0377 mRNA transcripts in all affected individuals by RT-PCR amplification and direct sequencing (the primers target exon 3 and exon 5) (fig 3A).

In summary, we have identified three families segregating an autosomal recessive contiguous gene deletion syndrome characterised by deafness and sperm dysmotility. This new syndrome is caused by the deletion of contiguous genes at 15q15.3.

LEARNING POINTS

• Deafness-infertility syndrome (DIS) is a new syndromic form of deafness caused by large contiguous gene deletions at 15q15.3.

• Contiguous gene deletions are related to genomic architecture (e.g. repetitive sequences)

• Fertility problems in DIS can be treated with intra-cytoplasmic sperm injection.