Hypogonadism - when does genetic diagnosis help in therapy?

OBJECTIVES: The objective of the study was to describe phenotype-genotype correlation in selected cases with infertility and emphasise the importance of genetic testing as useful tool for proper treatment decision making MATERIAL AND METHODS: Genetic tests were performed in four patients as a part of diagnostic procedure by Sanger sequencing or targeted next generation sequencing (NGS gene panel). RESULTS: We found the genetic causes of hypogonadotropic hypogonadism in 3 males and female with infertility. CONCLUSIONS: Genetic testing is carried out when searching for the genetic causes of clinically identified disorders. Genetic diagnostics may also be extremely helpful in treating hypogonadism but requires the assistance of a clinician endocrinologist or andrologist, as well as a geneticist.

Congenital coenzyme Q5-linked pathology: causal genetic association, core phenotype, and molecular mechanism.

Coenzyme Q5 (COQ5), a C-methyltransferase, modifies coenzyme Q10 (COQ10) during biosynthesis and interacts with polyA-tail regulating zinc-finger protein ZC3H14 in neural development. Here, we present a fifth patient (a third family) worldwide with neurodevelopmental and physiological symptoms including COQ10 deficiency. Our patient harbors one novel c.681+1G>A and one recurrent p.Gly118Ser variant within COQ5. The patient's mRNA profile reveals multiple COQ5 splice-variants. Subsequently, we comprehensively described patient's clinical features as compared to phenotype and symptoms of other known congenital coenzyme Q5-linked cases. A core spectrum of COQ5-associated symptoms includes reduced COQ10 levels, intellectual disability, encephalopathy, cerebellar ataxia, cerebellar atrophy speech regression/dysarthria, short stature, and developmental delays. Our patient additionally displays dysmorphia, microcephaly, and regressive social faculties. These results formally establish causal association of biallelic COQ5 mutation with pathology, outline a core COQ5-linked phenotype, and identify mRNA mis-splicing as the molecular mechanism underlying all COQ5 variant-linked pathology to date.

The Epidermal Transcriptome Analysis of a Novel c.639_642dup LORICRIN Variant-Delineation of the Loricrin Keratoderma Pathology.

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.

Prevalence of DDC genotypes in patients with aromatic L-amino acid decarboxylase (AADC) deficiency and in silico prediction of structural protein changes.

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive genetic disorder affecting the biosynthesis of dopamine, a precursor of both norepinephrine and epinephrine, and serotonin. Diagnosis is based on the analysis of CSF or plasma metabolites, AADC activity in plasma and genetic testing for variants in the DDC gene. The exact prevalence of AADC deficiency, the number of patients, and the variant and genotype prevalence are not known. Here, we present the DDC variant (n = 143) and genotype (n = 151) prevalence of 348 patients with AADC deficiency, 121 of whom were previously not reported. In addition, we report 26 new DDC variants, classify them according to the ACMG/AMP/ACGS recommendations for pathogenicity and score them based on the predicted structural effect. The splice variant c.714+4A>T, with a founder effect in Taiwan and China, was the most common variant (allele frequency = 32.4%), and c.[714+4A>T];[714+4A>T] was the most common genotype (genotype frequency = 21.3%). Approximately 90% of genotypes had variants classified as pathogenic or likely pathogenic, while 7% had one VUS allele and 3% had two VUS alleles. Only one benign variant was reported. Homozygous and compound heterozygous genotypes were interpreted in terms of AADC protein and categorized as: i) devoid of full-length AADC, ii) bearing one type of AADC homodimeric variant or iii) producing an AADC protein population composed of two homodimeric and one heterodimeric variant. Based on structural features, a score was attributed for all homodimers, and a tentative prediction was advanced for the heterodimer. Almost all AADC protein variants were pathogenic or likely pathogenic.

Chronic pancreatitis caused by a Homozygous SPINK1 c.194 + 2T > C variant and Pancreas Divisum in a 3-year-old child-case report.

Chronic pancreatitis (CP) is a rare disease in children. We describe the first case of a 3-year-old Caucasian patient with CP with the presence of a homozygous pathogenic variant c.194 + 2T > C in serine protease inhibitor, Kazal type 1 ( SPINK1 ) and pancreas divisum.

Loss-of-function variant in chymotrypsin like elastase 3B (CELA3B) is associated with non-alcoholic chronic pancreatitis.

