Diagnostics of CFTR-negative patients with congenital bilateral absence of vas deferens: which mutations are of most interest?

KEYWORDS:

• Azoospermia
• congenital absence of vas deferens
• CBAVD
• CFTR
• cystic fibrosis
• male infertility

Male infertility is one of the best examples of multifactorial diseases in which the genetic contribution is substantial [1]. Male reproductive function, including spermatogenesis, testicular development, endocrine regulation of testicular function, and sperm fertilizing ability are under the control of thousands of genes [2–7]. Basic and clinical research sustained by new technologies for genetic analysis (whole-genome association studies, arrays, next-generation sequencing, and exome sequencing) suggested that a very high number of genetic tests could potentially be introduced in the clinical practice to correctly identify male infertility of genetic origin [1,5,7]. However, only a few genetic analyses are currently recommended in standard clinical practice so far [1].

In this context, it is of primary importance to correctly assess infertile males with an adequate clinical workup, in order to clearly address the appropriate genetic tests in a personalized way [1]. Therefore, experts in reproductive medicine and andrology should strictly cooperate with clinical geneticists to perform the correct tests in the correct patient. Only the correct identification of subjects to be tested and the right application of genetic tests based on clear clinical data could be useful in the management of the infertile couple and genetic counseling, especially when assisted reproduction techniques should be used to overcome infertility.

Basically, genetic tests recommended in clinical practice for male infertility [1] could be grouped on the basis of the clinical diagnosis in those recommended for 1) primary testicular failure (karyotype, analysis of Y chromosome long arm microdeletions, and eventually analysis of TEX11 and NR5A1 gene mutations); 2) endocrine dysfunction (panel of genes for hypogonadotropic hypogonadism, androgen receptor gene mutations); 3) obstructive forms (CFTR gene mutations for congenital absence of vas deferens); 4) sperm morphological and functional defects (dynein genes DNAI1, DNAH5, DNAH11 for asthenozoospermia, DPY19L2 gene for globozoospermia, AURKC gene for macrocephaly).

Azoospermia with low seminal volume and acid pH, normal testicular volumes, and normal plasma levels of FSH, LH, and testosterone clearly suggest an obstructive form [1], which can be due to congenital bilateral absence of vas deferens (CBAVD) in up to 25% of cases [8,9]. The diagnosis of non-palpable vas deferens should be confirmed by scrotal ultrasound to confirm the absence of vas deferens and transrectal ultrasound to detect agenesis of the seminal vesicles, which is associated with CBAVD in about half of the cases. In other cases, hypoplasia of seminal vesicles could be found, and the degree of hypoplasia probably reflects the severity of the genetic abnormalities [10]. CBAVD could occur as an isolated symptom or as an atypical symptom of cystic fibrosis, one of the most frequent autosomal recessive conditions. More than 2000 mutations have been identified in CFTR gene (Cystic Fibrosis Mutation Database, CFMD, 2017) and classified in severe and mild based on their functional and phenotypic effect. Two severe mutations lead to cystic fibrosis, whereas two mild mutations or one severe plus one mild mutation lead to CBAVD. Nearly all patients with cystic fibrosis have CBAVD, and nearly all patients with isolated CBAVD have a mutation in the CFTR gene [9,11,12].

Genetic analysis of CFTR gene is therefore recommended in CBAVD patients. However, standard protocols of analysis allow detection of at least one CFTR mutation in ‘only’ about 80% of cases, with less than 50% of patients found to carry mutations in both alleles (compound heterozygotes) [1,9]. Therefore, a not negligible percentage of CBAVD patients are classified as CFTR negative. When two mutations are found, the diagnosis is made, but when just one mutation is found we cannot distinguish between the carrier status (1/30 subjects in western countries) from a condition actually related to CFTR but in which the second mutation is not found. This latter condition could be due to several factors, such as inadequate selection of mutations to be tested in the panel, incomplete gene coverage, or mutations in genes other than CFTR. Furthermore, in CBAVD patients’ large rearrangements and deletions in the CFTR gene have been described, which could be detected only by novel molecular techniques of analysis [13–15]. For example, by applying different techniques (reverse dot-blot analysis, multiple ligation-dependent probe amplification assays, denaturing high-performance liquid chromatography) in 15 CBAVD patients in whom a single CFTR mutation was found after screening for 36 mutations and the 5T allele (from a cohort of 23 patients), the second CFTR mutation was detected in six patients, which increased the final detection rate to 60.8% [16].

