NEXT-GENERATION SEQUENCING INFERTILITY PANEL IN TURKEY: FIRST RESULTS
Ikbal Atli E1*, Yalcintepe S1, Atli E1, Demir S1, Gurkan H1
*Corresponding Author: Corresponding Author: Associate Prof. Emine Ikbal Atli, Trakya University, Faculty of Medicine,
Department of Medical Genetics, Edirne, Turkey Balkan Campus, Highway D100 ORCID ID: 0000-
0001-9003-1449; Postal code: 22030; Phone: 0(284) 235-76-41/2330;
Email: emine.ikbal@gmail.com / eikbalatli@trakya.edu.tr
page: 49
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DISCUSSION
Highly diverse phenotypic representation and a com-
plicated multifactorial etiology, including environmental
and genetic factors, characterize the condition of male
infertility. In most cases, it is challenging to identify a
genetic cause of infertility due to the large number of can-
didate genes (9, 10). In any case, a multi-disease gene panel
can help identify the cause of male infertility. In order
to categorize genetic variants, a multifactorial likelihood
model can be used to assess the likelihood that a variant
is pathogenic based on a previous likelihood of patho- genicity based on in silico research and the genetic and
epidemiological data that are currently available (11-13).
Genetic variants can be categorized into five categories
according to the American College of Medical Genetics
and Genomics’ references: pathogenic, likely pathogenic,
variant of unknown consequence, likely benign, or benign
(14). A genetic alteration known as a VUS has ambiguous
effects on gene function. The interpretation of VUS is a
challenging task for the clinical management of infertile
male patients and genetic counseling. Since VUS are not
clearly related with a phenotype currently, but could be
categorized as pathogenic in the future, it is crucial to
detect and assess them. An example of this situation is
the NM_000071.3(CBS):c.833T>C (p.Ile278Thr) variant
detected in our patient group. This variant, which was
evaluated as VUS in the databases at the beginning of our
study, was later classified as pathogenic.
Variants in PROS1 and CFTR were detected in our
patient number 2. PROS1 variants show autosomal domi-
nant inheritance. Even though the detected variant was
evaluated as VUS, the classification of this variant should
be followed in the future.
Similarly, in our patient number 5, 2 pathogenic vari-
ants belonging to the F11 gene and a variant evaluated as
VUS in CFTR were detected. F11 variants show autosomal
dominant inheritance. The detected variants were evalu-
ated as pathogenic. OMIM has been associated with Factor
XI deficiency.
In another patient, case number 7, variants were de-
tected in 2 separate genes. VUS evaluation was performed
for TEX15. TEX15 is associated with the Spermatogenic
failure 25 phenotype in the OMIM database, and exhibits
an autosomal recessive pattern of inheritance. The other
variant detected in the patient is a possible pathogenic
variant belonging to the GP1BA gene. This gene, which
is associated with different types of diseases in the OMIM
database, can show autosomal dominant and recessive
inheritance. Particularly notable among these diseases are
Bernard-Soulier syndrome, type A2 (dominant) and von
Willebrand disease, platelet-type (dominant).
PROC variant was detected in patient number 9 and
was reported as possibly pathogenic. PROC variants have
been associated with autosomal recessive and dominant
forms of Thrombophilia 3 due to protein C deficiency in
OMIM. Another variant in the patient is the pathogenic
variant in the ITGA2 gene. The inheritance pattern and phe-
notype of variant of this gene have not yet been elucidated.
We detected variants in SEPT 12 and CFTR genes
in patient number 15. SEPT 12 variants cause autosomal
dominant Spermatogenic failure 10.
In patient number 19, we detected compound hetero-
zygous variants of the CFTR gene and an additional variant
of the CLCA4 gene. While CFTR gene variants exhibit
autosomal recessive inheritance, there are no entries in
the databases yet for the CLCA4 gene.
We detected F11 and CFTR pathogenic variants in
patient number 21. F11 was found to be associated with
autosomal dominant and recessive forms of Factor XI
deficiency in the OMIM database.
We detected DPY19L2 and CFTR pathogenic variants
in patient number 22. DPY19L2 was found to be associated
with autosomal recessive forms of spermatogenic failure
9 in the OMIM database. Similarly, patient number 40 has
variants in these two genes that were evaluated as VUS.
