Agnieszka Magdalena Rygiel

Aromatic L-amino acid decarboxylase (AADC), encoded by DDC gene, catalyzes the final step in dopamine and serotonin biosynthesis. Pathogenic DDC variants cause AADC deficiency, a severe neurometabolic disorder. This study aimed to characterize previously uninvestigated DDC variant combinations: p.Pro47Ala/p.Arg447Cys and p.Cys261Phe/p.Gly354Ser, with respect to structural and functional characteristics. Recombinant heterodimeric and homodimeric AADC proteins were expressed in E. coli and purified. Enzymatic activity was quantified via ion-pair reversed-phase HPLC with photodiode detection, while structural changes were analyzed using circular dichroism and molecular modelling. Functional characterization revealed that p.Pro47Ala, p.Gly354Ser, and p.Arg447Cys homodimers showed marked loss of activity towards both L-DOPA (88%, 97% and >99%, respectively) and L-5-hydroxytryptophan (88%, 99%, >99%, respectively), whereas p.Cys261Phe retained substantial activity (82% for L-DOPA and 75% for L-5-hydroxytryptophan). Kinetic analysis indicated a mild to severe decrease in substrate affinity for all homodimers, particularly for p.Arg447Cys. Furthermore, heterodimeric combinations showed positive complementation effects on decarboxylase reaction for both heterodimers. Surprisingly, p.Cys261Phe/p.Gly354Ser also restored the wild-type activity with L-DOPA and regained 43% of L-5-hydroxytryptophan activity, whereas p.Pro47Ala/p.Arg447Cys was 15% and 10% active towards L-DOPA and L-5-hydroxytryptophan with respect to the wild-type. These results are discussed in the light of patient’s AADC residual activity and phenotype, highlighting the complexity of polypeptide chain interactions in compound heterozygotes. The pathogenicity of AADC variants in this context depends on structural combination, complementation effects in heterodimeric species, and residual activity of homodimers.

Recently, the knowledge of the genetic basis of fertility disorders has expanded enormously, mainly thanks to the use of next-generation sequencing (NGS). However, the genetic cause of infertility, in the majority of patients, is still undefined. The aim was to identify novel and recurrent pathogenic/likely pathogenic variants in patients with isolated infertility or puberty delay using a targeted NGS technique. We have enrolled 41 patients (36 males and 5 females) with infertility problems or delayed puberty. We included the patients with hypogonadotropic hypogonadism (n = 12), hypergonadotropic hypogonadism (n = 15), abnormal sperm parameters (n = 10), androgen insensitivity syndrome (n = 3) and 46,XY gonadal dysgenesis (n = 1). Genetic tests were performed using targeted NGS panel of 35 genes implicated in fertility. Pathogenic or likely pathogenic variants potentially explaining the clinical phenotype were identified in 12 of 41 patients (29%). These included 9 of 12 patients (75%) with hypogonadotropic hypogonadism, 2 of 3 patients (66%) with androgen insensitivity syndrome, and the single patient with 46,XY gonadal dysgenesis. Among the 18 identified variants, 4 were novel (FGF8:p.Ala147Thr; SEMA3A:p.Arg544Cys; FGFR1:p.Thr141IlefsTer10; NSMF: p.Tyr242Cys), while 14 were recurrent. Our study expands the knowledge of the genetic basis of the infertility disorders and highlights the importance of genetic testing for proper diagnosis making and genetic counselling.

Recently, the knowledge of the genetic basis of fertility disorders has expanded enormously, mainly thanks to the use of next generation sequencing (NGS). However, the genetic cause of the infertility in majority patients is still undefined. Aim: The aim was to identify genetic variants associated with infertility disorders using targeting NGS technique and to describe phenotype - genotype correlation. Methods: We have enrolled 41 patients (36 males and 5 females) with infertility problems or delayed puberty including the patients with hypogonadism hypogonadotropic (HH, n = 12), primary hypogonadism (n = 15), abnormal semen parameters or gonadal dysgenesis (n = 11), androgen insensitivity (n = 3). Genetic tests were performed using NGS panel of 35 genes. Results: Overall, 14 pathogenic (P) or likely pathogenic (LP) variants including 3 novel and 11 recurrent variants were identified. Novel variants were found in genes associated with HH (FGF8, FGFR1, SEMA3). The genetic cause of the HH was determined in 58% (7/12) of the cases. Overall, The genetic testing enabled identification of the cause of the clinical phenotype in 26% (11/41) of the patients. Conclusions: Our study expands the knowledge of the genetic basis of the infertility disorders and highlights the importance of genetic testing for proper diagnosis making and genetic counselling.

Loricrin keratoderma (LK) is a rare autosomal dominant genodermatosis caused by LORICRIN gene mutations. The pathogenesis of the disease is not yet fully understood. So far, only 10 pathogenic variants in LORICRIN have been described, with all of them but one being deletions or insertions. The significance of rare nonsense variants remains unclear. Furthermore, no data regarding the RNA expression in affected patients are available. The aim of this study is to describe the two variants in the LORICRIN gene found in two distinct families: the novel pathogenic variant c.639_642dup and a rare c.10C > T (p.Gln4Ter) of unknown significance. We also present the results of the transcriptome analysis of the lesional loricrin keratoderma epidermis of a patient with c.639_642dup. We show that in the LK lesion, the genes associated with epidermis development and keratocyte differentiation are upregulated, while genes engaged in cell adhesion, differentiation developmental processes, ion homeostasis and transport, signaling and cell communication are downregulated. In the context of the p.Gln4Ter clinical significance evaluation, we provide data indicating that LORICRIN haploinsufficiency has no skin consequences. Our results give further insight into the pathogenesis of LK, which may have therapeutic implications in the future and important significance in the context of genetic counseling.

Large and giant congenital melanocytic nevi (CMN), benign naevomelanocytic proliferations derived from neural crests, with a projected adult size (PAS) ≥ 20 cm, are connected to a high risk of melanoma and neurocutaneous melanosis. Among several factors, genetic alterations seem to be involved in tumorigenesis. The aim of the present study was to analyse the mutation status of NRAS and BRAF genes in resection specimens from large or giant CMN in a group of Polish patients. The formalin-fixed, paraffin-embedded resection specimens from 18 patients, fixed in the years of 2006 to 2017, were included in the study. The regions containing the highest load of melanocytes were macrodissected prior to DNA isolation. The NRAS and BRAF mutation status was evaluated using qPCR. We detected activating mutations in NRAS gene (codons: 12 and 61) in 7 out of the 18 (38.9%) patients. No BRAF mutations were found. Our study, the first molecular analysis of large/giant CMN in Polish patients, supports the hypothesis that NRAS mutation in codon 61 are frequent, recurrent mutations in large/giant CMN. Moreover, we show, for the first time, that NRAS mutations in codon 12 (p.Gly12Asp) can be also detected in giant CMN. The exact role of these genetic alterations in CMN formation remains to be elucidated.

Chronic pancreatitis (CP) is a progressive, irreversible inflammatory disorder of the pancreas, which results from interrelations between different genetic and environmental factors. Genetic variants are the primary cause of the disease in early-onset nonalcoholic CP patients. Novel CP-associated genes are continuously emerging from genetic studies on CP cohorts, providing important clues for distinct mechanisms involved in CP development. On the basis of functional studies, the genetic alterations have been sub-grouped into CP-driving pathological pathways. This review focuses on the concept of CP as a complex disease driven by multiple genetic factors. We will discuss only well-defined genetic risk factors and distinct functional pathways involved in CP development, especially in the context of the early-onset nonalcoholic CP group. The diagnostic implications of the genetic testing will be addressed as well.