BACKGROUND: Genetic alterations in digestive enzymes have been associated with chronic pancreatitis (CP). Recently, chymotrypsin like elastase 3B (CELA3B) emerged as a novel risk gene. Thus, we evaluated CELA3B in two European cohorts with CP. METHODS: We analyzed all 8 CELA3B exons in 550 German non-alcoholic CP (NACP) patients and in 241 German controls by targeted DNA sequencing. In addition, we analyzed exons 6 and 7 by Sanger sequencing and the c.129+1G>A variant by melting curve analysis in 1078 further German controls. As replication cohort, we investigated up to 243 non-German European NACP patients and up to 1665 controls originating from Poland, Hungary, and Sweden. We assessed the cellular secretion and the elastase activity of recombinant CELA3B variants. RESULTS: In the German discovery cohort, we detected a splice-site variant in intron 2, c.129+1G>A, in 9/550 (1.64%) CP patients and in 5/1319 (0.38%) controls (P=0.007, OR=4.4, 95% CI=1.5-13.0). In the European replication cohort, this variant was also enriched in patients (9/178 [5.06%]) versus controls (13/1247 [1.04%]) (P=0.001, OR=5.1, 95% CI=2.1-12.0). We did not find the two previously reported codon 90 variants, p.R90C and p.R90L. CONCLUSIONS: Our data indicate that CELA3B is a susceptibility gene for CP. In contrast to previous reports suggesting that increased CELA3B activity is associated with CP risk, the splice-site variant identified here is predicted to cause diminished CELA3B expression. How reduced CELA3B function predisposes to pancreatitis remains to be elucidated.

The novel P330L pathogenic variant of aromatic amino acid decarboxylase maps on the catalytic flexible loop underlying its crucial role.

Aromatic amino acid decarboxylase (AADC) deficiency is a rare monogenic disease, often fatal in the first decade, causing severe intellectual disability, movement disorders and autonomic dysfunction. It is due to mutations in the gene coding for the AADC enzyme responsible for the synthesis of dopamine and serotonin. Using whole exome sequencing, we have identified a novel homozygous c.989C > T (p.Pro330Leu) variant of AADC causing AADC deficiency. Pro330 is part of an essential structural and functional element: the flexible catalytic loop suggested to cover the active site as a lid and properly position the catalytic residues. Our investigations provide evidence that Pro330 concurs in the achievement of an optimal catalytic competence. Through a combination of bioinformatic approaches, dynamic light scattering measurements, limited proteolysis experiments, spectroscopic and in solution analyses, we demonstrate that the substitution of Pro330 with Leu, although not determining gross conformational changes, results in an enzymatic species that is highly affected in catalysis with a decarboxylase catalytic efficiency decreased by 674- and 194-fold for the two aromatic substrates. This defect does not lead to active site structural disassembling, nor to the inability to bind the pyridoxal 5'-phosphate (PLP) cofactor. The molecular basis for the pathogenic effect of this variant is rather due to a mispositioning of the catalytically competent external aldimine intermediate, as corroborated by spectroscopic analyses and pH dependence of the kinetic parameters. Altogether, we determined the structural basis for the severity of the manifestation of AADC deficiency in this patient and discussed the rationale for a precision therapy.

Risk of chronic pancreatitis in carriers of loss-of-function CTRC variants: A meta-analysis.

The digestive protease chymotrypsin C (CTRC) protects the pancreas against pancreatitis by degrading potentially harmful trypsinogen. Loss-of-function genetic variants in CTRC increase risk for chronic pancreatitis (CP) with variable effect size, as judged by the reported odds ratio (OR) values. Here, we performed a meta-analysis of published studies on four variants that alter the CTRC amino-acid sequence, are clinically relatively common (global carrier frequency in CP >1%), reproducibly showed association with CP and their loss of function phenotype was verified experimentally. We found strong enrichment of CTRC variants p.A73T, p.V235I, p.K247_R254del, and p.R245W in CP cases versus controls, yielding OR values of 6.5 (95% confidence interval (CI) 2.4-17.8), 4.5 (CI 2.2-9.1), 5.4 (CI 2.6-11.0), and 2.6 (CI 1.6-4.2), respectively. Subgroup analysis demonstrated disease association of variants p.K247_R254del and p.R245W in alcoholic CP with similar effect sizes as seen in the overall CP group. Homozygosity or compound heterozygosity were rare and seemed to be associated with higher risk. We also identified a so far unreported linkage disequilibrium between variant p.K247_R254del and the common c.180C>T (p.G60 =) haplotype. Taken together, the results indicate that heterozygous loss-of-function CTRC variants increase the risk for CP approximately 3-7-fold. This meta-analysis confirms the clinical significance of CTRC variants and provides further justification for the genetic screening of CP patients.