F13B and CFTR variants were detected in another of
our patients, case number 32. F13B has been associated
with Factor XIIIB deficiency in OMIM. F13B variants
show autosomal recessive inheritance.
Interestingly, variants in 3 different genes were de-
tected in our last 2 patients. Variants considered to be
VUS were detected in the DNAH1, KLHL10 and SEPT12
genes in the first patient. KLHL10 and SEPT12 variants
have been associated with autosomal dominant spermato-
genic failure. DNAH1 has been found to be associated
with autosomal recessive Ciliary dyskinesia, primary and
Spermatogenic failure. These findings were associated
with the patient’s phenotype. In the second patient, vari-
ants considered to be VUS were detected in the DNAH1,
F10 and ITGB3 genes. The F10 gene has been associated
with autosomal recessive Factor X deficiency. ITGB3 has
been associated with autosomal recessive Bleeding dis-
order, platelet-type and autosomal dominant Glanzmann
thrombasthenia.
Although it is difficult to reconcile those with reces-
sive inheritance in the detected variants with the patient
clinics, those with dominant inheritance were compatible
with the patient clinics.
DNAH1 gene is the most frequently detected VUS
variant in our patient group. A diverse range of patients
with aberrant flagellar structures have been shown to have
mutant DNAH1 in the majority of current research. Male
infertility has been linked to numerous morphologic de-
fects of the sperm flagella caused by mutations in DNAH1.
After intracytoplasmic sperm injection, patients with mul-
tiple morphologic abnormalities of the flagella (MMAF)
caused by mutations in the DNAH1 gene have a favorable
prognosis. These investigations have demonstrated that
dysplasia of the sperm fibrous sheath (DFS) and infertility
are directly related to abnormalities in the DNAH1 gene
(15-18).
The most frequently detected CFTR variants in our
patient group were seen in all 3 groups (pathogenic, likely
pathogenic, VUS). One of the most researched genes for
male infertility, the CFTR gene, has 27 exons and more genicity based on in silico research and the genetic and
epidemiological data that are currently available (11-13).
Genetic variants can be categorized into five categories
according to the American College of Medical Genetics
and Genomics’ references: pathogenic, likely pathogenic,
variant of unknown consequence, likely benign, or benign
(14). A genetic alteration known as a VUS has ambiguous
effects on gene function. The interpretation of VUS is a
challenging task for the clinical management of infertile
male patients and genetic counseling. Since VUS are not
clearly related with a phenotype currently, but could be
categorized as pathogenic in the future, it is crucial to
detect and assess them. An example of this situation is
the NM_000071.3(CBS):c.833T>C (p.Ile278Thr) variant
detected in our patient group. This variant, which was
evaluated as VUS in the databases at the beginning of our
study, was later classified as pathogenic.
Variants in PROS1 and CFTR were detected in our
patient number 2. PROS1 variants show autosomal domi-
nant inheritance. Even though the detected variant was
evaluated as VUS, the classification of this variant should
be followed in the future.
Similarly, in our patient number 5, 2 pathogenic vari-
ants belonging to the F11 gene and a variant evaluated as
VUS in CFTR were detected. F11 variants show autosomal
dominant inheritance. The detected variants were evalu-
ated as pathogenic. OMIM has been associated with Factor
XI deficiency.
In another patient, case number 7, variants were de-
tected in 2 separate genes. VUS evaluation was performed
for TEX15. TEX15 is associated with the Spermatogenic
failure 25 phenotype in the OMIM database, and exhibits
an autosomal recessive pattern of inheritance. The other
variant detected in the patient is a possible pathogenic
variant belonging to the GP1BA gene. This gene, which
is associated with different types of diseases in the OMIM
database, can show autosomal dominant and recessive
inheritance. Particularly notable among these diseases are
Bernard-Soulier syndrome, type A2 (dominant) and von
Willebrand disease, platelet-type (dominant).
PROC variant was detected in patient number 9 and
was reported as possibly pathogenic. PROC variants have
been associated with autosomal recessive and dominant
forms of Thrombophilia 3 due to protein C deficiency in
OMIM. Another variant in the patient is the pathogenic
variant in the ITGA2 gene. The inheritance pattern and phe-
notype of variant of this gene have not yet been elucidated.
