1. NGS panel - Genetic testing for Alport syndrome

Alport syndrome

May 30, 2017

Clinical features

Alport syndrome (ATS) is a rare disease caused by genetic mutations in one of the type IV collagen genes that encode connective tissue proteins localized within basement membranes of the kidneys, ears, and eyes. Thus, the disease is characterized by renal, cochlear, and ocular involvement. Pathogenic variants in COL4A3, COL4A4, or COL4A5 result in a spectrum of phenotypes, from Alport syndrome to thin basement membrane nephropathy (TBMN), including X-linked Alport syndrome (XLAS), autosomal recessive Alport syndrome (ARAS), or autosomal dominant collagen IV disorders (ADAS and TBMN).

The most characteristic clinical features of Alport syndrome include the following:

Renal findings:

  • Hematuria (blood in urine) is present in almost 100% of all X-linked Alport syndrome male patients and more than 90% of affected females have microhematuria 1. Hematuria is also present in almost all autosomal recessive cases of Alport syndrome.
  • Proteinuria usually progresses with age and can occur in 30% of patients, mostly males 1, 2
  • Hypertension is detectable in the second decade of life, followed by edema and nephrotic syndrome
  • Renal insufficiency progress with the age of patient (all males with X-linked and all patients with autosomal recessive Alport syndrome).

Hearing impairment and cochlear findings:

  • Bilateral high-frequency sensorineural hearing loss presents in late childhood in 80-90% of males with X-linked ATS and all patients with autosomal recessive disease 1
  • In some families with X-linked ATS hearing loss is detectable at adult ages and it can progress to complete deafness
  • About 50% of males affected with X-linked Alport syndrome develop sensorineural deafness by the age of 25 years, and about 90% are deaf by age 401

Ocular findings:

  • Anterior lenticonus is present in 15-20% of X-linked ATS or autosomal recessive ATS cases 1
  • Perimacular flecks occur in approximately 30% of individuals with Alport syndrome 1, 2.

Family history

  • Patients affected with ATS usually have a positive family history for hematuria, deafness, and/or end stage renal
  • Family history of hematuria or renal failure may also be negative because 10-15% of males with X-linked ATS have the disorder as the result of a de novo pathogenic variant.

Alport syndrome includes a specific condition known as Thin basement membrane nephropathy (TBMN). TBMN is characterized by persistent microscopic hematuria that presents in childhood. Progressive renal disease is usually absent. TBMN should be suspected in patients with the following clinical signs:

  • Persistent microhematuria without proteinuria
  • Extrarenal abnormalities (very rare)
  • Family history negative for renal failure

The prevalence of Alport syndrome has been estimated at 1:50,000 live births1, 2, however, the incidence of Alport syndrome was found to be 1;53,000 in Finland, and 1:17,000 in Sweden 3,4 while TBMN is estimated to affect 1% of the population 1, 2.

The pathology and clinical features of Alport syndrome result from abnormalities of collagen proteins expressed in the basement membrane of kidneys, eyes, and ears. Most commonly, collagen α3, α4, and α5 chains are absent or less expressed in the basement membranes of individuals with Alport syndrome, resulting in the abnormal structure and function of the particular tissue.

The COL4A3 gene encodes alpha-3 collagen 4 protein. Mutations in the COL4A3 and COL4A4 genes are associated with 15% of all autosomal recessive forms of Alport syndrome and 20% of autosomal dominant Alport syndrome cases 3, 5. Several benign and more than 220 pathological variants have been reported in this gene to date (HGMD® Professional 2016.4), including the following:

  • Missense variants (~45%). The majority of missense variants in COL4A3 (~85%) are glycine substitutions in the conserved Gly-Xaa-Yaa repeat sequence
  • Frame shifts (small deletions, small insertions/duplications) (~20%)
  • Splice site variants (~15%)
  • Large deletions (5%).

COL4A4 encodes collagen alpha-4 chain of collagen protein complex. More than 170 mutations have been reported in this gene to date (HGMD® Professional 2016.4), including the following:

  • Missense variants (99/170), about 75% of which are glycine substitutions in the conserved Gly-Xaa-Yaa repeat sequence in the collagenous domain of the alpha4(IV)-chain)
  • Frame shifts (small deletions, small insertions/duplications) (~30%)
  • Splice site variants (~10%)
  • Nonsense variants (~9%) and
  • Large deletions (4%).

