Sexual development (SD) is a complex process with strict spatiotemporal regulation of gene expression. Despite advancements in molecular diagnostics, disorders of sexual development (DSD) have a ...diagnostic rate of ~50%. Androgen insensitivity syndrome (AIS) represents the most common form of 46,XY DSD, with a spectrum of defects in androgen action. Considering the importance of very strict regulation of the SD, it is reasonable to assume that the genetic cause for proportion of the DSD lies in the non-coding part of the genome that regulates proper gene functioning. Here we present a patient with partial AIS (PAIS) due to a mosaic
c.-547C>T pathogenic variant in the 5′UTR of androgen receptor (
) gene. The same mutation was previously described as inherited, in two unrelated patients with complete AIS (CAIS). Thus, our case further confirms the previous findings that variable gene expressivity could be attributed to mosaicism. Mutations in 5′UTR could create new upstream open reading frames (uORFs) or could disrupt the existing one. A recent systematic genome-wide study identified
as a member of a subset of genes where modifications of uORFs represents an important disease mechanism. Only a small number of studies are reporting non-coding mutations in the
gene and our case emphasizes the importance of molecular testing of the entire
locus in AIS patients. The introduction of new methods for comprehensive molecular testing in routine genetic diagnosis, accompanied with new tools for in sillico analysis could improve the genetic diagnosis of AIS, and DSD in general.
Summary
Spermatogenesis is a complex process that involves thousands of genes whose expression during different stages is strictly regulated. Small non‐coding microRNAs play an important role in the ...posttranscriptional regulation of mRNA processing during spermatogenesis. Using Agilent SurePrint v16 microRNA 8 × 60 K microarray kit, we investigated the microRNA expression profiles of 24 formalin‐fixed paraffin‐embedded testicular biopsies from patients with hypospermatogenesis (n = 10), hypospermatogenesis and azoospermia factor c region on the Y chromosome (AZFc) deletion (n = 3), Sertoli cell‐only syndrome (n = 3) and maturation arrest (n = 2), in comparison with subjects with normal spermatogenesis (n = 6). After adjusting for multiple testing, six deregulated miRNAs were detected in the patients with AZFc deletion, 30 in maturation arrest group, 52 in Sertoli cell‐only syndrome group of patients, and none in the group of patients with hypospermatogenesis. Some of the deregulated microRNAs were shared between groups, resulting in 58 unique differentially expressed microRNAs. The expression of five microRNAs (hsa‐miR‐34b, hsa‐miR‐449b, hsa‐miR‐517c, hsa‐miR‐181c, and hsa‐miR‐605) was validated by qRT‐PCR in a total of 74 samples. Using mRNA expression profiles of subjects with matching histopathological patterns of impaired spermatogenesis from publically available Gene Expression Omnibus data sets, we have performed integrated mRNA–microRNA regulatory network analysis. Pathway analysis revealed significantly enriched set of genes for tumor necrosis factor‐related apoptosis‐inducing ligand signaling pathway, previously shown to be involved in regulation of apoptosis in normal functioning testis. Our results should be considered as preliminary as we have analyzed only a small number of patients in each studied group. Further studies with larger number of patients with impaired spermatogenesis as well as more targeted approaches with parallel microRNA and mRNA expression profiling in isolated subpopulations of somatic or germ cells from different stages of spermatogenesis are needed to clarify the role of the microRNAs in the process of spermatogenesis.
Chromosomal abnormalities are the most common causes of early pregnancy losses (EPLs). In this study, we aimed to evaluate the incidence and spectrum of chromosomal abnormalities in EPLs and ...correlate them with different clinical characteristics. We performed Quantitative Fluorescent PCR (QF-PCR), followed by subtelomeric Multiplex Ligation Probe Amplification (MLPA) analysis to detect chromosomal abnormalities in 900 products of conceptions (POCs) from EPLs collected over a period of 10 years.
Chromosomal abnormalities were present in 56.25% of uncontaminated EPLs, with significantly higher incidence in women ≥36 years (71.37%, p<0.0001) in comparison to women ≤30 years of age (43.40%). Trisomies were also more common in women ≥36 years (79.68%, p<0.0001) than in those ≤30 years of age (48.70%). In contrast, triploidy and monosomies were more prevalent in women ≤30 years of age (26.09%, p<0.0001 and 16.52%, p=0.0066 respectively) than in women ≥36 years of age (6.42% and 6.42% respectively). Trisomy 16 was more common in women ≤30 (39.29%, p=0.0009) than in those ≥36 years of age (16.78%), while trisomy 22 was predominant among women ≥36 (23.49%, p=0.013), and was not present in the group of women ≤30 years of age. The frequency of chromosomal abnormalities in POCs from women with sporadic (61.19%) was higher than in those with recurrent EPLs (55.21%). This difference, however, was not statistically significant (p=0.164). Although some differences in the chromosomal aneuploidy rates among women with different ABO blood groups, as well as among 6–8 and 9–11 gestational week EPLs were observed, further larger studies are required to confirm these findings.