We detected variants in SEPT 12 and CFTR genes
in patient number 15. SEPT 12 variants cause autosomal
dominant Spermatogenic failure 10.
In patient number 19, we detected compound hetero-
zygous variants of the CFTR gene and an additional variant
of the CLCA4 gene. While CFTR gene variants exhibit
autosomal recessive inheritance, there are no entries in
the databases yet for the CLCA4 gene.
We detected F11 and CFTR pathogenic variants in
patient number 21. F11 was found to be associated with
autosomal dominant and recessive forms of Factor XI
deficiency in the OMIM database.
We detected DPY19L2 and CFTR pathogenic variants
in patient number 22. DPY19L2 was found to be associated
with autosomal recessive forms of spermatogenic failure
9 in the OMIM database. Similarly, patient number 40 has
variants in these two genes that were evaluated as VUS.
F13B and CFTR variants were detected in another of
our patients, case number 32. F13B has been associated
with Factor XIIIB deficiency in OMIM. F13B variants
show autosomal recessive inheritance.
Interestingly, variants in 3 different genes were de-
tected in our last 2 patients. Variants considered to be
VUS were detected in the DNAH1, KLHL10 and SEPT12
genes in the first patient. KLHL10 and SEPT12 variants
have been associated with autosomal dominant spermato-
genic failure. DNAH1 has been found to be associated
with autosomal recessive Ciliary dyskinesia, primary and
Spermatogenic failure. These findings were associated
with the patient’s phenotype. In the second patient, vari-
ants considered to be VUS were detected in the DNAH1,
F10 and ITGB3 genes. The F10 gene has been associated
with autosomal recessive Factor X deficiency. ITGB3 has
been associated with autosomal recessive Bleeding dis-
order, platelet-type and autosomal dominant Glanzmann
thrombasthenia.
Although it is difficult to reconcile those with reces-
sive inheritance in the detected variants with the patient
clinics, those with dominant inheritance were compatible
with the patient clinics.
DNAH1 gene is the most frequently detected VUS
variant in our patient group. A diverse range of patients
with aberrant flagellar structures have been shown to have
mutant DNAH1 in the majority of current research. Male
infertility has been linked to numerous morphologic de-
fects of the sperm flagella caused by mutations in DNAH1.
After intracytoplasmic sperm injection, patients with mul-
tiple morphologic abnormalities of the flagella (MMAF)
caused by mutations in the DNAH1 gene have a favorable
prognosis. These investigations have demonstrated that
dysplasia of the sperm fibrous sheath (DFS) and infertility
are directly related to abnormalities in the DNAH1 gene
(15-18).
The most frequently detected CFTR variants in our
patient group were seen in all 3 groups (pathogenic, likely
pathogenic, VUS). One of the most researched genes for
male infertility, the CFTR gene, has 27 exons and more genicity based on in silico research and the genetic and
epidemiological data that are currently available (11-13).
Genetic variants can be categorized into five categories
according to the American College of Medical Genetics
and Genomics’ references: pathogenic, likely pathogenic,
variant of unknown consequence, likely benign, or benign
(14). A genetic alteration known as a VUS has ambiguous
effects on gene function. The interpretation of VUS is a
challenging task for the clinical management of infertile
male patients and genetic counseling. Since VUS are not
clearly related with a phenotype currently, but could be
categorized as pathogenic in the future, it is crucial to
detect and assess them. An example of this situation is
the NM_000071.3(CBS):c.833T>C (p.Ile278Thr) variant
detected in our patient group. This variant, which was
evaluated as VUS in the databases at the beginning of our
study, was later classified as pathogenic.
Variants in PROS1 and CFTR were detected in our
patient number 2. PROS1 variants show autosomal domi-
nant inheritance. Even though the detected variant was
evaluated as VUS, the classification of this variant should
be followed in the future.
Similarly, in our patient number 5, 2 pathogenic vari-
ants belonging to the F11 gene and a variant evaluated as
VUS in CFTR were detected. F11 variants show autosomal
dominant inheritance. The detected variants were evalu-
ated as pathogenic. OMIM has been associated with Factor
XI deficiency.
