Unveiling Maternal Germline Mosaicism in X-Linked Alport Syndrome by Advanced Genetic Testing

Introduction

Alport syndrome is an inherited disease caused by mutations in the COL4A3/4/5 genes, resulting in structural and functional abnormalities in the α3/α4/α5 chains encoded by these genes. This leads to the loss of type IV collagen in the basement membrane, causing dysfunction in organs such as the glomerulus, retina, and cochlea [1]. Alport syndrome manifests a spectrum of renal abnormalities, characterized by hematuria, proteinuria, and elevated serum creatinine levels, even progressing to end-stage renal disease. Additional symptoms may include sensorineural hearing loss, lenticonus, and fleck retinopathy [2]. X-linked Alport syndrome (XLAS) due to COL4A5 disease-causing variants is the most common form and accounts for 80% of all Alport syndrome cases [3]. Germline mosaicism, which is the presence of 2 or more different genotypes in an individual, is suspected when 2 or more affected children are born to apparently unaffected parents. When both boys and girls are affected, germline mosaic is strongly suspected to exist in the mother’s ova [4]. In this report, we present an identified variant in the COL4A5 gene and maternal germline mosaicism in a family with XLAS.

Case Report

The proband (II-1) was a 16-year-old male who presented with foamy urine for the past 4 months before admission. Urine analysis revealed 3+ proteinuria, with a 24-hour urinary protein concentration of 2.08 g, and 3–5 red blood cells per high-power field on microscopic examination. His serum creatinine level was elevated at 130.60 μmol/L, and renal ultrasound did not reveal any abnormalities. Blood pressure monitoring indicated hypertension at 150/90 mmHg. Renal pathology revealed irregular thickness, curvature, splitting, and lamination of the glomerular basement membrane, along with thickening of Bowman’s capsule and pericapsular fibrosis, which suggested a suspected diagnosis of hereditary kidney disease. Other tests such as auditory impedance, pure tone audiometry, and eye examination revealed no abnormalities.

Through next-generation sequencing (NGS) targeting glomerular disease-associated genes, a hemizygous missense variant of the COL4A5 gene (NM_000495.5: exon 39: c.G3508A: p.G1170S) was detected (Figure 1A). This variant was classified as “pathogenic” based on the criteria defined by the American College of Medical Genetics and Genomics (ACMG) [5]. As a result, the patient was diagnosed with XLAS and we treated him with angiotensin-converting enzyme inhibitors.

The proband’s father (I-1) died in an accident a few years ago and had no history of kidney abnormalities such as proteinuria or hematuria. To further investigate the mode of inheritance in the proband, urine analysis and blood tests were conducted for the proband’s mother (I-2) and sister (II-2) after informed consent. Interestingly, the proband’s sister showed minimal microscopic hematuria, with normal serum creatinine levels. Peripheral blood NGS targeting renal disease-associated genes revealed the presence of the same genetic variant (NM_000495.5: exon 39: c. G3508A: p. G1170S) in the proband’s sister (Figure 1B). However, the proband’s mother had normal urine analysis and renal function, and no variant was detected at this specific location (Figure 1C).

Discussion

The COL4A5 gene is located at Xq22 and consists of 51 exons. It encodes the α5 chain of type IV collagen, which is made up of 1685 amino acid residues [6]. Mutations in the COL4A5 gene result in abnormal expression of type IV collagen α5. In Alport syndrome, the kidneys exhibit characteristic ultrastructural changes under an electron microscope, including an irregularly thickened glomerular basement membrane, splitting, lamination, and a basket-weave appearance. Immunohistochemical analysis indicates a complete absence of the α5 chain of type IV collagen in the glomerular basement membrane and Bowman’s capsule for male patients with XLAS, while showing a mosaic pattern with segmental loss for females with XLAS [7].

The clinical phenotype of XLAS is influenced by the mode of inheritance and the sex of the affected individuals. The COL4A5 gene is located at Xq22, so females in the family are heterozygous carriers, while males are hemizygous for the gene [8]. XLAS males typically experience more severe clinical manifestations and have a greater risk of developing renal failure. Almost all XLAS males present with hematuria [9], among whom approximately 90% develop renal failure before the age of 40. Additionally, hearing loss and ocular abnormalities are common in this population. In heterozygous XLAS females, 96% patients exhibit microscopic hematuria, 75% have proteinuria, and renal impairment is usually less severe [10]. The proband (II-1) in this family had a hemizygous variant and elevated serum creatinine levels, indicating a higher risk of progressing to end-stage renal disease. However, the proband’s sister (II-2) had a heterozygous variant and presented with only isolated microscopic hematuria.