Variants in the pancreatic CUB and zona pellucida-like domains 1 (CUZD1) gene in early-onset chronic pancreatitis - A possible new susceptibility gene.

OBJECTIVE: Non-alcoholic chronic pancreatitis (NACP) frequently develops in the setting of genetic susceptibility associated with alterations in genes that are highly expressed in the pancreas. However, the genetic basis of NACP remains unresolved in a significant number of patients warranting a search for further risk genes. DESIGN: We analyzed CUZD1, which encodes the CUB and zona pellucida-like domains 1 protein that is found in high levels in pancreatic acinar cells. We sequenced the coding region in 1163 European patients and 2018 European controls. In addition, we analyzed 297 patients and 1070 controls from Japan. We analyzed secretion of wild-type and mutant CUZD1 from transfected cells using Western blotting. RESULTS: In the European cohort, we detected 30 non-synonymous variants. Using different prediction tools (SIFT, CADD, PROVEAN, PredictSNP) or the combination of these tools, we found accumulation of predicted deleterious variants in patients (p-value range 0.002-0.013; OR range 3.1-5.2). No association was found in the Japanese cohort, in which 13 non-synonymous variants were detected. Functional studies revealed >50% reduced secretion of 7 variants, however, these variants were not significantly enriched in European CP patients. CONCLUSION: Our data indicate that CUZD1 might be a novel susceptibility gene for NACP. How these variants predispose to pancreatitis remains to be elucidated.

The retrospective molecular analysis of large or giant congenital melanocytic nevi in a group of Polish children.

BACKGROUND: 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. MATERIAL AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Loss of function TRPV6 variants are associated with chronic pancreatitis in nonalcoholic early-onset Polish and German patients.

PURPOSE: Loss of function variants of the transient receptor potential cation channel, subfamily V, member 6 (TRPV6) have been recently associated with chronic pancreatitis (CP) in Japanese, German and French patients. Here, we investigated the association of TRPV6 variants with CP in independent European cohorts of early-onset CP patients from Poland and Germany. PATIENTS AND METHODS: We enrolled 152 pediatric CP patients (median age 8.6 yrs) with no history of alcohol/smoking abuse and 472 controls from Poland as well as 157 nonalcoholic young CP patients (median age 20 yrs) and 750 controls from Germany. Coding regions of TRPV6 were screened by Sanger and next generation sequencing. Selected, potentially pathogenic TRPV6 variants were expressed in HEK293T cells and TRPV6 activity was analyzed using ratiometric Ca2+ measurements. RESULTS: Overall, we identified 10 novel (3 nonsense and 7 missenses) TRPV6 variants in CP patients. TRPV6 p.V239SfsX53 nonsense variant and the variants showing significant decrease in intracellular Ca2+ concentration in HEK293T cells (p.R174X, p.L576R, p.R342Q), were significantly overrepresented in Polish patients as compared to controls (6/152, 3.9% vs. 0/358, 0%; P = 0,0007). Nonsense TRPV6 variants predicted as loss of function (p.V239SfsX53 and p.R624X) were also significantly overrepresented in German patients (3/157; 2.0% vs 0/750; 0%, P = 0.005). CONCLUSIONS: We showed that TRPV6 loss of function variants are associated with elevated CP risk in early-onset Polish and German patients confirming that TRPV6 is a novel CP susceptibility gene.

Genetic Risk Factors in Early-Onset Nonalcoholic Chronic Pancreatitis: An Update.

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.

The genetic basis of classical galactosaemia in Polish patients.

Classic galactosemia (OMIM #230400) is an autosomal recessive disorder caused by homozygous or compound heterozygous pathogenic variants in the galactose-1-phosphate uridylyltransferase gene (GALT; 606999) on chromosome 9p13. Its diagnosis is established by detecting elevated erythrocyte galactose-1-phosphate concentration, reduced erythrocyte galactose-1-phosphate uridylyltransferase (GALT) enzyme activity. Biallelic pathogenic variants in the GALT gene is confirmed by DNA analysis. Our paper presents molecular characteristics of 195 Polish patients diagnosed with galactosemia I, intending to expand the current knowledge of this rare disease's molecular etiology. To the best of our knowledge, the described cohort of galactosemia patients is the largest single-center cohort presented so far.

The hybrid allele 1 of carboxyl-ester lipase (CEL-HYB1) in Polish pediatric patients with chronic pancreatitis.