In another patient, case number 7, variants were de-
tected in 2 separate genes. VUS evaluation was performed
for TEX15. TEX15 is associated with the Spermatogenic
failure 25 phenotype in the OMIM database, and exhibits
an autosomal recessive pattern of inheritance. The other
variant detected in the patient is a possible pathogenic
variant belonging to the GP1BA gene. This gene, which
is associated with different types of diseases in the OMIM
database, can show autosomal dominant and recessive
inheritance. Particularly notable among these diseases are
Bernard-Soulier syndrome, type A2 (dominant) and von
Willebrand disease, platelet-type (dominant).
PROC variant was detected in patient number 9 and
was reported as possibly pathogenic. PROC variants have
been associated with autosomal recessive and dominant
forms of Thrombophilia 3 due to protein C deficiency in
OMIM. Another variant in the patient is the pathogenic
variant in the ITGA2 gene. The inheritance pattern and phe-
notype of variant of this gene have not yet been elucidated.
We detected variants in SEPT 12 and CFTR genes
in patient number 15. SEPT 12 variants cause autosomal
dominant Spermatogenic failure 10.
In patient number 19, we detected compound hetero-
zygous variants of the CFTR gene and an additional variant
of the CLCA4 gene. While CFTR gene variants exhibit
autosomal recessive inheritance, there are no entries in
the databases yet for the CLCA4 gene.
We detected F11 and CFTR pathogenic variants in
patient number 21. F11 was found to be associated with
autosomal dominant and recessive forms of Factor XI
deficiency in the OMIM database.
We detected DPY19L2 and CFTR pathogenic variants
in patient number 22. DPY19L2 was found to be associated
with autosomal recessive forms of spermatogenic failure
9 in the OMIM database. Similarly, patient number 40 has
variants in these two genes that were evaluated as VUS.
F13B and CFTR variants were detected in another of
our patients, case number 32. F13B has been associated
with Factor XIIIB deficiency in OMIM. F13B variants
show autosomal recessive inheritance.
Interestingly, variants in 3 different genes were de-
tected in our last 2 patients. Variants considered to be
VUS were detected in the DNAH1, KLHL10 and SEPT12
genes in the first patient. KLHL10 and SEPT12 variants
have been associated with autosomal dominant spermato-
genic failure. DNAH1 has been found to be associated
with autosomal recessive Ciliary dyskinesia, primary and
Spermatogenic failure. These findings were associated
with the patient’s phenotype. In the second patient, vari-
ants considered to be VUS were detected in the DNAH1,
F10 and ITGB3 genes. The F10 gene has been associated
with autosomal recessive Factor X deficiency. ITGB3 has
been associated with autosomal recessive Bleeding dis-
order, platelet-type and autosomal dominant Glanzmann
thrombasthenia.
Although it is difficult to reconcile those with reces-
sive inheritance in the detected variants with the patient
clinics, those with dominant inheritance were compatible
with the patient clinics.
DNAH1 gene is the most frequently detected VUS
variant in our patient group. A diverse range of patients
with aberrant flagellar structures have been shown to have
mutant DNAH1 in the majority of current research. Male
infertility has been linked to numerous morphologic de-
fects of the sperm flagella caused by mutations in DNAH1.
After intracytoplasmic sperm injection, patients with mul-
tiple morphologic abnormalities of the flagella (MMAF)
caused by mutations in the DNAH1 gene have a favorable
prognosis. These investigations have demonstrated that
dysplasia of the sperm fibrous sheath (DFS) and infertility
are directly related to abnormalities in the DNAH1 gene
(15-18).
The most frequently detected CFTR variants in our
patient group were seen in all 3 groups (pathogenic, likely
pathogenic, VUS). One of the most researched genes for
male infertility, the CFTR gene, has 27 exons and more than 180,000 base pairs of DNA. A membrane ion chan-
nel protein called CFTR, which is encoded on chromo-
some 7p, controls the vas deferens in the male genital
tract. Considered a moderate type of cystic fibrosis (CF),
CBAVD is a major contributing factor to obstructive azo-
ospermia (OA) and is one of the primary factors leading
to male reproductive abnormalities. Previous research has
shown a positive correlation between CFTR mutations and
CBAVD. There are about 1,500 variants listed in the CFTR
database. Mutations in the F508 and IVS8-5T genes may
be important in nonobstructive male infertility disorders
such oligozoospermia and nonobstructive azoospermia
(NOA). According to our findings, there is a chance that
the genetic variant IVS8-5T could serve as a biomarker
for nonobstructive male infertility. Three of the pathogenic
variants we detected in the patient group are IVS8-5T. We
also detected a compound heterozygous CFTR variant
in one of our patients. The condition of the patient was
evaluated clinically. CFTR variant rates are also signifi-
cantly higher than the carrier rate reported in our patient
group (19-22).