The clinical presentation of XLAS is also influenced by the specific location and type of genetic variants [11]. COL4A5 deletions, insertions, and nonsense variants are associated with early-onset renal failure. This genotype–phenotype correlation is particularly notable in XLAS males. XLAS females, even with severe mutations, can remain completely asymptomatic or only show persistent microscopic hematuria throughout their lives. In female patients with XLAS, there is a limited correlation between the characteristics of the mutation and the risk of renal failure [12].

Interestingly, as shown in Figure 2, a variant in the COL4A5 gene was not detected in the proband’s mother (I-2). However, both the proband (II-1) and his sister (II-2) had a genetic variant in COL4A5. Germline mosaicism is often suspected when more than 1 affected child is born to parents who do not show any signs of the disorder. It is particularly strong when both boys and girls are affected. To date, numerous cases of germline mosaicism have been reported in patients with XLAS.

Mosaicism refers to the presence of 2 or more different genotypes in an individual. Mosaicism arises when a mutation emerges in the early stages of embryonic development, resulting in a subset of cells possessing a distinct genetic makeup from the rest of the organism’s cells. Mosaicism can be categorized into somatic mosaicism, germline mosaicism, and mixed mosaicism, depending on the timing and distribution of genomic changes. Various mechanisms, such as chromosome nondisjunction, postzygotic lagging, and endoreduplication, can give rise to mosaicism. If the mutation occurs before the differentiation of primordial germ cells, it can be present in both germ cells and somatic cells. Due to different tissue involvement and varying degrees of mosaicism in each tissue, XLAS often exhibits different expression rates and incomplete penetrance [13]. Mosaicism is considered to be an important factor contributing to the milder phenotype observed in XLAS [14]. In the case mentioned, the proband’s mother did not display any signs of renal impairment, such as hematuria, proteinuria, or abnormal blood creatinine levels.

With advancements in genetic testing technologies, mosaic phenomena have been observed in various genetic diseases. Commonly used genetic testing methods include Sanger sequencing, targeted next-generation sequencing, and droplet digital PCR. These testing techniques analyze the proportion of mosaicism in somatic cells and can be performed using genomic DNA extracted from various sources, such as peripheral blood leukocytes, urinary sediments, hair roots, and skin. The presence of mosaic characteristics in the skin or kidneys of XLAS males may suggest the existence of mosaic phenomena. Identifying asymptomatic or mildly symptomatic females as carriers of germline mosaicism in XLAS patients is crucial for genetic screening in subsequent generations and assessing the risk of transmitting the disease during renal transplantation between parents [15]. However, detecting germline mosaicism is challenging because it requires testing germ cells. Current research is limited to studying germline mosaicism in males. Sampling for female germline mosaicism detection is invasive and ethically complex, making it difficult to determine the actual rate of germline mosaicism and the true recurrence risk.

Some scholars have attempted to infer the presence of germline mosaicism by screening for somatic mosaicism when suspecting its existence [16,17]. To date, only a few reports exist on sperm germline mosaicism in different genetic diseases. Using droplet digital PCR detection technology, Frisk et al discovered germline mosaicism in semen samples from patients without previously detected mosaicism in blood and observed that the level of mosaicism in sperm was always greater than that in blood [18]. However, there is no direct correlation between germline mosaicism and somatic mosaicism, and the detection of somatic mosaicism cannot directly predict the presence of germline mosaicism, nor can the abundance of somatic mosaicism be directly used to assess the abundance of germline mosaicism. In addition, researchers are working on the relationship between mosaicism modes and clinical phenotypes [19]. In XLAS patients, a correlation between the percentage of gene mosaicism and renal symptoms revealed a trend associating lower frequency of variant alleles with a lighter phenotype. In XLAS male patients with mosaicism, the renal immunohistochemistry showed a mosaic pattern, and the phenotype of renal symptoms was mild. XLAS women with germline mosaicism may not even have any associated clinical symptoms. Although their symptoms are mild, identification of gene mosaicism is crucial for family planning, as the risk for their offspring depends on the proportion of chimeras in the germline precursor cells. The susceptibility to mutations during germ cell development and the timing of mutations during parental embryogenesis may affect the abundance of mutations in both germline and somatic tissues, as well as the overall recurrence risk [20]. Analysis of tissues representing all 3 germ layers during embryonic development will aid in understanding when mutations occur and further deciphering the mechanism of mosaicism. Larger-scale studies of germline mosaicism in XLAS and other genetic diseases are needed to determine the exact recurrence risk.

Conclusions

We reported an identified variant (NM_000495.5: exon 39: c.G3508A: p.G1170S) in the COL4A5 gene from maternal germline mosaicism in a family of X-linked Alport syndrome patients. Even if female carriers are asymptomatic, timely identification of germline mosaicism is essential to avoid the transmission of the same variant to other offspring, and novel laboratory tools with high specificity, sensitivity, and minimal invasiveness for detecting mosaic phenomena are needed.