Generally, first-level CFTR gene mutation screening in patients with CBAVD should be performed as the targeted variant panel that includes causing variants based on geographic and ethnic origin of the patient. The panel usually includes 30–50 mutations able to detect >80% of carriers of classic cystic fibrosis mutations and should also include the 5T allele, a variant typically associated with CBAVD, and TG(n) repeat polymorphism, which influences its penetrance and clinical effect. When no mutation or one mutation is found, second step analysis should be performed by Sanger sequencing of exons, 5ʹ flanking regions and intron-exon boundaries or, preferably, by next-generation sequencing of the entire gene. Also, after this in-depth analysis, about 20% of CBAVD patients remain undiagnosed (no mutations or one mutation detected). However, the application of Next Generation Sequencing analysis in the diagnosis of CFTR mutations in CBAVD patients will likely improve our ability to identify both CFTR mutations, and as a consequence, the number of CFTR real negative CBAVD will decrease.

To this regard, it has to be noted that the main difference between typical cystic fibrosis and CBAVD is the identification of different and rare CFTR mutations and variants in high frequency in individuals with CBAVD as compared to the typical cystic fibrosis forms, and only a few typical cystic fibrosis mutations, such as the p.F508del, are found in individuals with CBAVD. The panels used for CFTR gene screening have been developed for the identification of the most frequent mutations associated with cystic fibrosis, whereas there is no consensus as to whether a CBAVD-specific panel could have better diagnostic potential.

Finally, it has been suggested that CFTR-negative CBAVD patients could harbor mutations in ADGRG2, an X-linked gene, for which therefore the identification of only one causative mutation is sufficient for genetic diagnosis. Four studies reported screening for ADGRG2 gene mutations in CFTR-negative CBAVD patients: the original study [17] was performed on 26 patients and replication studies have been performed only in 18 Chinese men [18], 17 Chinese men [19] and in a Pakistani family [20]. Altogether, these studies suggested that ADGRG2 gene mutations could account for about 10–15% of the CBAVD patients who are CFTR negative. Although these results are very promising, unless further studies will be performed in a higher number of patients of different ethnic origin, we suggest that screening for ADGRG2 gene mutation should be limited to research purpose and cannot be suggested yet in the routine clinical management of men with CBAVD.

Congenital absence of vas deferens could also be unilateral (CUAVD) as a consequence of mild CFTR mutation. This condition is rare, with a 1:100 proportion with CBAVD. In CUAVD cases the clinical suspicion could be extremely difficult. In fact, these cases might present with mild oligozoospermia and even normozoospermia (if the contralateral testis is normally functioning), and seminal volume and pH can be unaffected [1]. The only way to have a correct diagnosis is performing scrotal ultrasound, but it is evident that a large number of patients remain undiagnosed. A proportion of CUAVD patients has an ipsilateral absence of the kidney, but this condition is not related to CFTR.

Expert opinion

In conclusion, the detection rate of CFTR gene mutations in patients with CBAVD (and CUAVD) could be ameliorated if the test is performed in the correct patient after a complete diagnostic workup, including, for example, also abdominal ultrasound to exclude patients with renal agenesis, a condition not related to CFTR. We recently showed that CFTR gene mutation screening in almost 500 azoospermic men identifies 3.1% of patients with two mutations, further highlighting the importance of correct and comprehensive clinical evaluation of infertile males in selecting patients who really need genetic analysis [21]. The detection rate could also be higher if next-generation sequencing of the entire gene (including analysis of Copy Number Variants) is used at first step analysis, allowing the detection of both mutations. However, genetic counseling for the couple and calculation of the risk of transmission are not affected and we should consider the cost of such analysis. ADGRG2 gene screening could be proposed to patients who are CFTR negative after in-depth analysis and without renal anomalies, however not yet as a routine procedure, and it has to be determined whether this test could also be useful in CBAVD patients with only one CFTR mutation detected and in CUAVD patients.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewers Disclosure

Peer reviewers on this manuscript have no relevant financial relationships or otherwise to disclose.

Additional information

Funding

This paper was not funded.