In conclusion, our study enriches the knowledge about chromosomal abnormalities as a cause of EPLs and confirms the higher incidence of foetal chromosomal abnormalities in EPLs in women of older reproductive age. Furthermore, it shows that using QF-PCR and MLPA methodologies, a high detection rate of chromosomal abnormalities in EPLs can be reached.
Gitelman syndrome (GS) is a rare renal tubulopathy with an autosomal recessive mode of inheritance, caused by biallelic pathogenic variants in the
gene. The clinical features may overlap with other ...disorders, such as Bartter syndrome type 3, HNF1B nephropathy or even mitochondrial disease, but can be distinguished by molecular genetic analysis.
Here we report on two preschool brothers, who presented with a several months’ history of episodes of carpopedal spasms and muscle aches.
The biochemical analyses revealed hypokalemia and hypomagnesemia without metabolic alkalosis. A 24-h urine sample demonstrated hypocalciuria.
The molecular analyses showed that both patients were heterozygous for 3 (likely) pathogenic variants in
: c.1805_1806del; p. (Tyr602Cysfs*31), c.2660+1G>A and c.2944 A>T; p. (Ile982Phe). Analysis of the parents showed that the mother was heterozygous for the c.2944 A>T p.(Ile982Phe) variant, and the father carried the other 2 variants (c.1805_1806del and c.2660+1G>A).
Herein we present two children in a family from N. Macedonia with clinical manifestations and electrolyte imbalances suggestive of GS. The results of the tubulopathy next generation sequencing (NGS) panel confirmed the diagnosis. The boys are treated with a high salt diet and oral potassium and magnesium supplements.
Abstract The most prevalent "rare" disease worldwide, cystic fibrosis (CF), is an autosomal recessive multisystem disease, caused by mutations in the CFTR gene. The knowledge of CFTR mutations ...present in certain population is important for designing a simple, fast and cost-effective genetic testing approach, also for better management of CF patients, including the administration of novel targeted therapies. Here, we present genetic results of 158 unrelated CF patients from the National CF Registry of the Republic of North Macedonia. Initially, patients were screened for the 11 most common CF mutations. Additional CF mutations and large deletions/duplications in the CFTR gene were analyzed using commercial kits. If the genotype was undetermined, all CFTR exons were analyzed using Sanger DNA sequencing or next generation sequencing (NGS) (since 2014). The most common CF mutation, c.l521_ 1523del (legacy name F508del), was found with an overall incidence of 75.9%. Additionally, 26 other pathogenic variants and three large deletions were identified in the CFTR gene as a genetic cause of CF. Two of these, c.1070 C>T (p.Ala357Val) and c.2779_2788dup CTTGCTATGG (p.Gly930AlafsTer48), were novel. According to the distribution and prevalence of the pathogenic variants detected in our patients, a fast and cost-effective method, based on a single base extension was designed as a first-line CF genetic test with a 90.0% detection rate within our population. Furthermore, the knowledge of CFTR mutation classes in our CF patients represents the first step toward personalized therapy for CF in our country.
Summary
Seminal plasma is a potential source of biomarkers for many disorders of the male reproductive system including male infertility. The identification and characterisation of differentially ...expressed proteins in seminal plasma of man with normal and impaired spermatogenesis can help in the elucidation of the molecular basis of male infertility. We compared the protein expression profiles of seminal plasma from four different groups of men as follows: normozoospermic, asthenozoospermic, oligozoospermic and azoospermic groups, using two‐dimensional differential gel electrophoresis (2‐D DIGE). We found eight proteins with statistically significant increased expression in azoospermia compared with at least one of the other studied groups. The differentially expressed spots were fibronectin, prostatic acid phosphatase (PAP), proteasome subunit alpha type‐3, beta‐2‐microglobulin, galectin‐3‐binding protein, prolactin‐inducible protein and cytosolic nonspecific dipeptidase. Notably, PAP was increased in patients with azoospermia compared with that of all other groups. We have observed no statistically significant differences in protein expression between three of the groups: normozoospermic, oligozoospermic and asthenozoospermic. We suggest that the identified panel of proteins in our study especially PAP have a strong potential to be used as azoospermia markers. However, further investigations will be necessary to validate these markers in samples of larger and independent patient cohorts and to clarify their role in the pathogenesis of male infertility.
Steroid 5-α-reductase-2 (5-ARD) deficiency is a result of mutations of the
gene. It causes the disorder of sexual differentiation (DSD) in 46,XY individuals with a variable genital phenotype. We ...present two siblings with female external genitalia at birth and bilateral inguinal testes, raised as females. These are the first molecularly characterized patients from the Republic of North Macedonia (RN Macedonia) with a different clinical course due to the time of the diagnosis. Diagnosis of Patient 1 was based upon the detection of bilateral inguinal testes and testosterone/dihidrotestosterone ratio. Sex reversal was initiated by testes removal at the age of 20 months. Breast implantation and vaginoplasty were performed in adolescence and the girl is comfortable with the female sex. Her sibling, Patient 2, raised as a girl, was clinically assessed at 11.5 years due to the growth of phalus, deep voice and Adam’s apple enlargement. No change of gender was accepted. Complex molecular analysis including multiplex quantitative fluorescent polymerase chain reaction (PCR) screening for sex chromosome aneuploidies and
presence, Sanger sequencing combined with multiplex ligation-dependent probe amplification (MLPA), microarray-based comparative genomic hybridization (aCGH), and real-time PCR analysis for detection of exon copy number changes confirmed a novel c.146C>A (p.Ala49Asp) point mutation in the first exon inherited from the mother, and complete deletion of the first exon and adjacent regions inherited from the father. Novel genotype causing 5-ARD is presented. Genetic analysis is useful for the diagnosis and timely gender assignment in patients with 5-ARD. However, final gender assignment is difficult and requires combined medical interventions.
