Nastaran Foyouzi, MD, Wayne W. Grody, M.D., Ph.D.
Department of Human Genetics, UCLA, Los Angeles, California 90095
Departments of Pathology and Laboratory Medicine, Pediatrics, and Human Genetics, UCLA, Los Angeles, California 90095
Congenital bilateral absence of the vas deferens (CBAVD) is considered a genital form of cystic fibrosis (CF). It is generally identified during the evaluation of male infertility and is associated with a high incidence of mutated cystic fibrosis transmembrane conductance regulator (CFTR) alleles. Diagnosis of CBAVD will be determined in males with azoospermia and absence of vas deferens or the presence of CBAVD-causing CFTR pathogenic variants. Currently genetic diagnosis or risk prediction of CBAVD is established by either expanded CFTR mutation testing or by carrier screening of the 23 most prevalent CFTR mutations comprising the minimal population screening panel recommended by ASRM, ACOG and ACMG. However, this approach raises some serious concerns including low and limited detection rates depending on the patient’s ethnicity, and inability to detect rare or novel CFTR mutations associated with CBAVD.
Widespread CF carrier screening involves only targeted CFTR mutation analysis, which is typically accomplished by allele-specific DNA probe hybridization or by scanning selected exons of the CFTR gene containing the pathogenic variants of interest by either Sanger sequencing or Next-Generation Sequencing (NGS). These approaches only detect 25% of the mutations in CBAVD males, because the existing CF screening panels were originally designed to detect the most common mutations associated with CF, and were largely derived from study of affected patients of Northern European and Caucasian decent. Additionally, current screening approaches do not interrogate tandem nucleotide repeats located in intron 8 of the CFTR gene unless the R117H mutation is detected. This underscores the need for a more optimal approach to CFTR mutation detection in men with obstructive azoospermia and their reproductive partners. To that end, we sought to address the value of whole CFTR gene sequencing in these individuals and discuss the possible etiologies for negative common CF mutation screening in these patients.
CBAVD usually results from compound heterozygous mutations in the CFTR gene, which usually includes a classic CF pathogenic variant and a mild pathogenic variant (e.g., R117H) (2). However, if a defective CFTR protein is responsible for CBAVD, why do not all of these patients have two detected mutations in the CFTR gene? Multiple factors have been shown to affect the genotype-phenotype correlation in CF and CBAVD:
- Presence of rare or unknown CF mutations in CBAVD: More than 1,800 mutations in the CFTR gene have been identified in individuals with cystic fibrosis (CF Genetic Analysis Consortium http://www.genet.sickkids.on.ca/cftr/). The most common mutation is a 3-bp deletion (CTT) in exon 10 of the gene, leading to the deletion of a phenylalanine at amino acid position 508 (ΔF508) (3). As a result, the protein does not undergo the normal posttranslational modification needed for delivery to the cell membrane and is degraded in the Golgi apparatus (4). The majority of the other CFTR mutations are very rare with only four other mutations (G542X, N1303K, G551D and W1282X) having overall frequencies above 1% (5). It is important to note that the frequency of known CFTR mutations is different in affected CF patients compared to those with CBAVD; For example, the frequency of ΔF508 is 21% in CBAVD versus 76% in classic CF patients (6). These differences in frequency of the regularly tested mutations in CBAVD compared to classic CF render most CF screening panels less useful for infertility investigations.
- Presence of intronic repeats: The heterogeneous phenotype of CBAVD and inability to identify the 2 mutations in >80 % of individuals with CBAVD has been attributed to variability in the number of thymidine nucleotide repeats (polythymidine polymorphism annotated as 5T, 7T, 9T) and the presence of TG repeats in intron 8 of CFTR (1, 7).
- For example, when TG repeat number is determined, the sensitivity for identifying affected individuals is increased to 91%, while the specificity for excluding those who are unaffected is 78% (8). Currently, none of the CF carrier screening panels will routinely test for these microsatellite repeats unless the R117H mutation is identified, in which case it is performed as a reflex test. Given the possibility of less common or novel CFTR pathogenic variants other than R117H in CBAVD patients, omission of these repeat polymorphisms from the carrier screening report will affect the interpretation and underestimate the risk to offspring. It must be acknowledged that the testing guidelines for CF carrier screening were designed to inform couples of the risk of classical CF in their offspring, not CBAVD. However, diagnostic work-up of infertile males with CBAVD usually will include analysis of the polyT and polyTG repeats in intron 8, regardless of whether the R117H mutation is detected.
