The aim, design and setting of the study
Sensorineural hearing loss (SNHL) was detected in four patients with GSD type I. The proper audiological diagnosis was postponed for each subject due to the development of the serious metabolic complications. Delayed speech development was noticed in all probands. Although speech therapy was implemented (for each child), no satisfactory improvement was observed. Therefore, the hearing tests were performed. Pure tone audiometry (PTA) revealed the ski-slope audiometric curves in all individuals. Although the diagnosis of hearing impairment was established at an early school age, the results of the newborn hearing screening indicating needs of audiological management and the speech impairment strongly suggest a pre-lingual origin of the hearing loss in our probands. Considering a similar pattern of the hearing tests results, together with the common inborn error of metabolism, molecular investigations were performed. Simultaneously, we assessed hearing status in the whole group of patients with GSD type I
The group of patients with GSD type I
The study group included 40 Polish patients with GSD type I diagnosed and treated in the Children’s Memorial Health Institute in Warsaw, Poland. The group comprises 20 cases with subtype Ia and 20 subjects with subtype Ib. The median age of probands was 12 years, range: 5-40 years. They all underwent audiological testing.
The characteristics of four patients with GDSI and hearing impairment:
Patient 1 (MB)
The first patient is a 14-year-old boy with GSDIa. His general health status was stable due to corn starch dietary supplementation. The main health problems were hypertension (treated with angiotensin-converting-enzyme inhibitor), hepatomegaly, and persistent alterations in the results of the laboratory tests: lactic acidosis, hyperuricamia, hypercholesterolemia. Delayed speech development was noticed. Appropriate intensive therapy was introduced at the age of 5 years due to speech illegibility. He has followed the mouth of the speaker, needed repetition and required loud TV sounds. At school he had many problems, especially with understanding teacher’s explanations and dictation.
A panel of hearing tests performed at the age of 8 displayed sensorineural hearing loss of the cochlear origin. Ski-slope shape of the audiogram meeting the criteria of partial deafness was revealed in PTA (Figure 1). The normal hearing threshold was observed only at low frequencies (125Hz-500Hz) in the better hearing ear, the hearing level at the mid-range and high frequencies (1kHz-8kHz) was below 90dBHL. Type A tympanograms were registered in both ears. Neither distortion products otoacoustic emissions (DPOAE) nor stapedial reflexes above 500Hz were registered. Brainstem evoked auditory potentials (BEAP) revealed profound hearing loss, which was consistent with the audiogram. Patient 1 was fitted with hearing aids, however, no satisfactory outcome was achieved, as suspected. A cochlear implant was recommended and remains under consideration due to the lack of the patient’s acceptance of the method.
Patient 2 (AS)
The second patient is a 12 years-old girl suffering from GSDIa. She was born at term in 38Hbd by CC (due to vaginal bleeding) after uneventful pregnancy. The birth weight was 3400g and she was assessed at 10 points according to the Apgar score.
At the 4. month of age an increasing lost of appetite accompanied by enlarged abdomen circumference and hepatomegaly was noticed. Laboratory tests revealed asymptomatic hypoglycaemia, hypertriglyceridemia, lactic acidosis and an increased level of liver function tests with normal bilirubin level and appropriate parameters of the clotting system. Liver core biopsy showed diffuse, non-zonal, predominantly macrovacuolar liver steatosis of moderate-severe degree, extending approximately 70-80% of the parenchyma. Mild degree of portal fibrosis without fibrous bridging were seen. There were no signs of inflammation, cholestasis, neither pathology of intrahepatic bile ducts. (Figure 2).