Another gene we detected among pathogenic gene
variants is the CBS gene. The CBS gene is the most com-
mon locus for mutations associated with homocystin-
uria. Cystathionine-β-synthase, also known as CBS, is
an enzyme that is encoded by the CBS gene in humans.
The trans-sulfuration pathway enzymes cystathionine
β-synthase (CBS) and cystathionine γ-lyase (CSE) are
recognized for their non-specific substrate recognition.
These enzymes provide their substrates an alternate CBS
and CSE pathway, allowing them to function somewhat
in reverse. In addition, these enzymes are involved in the
synthesis of hydrogen sulphide (H2S). This is a gaseous
transmitter with antioxidant and anti-inflammatory proper-
ties. Although both CSE and CBS are frequently present in
the testes—CSE is mostly found in immature germ cells
and Sertoli cells, while CBS is extensively distributed in
Leydig cells, germ cells, and Sertoli cells—it is unclear
how much each kind of cell contributes to the produc-
tion of H2 S in the testes. Numerous research studies have
demonstrated that male infertility is associated with failure
of one carbon metabolism, namely the imbalance of CBS
and CSE enzymes in the trans-sulfuration pathway, and
a specific deficit in H2 S output is documented (23, 24).
Another gene we detected as a pathogenic variant in
our patient group was FXI. Compound heterozygous FXI
pathogenic variant was detected in one of our patients.
In another patient, we detected a heterozygous FXI vari-
ant of CFTR accompanied by pathogenic variant. FXI, or
coagulation factor XI, inhibits fibrinolysis and encour-
ages the production of fibrin. One measurement only el-
evated plasma FXI levels are linked to an increased risk
of thrombosis. Hemophilia C, another name for factor XI
deficiency, is an autosomal recessive condition mostly
affecting Ashkenazi Jews. It is typically linked to variants
in bleeding characteristics. The majority of transmission is
autosomal recessive. Couples who are considered at-risk
(both individuals carry a mutation that causes the dis-
ease) should be informed through genetic counseling that
there is a 25% chance that each pregnancy will result in a
homozygous child who is affected. There have also been
reports of heterozygous patients with bleeding symptoms,
pointing to an autosomal dominant mode of transmission
with varying penetrance (25, 26).
Genes that code for hormones and hormone receptors
which are involved in the functioning of the human repro-
ductive system are included in the fertility panel design.
Numerous investigations have demonstrated the associa-
tion between specific polymorphisms in genes encoding
receptors, including those that bind to FSH and LH, and the
results of an ovarian hyperstimulation cycle under control
and in vitro fertilization treatment. The intended genetic
panel’s results will yield the data required to ascertain the
frequency of these variants in our community and assess
the panel’s usefulness in clinical settings.
The involvement of the clinicians who seek this ge-
netic investigation needs to be emphasized. If the gene
panel is able to pinpoint the underlying reason of infertility,
clinicians will need to have a comprehensive picture of the
patient’s phenotype. “Idiopathic infertility” affects a large
number of individuals, and while a genetic component
may be identified in certain cases, the absence of a distinct
phenotype may make it more difficult to interpret the data,
particularly variants with unclear significance. Clinicians
should also be aware that three factors play a major role
in how these investigations are interpreted: the patient’s
phenotypic characteristics, their medical history, and any
pertinent family history. For the diagnostic laboratory to
properly interpret variants found through testing, it is im-
perative that they have information about all observable
traits as well as the family’s medical history (27-30).
The first unique gene sequencing panel intended for
the diagnosis of hereditary infertility in males is presented
here, for the first time in Turkey. The use of this panel will
advance knowledge of the genetic causes of infertility, en-
hance genetic and reproductive counseling, and eventually
lead to more accurate assisted reproductive techniques.
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