COL4A5 encodes alpha-5 chain of collagen protein complex and mutations in this gene are responsible for 65% of X-linked Alport syndrome cases 1, 3, 5: More than 900 mutations have been reported in this gene to date (HGMD® Professional 2016.4), including:

  • Missense variants (~45% of the variants, 85% of which are glycine substitutions in the conserved Gly-Xaa-Yaa repeat sequence in the collagenous domain of the alpha5(IV)-chain). The pathogenic variants c.4692G>A, c.4946T>G, and c.5030G>A are particularly common in the United States 1.
  • Frame shifts (small deletions, small insertions/duplications) (~20%)
  • Splice site variants (~20%)
  • Large deletions and insertions/duplications (~12%) and other larger structural rearrangements (inversions and translocations).

Mutations in these three collagen-associated genes could also contribute to the increased risk of different diseases that are included in Alport syndrome. Risk of renal disease is specially increased in the presence of large rearrangements and pathogenic nonsense and frameshift variants in COL4A3, COL4A4, and COL4A5, with half of all affected patients developing end-stage renal disease before age of 20 years 6.

Furthermore, in affected individuals with splice-site variants, the probability of end-stage renal disease (ESRD) before age 30 is 70%, with half of the patients developing ESRD before age of 25 years 6. Also, missense variants are associated with 50% probability of ESRD before age 306. Risk of deafness is 50% at age ten years in patients with large rearrangements of COL4A5 or nonsense, frameshift, or splice site variants in the same gene 1. Risk of anterior lenticonus is ~15% in male patients with X-linked Alport syndrome, and lenticonus is significantly more common in individuals with a COL4A5 deletion or a small pathogenic variant resulting in a premature stop codon than in those with pathogenic missense or splice-site variants 6.

Alport syndrome is treated with angiotensin-converting enzyme inhibitor (ACE) and/or angiotensin receptor blocker, routine treatment of hypertension, and possible renal transplantation for ESRD. Routine treatment of hearing loss and ophtalmological problems is also an important part of the therapy.

CENTOGENE offers the Alport syndrome panel, including full gene sequencing and deletion/duplication analysis of selected genes (COL4A3, COL4A4, and COL4A5). In addition, any of the genes in the Alport syndrome panel can also be ordered individually, for full gene sequencing and deletion/duplication analysis.

Differential diagnosis

The differential diagnosis of Alport syndrome-related disorders – depending on the major symptoms in the initial case – includes the following diseases7:

  • Acute poststreptococcal glomerulonephritis
  • Medullary cystic disease
  • Polycystic kidney disease
  • Multicystic renal dysplasia
  • Nail-Patella syndrome
  • Nephritis

Testing strategy

CENTOGENE offers advanced, fast and cost-effective strategy to test large NGS panels and diagnose complex phenotypes based on the PCR-free Whole Genome Sequencing and NGS technology. This approach offers an unparalleled advantage by reducing amplification/capture biases and provides sequencing of entire gene at a more uniform coverage.

To confirm/establish the diagnosis, CENTOGENE offers the following testing strategy for Alport syndrome using NGS Panel Genomic targeted towards this specific phenotype:

Step 1: Whole genome sequencing from a single filter card. The sequencing covers the entire genic region (coding region, exon/intron boundaries, intronic and promoter) for all the genes included in the Alport syndrome panel. Copy Number Variants analysis derived from NGS data is also included.

Step 2: If no mutation is identified after analysis of the Alport syndrome panel, based on the approval and consent, we further recommend to continue the bioinformatics analysis of the data obtained by whole genome sequencing to cover genes that are either implicated in an overlapping phenotype or could be involved in a similar pathway but not strongly clinically implicated based on the current information in literature.

Referral reasons

The following individuals are candidates for Alport syndrome gene testing:

  • Individuals with a family history of Alport syndrome and presentation of the most common symptoms
  • Individuals without a positive family history of Alport syndrome, but with symptoms resembling this disease
  • Individuals with a negative but suspected family history of Alport syndrome, in order to perform proper genetic counseling (prenatal analyses are recommended in families with affected individuals).

Test utility

Sequencing, deletion/duplication of Alport syndrome-related genes should be performed in all individuals suspected for this particular phenotype. In parallel, other genes reported to be related with Alport syndrome clinical phenotype should also be analyzed for the presence of mutations, due to the overlap in many clinical features caused by those particular genes.

Confirmation of a clinical diagnosis through genetic testing can allow for genetic counseling and may direct medical management. Genetic counseling can provide a patient and/or family with the natural history of the condition, identify at-risk family members, provide reproductive risks as well as preconception/prenatal options, and allow for appropriate referral for patient support and/or resources.

More information on CENTOGENE´s Alport syndrome panel can be found in our genetic test catalogue.