Following the diagnosis, an immediate dietary treatment for GSDIa was commenced when she was 7 months of age. Her development was satisfactory. The main health problems were hypertension treated with angiotensin-converting-enzyme inhibitor (Tritace) and hepatomegaly accompanied with abdominal obesity. Lactic acidosis was a single blood marker of GSDIa. She started speech therapy due to an inappropriate articulation when she was five. No improvements were observed. Subsequently, she encountered school problems with a hearing synthesis, learning to read, writing and understanding details. For those reasons she was referred to an audiology unit when she was 7 years old. A battery of hearing tests was performed. PTA revealed the ski-slope hearing curve. The audiogram displayed normal hearing at low and mid-range frequencies, with a sloping curve from 1kHz to high frequencies - up to the level of 90dB (Figure 1).
Type A tympanograms were registered in both ears, stapedial reflexes were registered in all examined frequencies but 4kHz. DPOAE were registered only below the 1500Hz. BEAP confirmed the audiometric hearing thresholds and the cochlear origin of the hearing impairment. Patient 2 was fitted with hearing aids and since then she enjoys auditory benefits.
Patient 3 (SK)
The third patient is a 9-year-old boy suffering from GSDIb. He was the first child of unrelated parents. He was born through natural delivery at 34Hbd, the birth weight was 1870g, he obtained 8 points according to the Apgar score. The pregnancy was complicated by maternal diabetes mellitus. Recurrent upper respiratory tract infections, accompanied by episodes of severe hypoglycaemia, leukocytosis with neutropenia were noticed from the first months of age. The diagnosis of GSDIb was established at the 9. month of life and subsequent dietary therapy was introduced. Patient 3 has got many health problems including hypertension, hepatomegaly and hypertrophic cardiomyopathy. Right kidney surgery was preformed when he was seven. A tumoral mass clinically suspected for abscess was detected. Microscopically renal parenchyma showed complete effacement of architecture by non-specific, polymorphic, partly purulent inflammatory infiltrate with interstitial fibrosis. Only a few glomeruli and tubules were preserved and discernible in fibro-inflammatory mass (Figure 3). Alterations detected in the results of blood tests of Patient 3 included hypertransaminasemia, lactic acidosis, hyperuricemia, hypercholesterolemia, and hypertriglyceridemia. He was persistently treated with granulocyte colony-stimulating factor (G-CSF) – Neupogen due to leukopenia. Low white blood cells count was the key factor of recurrent infections, especially the recurrent otitis media. He was under ENT care from infancy. Hearing tests have been performed since he was 7 months. Both tympanometry and DPOAE tests indicated otitis media and BEAP confirmed hearing loss. Hearing aids were contradicted due to subsequent effusions from ears. A delayed speech development was observed. Adenoidectomy with insertion of the ventilation tubes into the tympanic membranes, equalizing pressure in the middle ear, was done when he was four years old. Audiological tests (BEAP, DPOAE, and tonal play audiometry) performed after surgery, displayed severe hearing impairment, while type B tympanograms confirmed the correct function of the tubes. Mastoidectomy of the left ear followed subsequent ear infections. PTA was done at the age of 5 years, the audiogram had a ski-slope shape, which was slightly improved in comparison to the previous ones (Figure 1). The normal hearing remained at low and the midrange frequencies 125Hz-1500Hz in the better hearing ear (the right ear). Mild sensorineural hearing impairment was found at mid-frequencies, and severe hearing loss was detected below 4kHz (at high frequencies). He was finally successfully fitted with hearing aids.
Patient 4 (MR)
The fourth patient with GSDIb associated with SNHL is already a 23-year-old man. His main health problems are short stature, hepatomegaly, glaucoma of the left eye, total retinal detachment of the left eye, and exotropia of the left eye. He underwent vitro-retinal surgery of the left eye.
He was born at term through spontaneous vaginal delivery, after uneventful pregnancy, with 3200g and 10 points of the Apgar score. Neonatal adaptation problems with hyperbilirubinemia and hypoglycaemia were thought to be related to his intrauterine infection. Hypoglycaemia, hypertriglyceridemia, hypercholesterolemia, hypertransaminasemia, hyperuricemia, lactic acidosis and leukopenia have been reported since he was 3 months old. The diagnosis and dietary treatment of GSD1b was established at the age of 9 months. At the age of 6.5 years sensorineural hearing loss was detected based on PTA. The audiogram presented the ski-slope audiometric curve. Normal hearing was observed only up to 250 Hz, severe impairment was detected already at mid-range frequencies and deafness above the 4kHz (Figure 1). He was fitted with hearing aids, immediately after the diagnosis, but no satisfactory speech improvement was observed. He was referred to a cochlear implant surgery.