There is a widely accepted consensus on the benefits of newborn screening (NBS) for cystic fibrosis (CF) in terms of reduced disease severity, improved quality of life, lower treatment burden, and ...reduced costs. More and more countries in the world are introducing NBS for CF as a national preventive health program. Newborn screening for CF was introduced in the Republic of North Macedonia (RNM) in April, 2019, after a pilot study of 6 months in 2018. A two-step immunoreactive trysinogen (IRT-IRT) algorithm is performed, and then a sweat test for confirmation/exclusion of the CF diagnosis when the IRT values were both over the cutoff (70.0 and 45.0 ng/mL, respectively). In cases with confirmed diagnosis of CF (a sweat chloride concentration >60.0 mmol/L) or with intermediate sweat test results (a sweat chloride concentration of between 30.0 and 59.0 mmol/L), CF transmembrane conductance regulator (CFTR) mutation analysis is performed. By the end of 2020, over a period of 27 months, including the pilot study period, a total number of 43,139 newborns were screened for CF. Seventeen (0.039%) newborns were diagnosed with CF. In all newly discovered CF cases by screening, the diagnosis was confirmed by determination of the CFTR mutations. The most common CFTR mutation, F508del, was found with an overall incidence of 70.6%. Other more frequent mutations were G542X (11.8%) and N1303K (5.9%). Four mutations were found in one CFTR allele each: G1349D, G126D, 457TAT>G and CFTRdupexon22, with the last one being newly discovered with unknown consequences. An incredibly large difference was found in the incidence of the disease between the Macedonian and Albanian neonatal population, with almost four time higher prevalence among Albanians (1:4530
. 1:1284).
Dyskeratosis congenita (DC) is a clinically and genetically heterogeneous, multisystem inherited syndrome with a very high risk for bone marrow failure (BMF) and cancer predisposition. The classical ...clinical form of DC is characterized by abnormal skin pigmentation, nail dystrophy, and oral leukoplakia. Bone marrow failure is considered to be an important and major complication of DC and the leading cause of death which develops in around 85% of cases. A number of genes involved in telomere maintenance are associated with DC, such as genes that encode the components of the telomerase complex (
,
,
,
10, and
2), T-loop assembly protein (
), telomere capping (
), telomere shelterin complex (
), and telomerase trafficking protein (
). Mutations in
have been reported in 11–20% of all patients with DC and have been associated with bone marrow failure. Here we report on a 19-month old boy with very early presentation of bone marrow failure as a first clinical manifestation of DC. Upon first admission, the patient presented with thrombocytopenia and macrocytic anemia. Soon after, his blood counts deteriorated with the development of pancytopenia and aplastic anemia. Four months later, he developed nail dystrophy and skin hyperpigmentation. A
heterozygous pathogenic variant c.845G>A, p.(Arg282His) was located in exon 6 of
gene and was identified via clinical exome sequencing. The findings confirmed the diagnosis of DC. This is the first case with DC due to
pathogenic variant reported in North Macedonia.
Metatropic dysplasia (MD) is a rare skeletal dysplasia associated with heterozygous mutations in the
gene. We describe a 28-month-old boy with knock-knees referred for metabolic investigation ...suspected of carrying vitamin D-resistant rickets. He has received regular vitamin D prophylaxis at the usual dose. Laboratory investigations revealed normal values for calcium, phosphorus and alkaline phosphatase. He was short (-3.5 SDS), his mental development was normal, and he started to walk at the age of 22 months. The diagnostic clue for the diagnosis of metatropic dysplasia was the presence of the hump back in the upper lumbar and lower thoracic vertebrae, in addition to a long and narrow chest. An X-ray survey of the skeleton revealed platyspondyly, dysplastic metaphyses with dumbbell appearance of the long bones, kyphoscoliosis, and narrow and elongated thorax with short ribs. This is the first patient with MD in the Republic of Macedonia. Knock-knees were the cause of his referral, as a peculiarity of his phenotype. The very presence of the hump back, and the dumbbell appearance of the long bones distinguished the MD from other bone dysplasias with similar characteristics. We believe that the presence of those two features can shorten the path to accurate diagnosis in the crowded field of overlapping skeletal dysplasias. The diagnosis of MD in this patient was further confirmed by the discovery of the mutation c.2396C>T; p.Pro799Leu (P799L) of the
gene.
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