- Ethnic differences: The frequency of CFTR mutations is not equally distributed across populations with CBADV. For instance, the ΔF508 mutation is more common in Caucasian (22%) rather than non-Caucasian (8%) populations, and the 5T allele is more common in non-Caucasian (31%) versus Caucasian (20%) populations (9) (Table-1). Additionally, the clinical sensitivity of current 23-mutation panel screening tests are not absolute for all ethnic groups and some mutations will not be detected: for example, CF carrier detection rates are estimated to be 97% for Ashkenazi Jewish, 88% for non-Hispanic Caucasian, 69% for African American, and 57% for Hispanic populations; the detection rate in Asian Americans is not precisely known but is undoubtedly much lower than in the other groups (10, 11) . Even a panel that includes more than 129 pathogenic variants would have a high yield 96% carrier detection rate only in individuals with Northern European ancestry (12). This is largely due to the dramatically lower proportion of carriers of ΔF508 in the other groups. Therefore, standard CFTR screening will tend to underscore CFTR mutation detection in men with obstructive azoospermia, especially among non-Caucasians.
- Presence of CF gene deletion: In 2% of patients with CBAVD, the mutation is a large deletion involving one or more CFTR exons (13), which will usually be invisible to standard sequence analysis, as the technique is inefficient at detecting large deletions and duplications. Hence, in CBAVD patients it is important to consider deletion/duplication analysis, either concurrent with CFTR gene sequencing or when sequencing is negative.
- Presence of modifier genes: Sometimes a mutation is not present in the CFTR gene but rather in other genes that are capable of modifying the phenotypic effect of CFTR mutations. For example, it is well established that transforming growth factor β (TGFβ-1) and endothelin receptor type A (ENDRA) are important modifiers of the CF pulmonary phenotype (14). Recently it has been shown that TGFβ-1 and EDNRA play a significant role in development of the vas deferens, and indicate that the defective gene products may contribute to development of CBAVD in the setting of CFTR insufficiency (15, 16). There is some evidence that other genes may cause CF-like disease in a small fraction of patients. These findings are too preliminary to be used routinely in a clinical context.
- Insufficient knowledge about CBAVD: The most studied genotype-phenotype correlation of CF is the classic form of the disease. It has been shown that the only symptom that correlates well with the mutational genotype in CFTR is pancreatic function. In CF patients from the same family, pancreatic function tends to “breed true”, indicating that exocrine pancreatic status is determined primarily by the genotype at the CFTR locus. However, pulmonary disease, liver disease and CBAVD do not show much correlation with the type of mutation and vary considerably within families, which represents the clinical heterogeneity of the disease.
In summary, the distinction between CF and CBAVD requires a proper clinical examination of the patient, personal and family history, and a different approach to molecular testing. The most thorough approach is complete CFTR mutation analysis and genetic counseling of CBAVD males who are planning to undergo assisted reproductive technologies (ART). Keep in mind that detailed genetic analysis (such as sequencing) of the CFTR gene is considered a diagnostic test rather than a screening procedure, and the panels used in routine carrier screening cover only the most common classic CF pathogenic variants, leaving less frequent mutations missed. For this reason, it is important to offer whole CFTR gene analysis by either Sanger or NGS and including intronic poly T and poly TG tract analysis, along with consideration of geographical and ethnic differences, to all CBAVD males who are planning to undergo fertility treatment. Sequencing and deletion analysis of all exons, intron/exon borders, promoter regions and specific intronic regions of the CFTR gene detects more than 98% of CFTR mutations (17). The cost comparison and insurance coverage between CF carrier screening and full CFTR gene sequencing is another challenge for patients and clinicians. Current CF carrier testing costs about $100–$300 per person compared to $500-$1500 for full gene sequencing. Insurance coverage differs according to policy, and it will be helpful to inform the insurance companies about the role of full CFTR gene sequencing in CBAVD as a diagnostic rather than screening strategy, as we have described here. Only in this way will the molecular dissection of male infertility due to CBAVD be optimized and accurate risk assessment to offspring’s could be determined.
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