Routine videootoscopy and following audiological tests were performed in all examined patients. Hearing threshold level was assessed with PTA (Interacoustics, AC40). The clinical interpretation of the degree of hearing impairment was based on ANSI (American National Standards Institute) and ISO (International Standards Organization) standards. The inner ear functions were checked with clinical DPOAE (Interacoustics, Titan). Middle ear functions were assessed with tympanometry and stapedial muscle reflexes (Interacoustics, AT235H).
The auditory pathways in the brainstem were checked with BEAP (GSI, Audera) in the four probands with hearing impairment. Chirp stimulus was used for detection of high frequencies (2-4kHz) and tone-burst stimuli to examine tones of 500Hz and 1kHz, respectively. BEAP were used to confirm the diagnosis of hearing impairment.
Genetic analyses were performed after obtaining informed consent from the Patient 4 and the parents of Patients 1-3. Genomic DNA was extracted by automated method (MagnaPure, Roche) or manual (phenol-chloroform) method from peripheral blood samples of the probands and relatives. Next-generation sequencing (NGS) of Trusight One sequencing panel, created by Illumina for the simultaneous sequencing of 4,813 clinically relevant genes was used as a genetic target. NGS was performed on a HiSeq 1500 platform using an exome enrichment kit (Illumina) according to the manufacturer's protocol. Generated reads were first merged and low-quality reads removed. Then the reads were aligned to the hg19/hg38 (GRCh37/GRCh38) reference human genome. Potential PCR duplicates and reads mapping to multiple genomic locations were removed. In variant calling, any call with the ratio smaller than 0.2 was assumed to be homozygous, and the rest - heterozygous. Alignments were viewed with Integrative Genomics Viewer v. 2.3.82 (7). The detected variants were annotated using Annovar and converted to MS Access format for final manual analysis.
All the non-coding and common variants (minor allele frequency (MAF) above 0.01 in the general population) were discarded. The rare variants affecting the coding regions were filtered basing on the autosomal recessive mode of inheritance and predicted consequences at the transcript level. The variants were prioritized according to the population frequency and the predicted effect on the protein. The potential consequences were defined according to the conservation of the affected amino acids and in silico predictions by using different algorithms.
To identify the molecular basis of the glycogen storage disease, first we analyzed GSDI related genes. Additionally, in order to detect sensorineural hearing impairment potentially causative variants from TruSight One target, we applied a computational algorithm termed Phenotypic Interpretation of eXomes (PhenIX) . The software evaluates and ranks detected gene variants based on pathogenicity and semantic similarity of hearing loss described by Human Phenotype Ontology (HPO) terms to those of known Mendelian diseases.
Additionally, for Patients 2 and 3 as well as for their parents, the whole exome analysis of case-parents’ trios (trio-WES) using SureSelect Human All Exon Kit (Agilent, 60mb V6) were performed. The prepared libraries were subsequently sequenced as 100 bp paired - end reads on the Illumina Hiseq 4000 (trio-WES) to an average 141-fold coverage with more than 98% of target sequences being covered over 20-fold.
NGS data were analyzed using an in-house procedure, described in detail previously . Additionally, for WES data, the allele frequency was assessed in an in-house database (Munich-Exome-Server which include data from 6,000 exomes).
The interpretation of identified variants was based also on information reported in public databases: Human Gene Mutation Database (HGMD), Decipher, ClinVar, Online Mendelian Inheritance in Man (OMIM) and Varsome The Human Genomics Community.
The candidate pathogenic variants were verified in the probands and relatives, by Sanger sequencing using BigDye Chemistry (Applied Biosystems).