OBJECTIVES: It has previously been reported in a European case-control study with patients from Germany and France that CEL-HYB1, a hybrid allele of the carboxyl ester lipase (CEL) gene and its pseudogene CELP, increases susceptibility to chronic pancreatitis (CP). Here, we aimed to replicate this finding in Polish pediatric patients with CP. METHOD: The distribution of the CEL-HYB1 allele in a CP pediatric cohort (n = 147, median age at CP onset 7.6 years) with no history of alcohol/smoking abuse was compared with ethnically matched healthy controls (n = 500, median age 46 years). Screening was performed using long-range PCR followed by agarose gel-electrophoresis. RESULTS: We observed no significant difference in the carrier frequency of the CEL-HYB1 allele between CP patients (7/147, 4.8%) and controls (12/500, 2.4%; P = 0.16). CONCLUSIONS: This study found no statistically significant association between CEL-HYB1 and chronic pancreatitis in a cohort of Polish pediatric CP patients.

Novel Pathogenic PRSS1 Variant p.Glu190Lys in a Case of Chronic Pancreatitis.

Mutations in the PRSS1 (serine protease 1) gene encoding human cationic trypsinogen cause hereditary pancreatitis or may be associated with sporadic chronic pancreatitis. The mutations exert their pathogenic effect either by increasing intra-pancreatic trypsinogen activation (trypsin pathway) or by causing proenzyme misfolding and endoplasmic reticulum stress (misfolding pathway). Here we report a novel heterozygous c.568G>A (p.Glu190Lys) variant identified in a case with chronic pancreatitis. The parents of the index patient had no history of pancreatitis but were unavailable for genetic testing. Functional characterization revealed 2.5-fold increased autoactivation of the mutant trypsinogen relative to wild type. Unlike many other clinically relevant PRSS1 mutations, p.Glu190Lys did not alter the chymotrypsin C (CTRC)-dependent degradation of trypsinogen nor did it increase CTRC-mediated processing of the trypsinogen activation peptide. Cellular secretion of the mutant protein was unchanged indicating normal folding behavior. Based on the genetic and functional evidence, we classify the p.Glu190Lys PRSS1 variant as likely pathogenic, which stimulates autoactivation of cationic trypsinogen independently of CTRC.

Chymotrypsinogen C Genetic Variants, Including c.180TT, Are Strongly Associated With Chronic Pancreatitis in Pediatric Patients.

OBJECTIVES: Genetic studies in adults/adolescent patients with chronic pancreatitis (CP) identified chymotrypsinogen C (CTRC) genetic variants but their association with CP risk has been difficult to replicate. To evaluate the risk of CP associated with CTRC variants in CP pediatric patients-control study. METHODS: The distribution of CTRC variants in CP pediatric cohort (n = 136, median age at CP onset 8 years) with no history of alcohol/smoking abuse was compared with controls (n = 401, median age 45). RESULTS: We showed that p.Arg254Trp (4.6%) and p.Lys247_Arg254del (5.3%) heterozygous mutations are frequent and significantly associated with CP risk in pediatric patients (odds ratio [OR] = 19.1; 95% CI 2.8-160; P = 0.001 and OR = 5.5; 95% CI 1.6-19.4; P = 0.001, respectively). For the first time, we demonstrated that the c.180TT genotype of common p.Gly60Gly variant is strong, an independent CP risk factor (OR = 23; 95% CI 7.7-70; P < 0.001) with effect size comparable to p.Arg254Trp mutation. The other novel observation is that common c.493+51C>A variant, both CA and AA genotype, is significantly underrepresented in CP compared with controls (15% vs 35%; OR = 0.33; 95% CI 0.19-0.59; P < 0.001 and 2.8% vs 11%; OR = 0.24; 95% CI 0.06-0.85; P = 0.027, respectively). CONCLUSIONS: Our study provides evidence that CTRC variants, including c.180TT (p.Gly60Gly) are strong CP risk factors. The c.493+51C>A variant may play a protective role against CP development.

The Etiology and Clinical Course of Chronic Pancreatitis in Children With Early Onset of the Disease.

OBJECTIVES: The etiological factors of chronic pancreatitis (CP) in children differ from those in adults. To date, no study has assessed the clinical course of CP in young children. The aim of our study was to evaluate the etiology and the clinical presentation of the disease in children with disease onset before 5 years of age in comparison to later-onset of CP. METHODS: A total of 276 children with CP, hospitalized from 1988 to 2015, were enrolled in the study. Data on presentation, diagnostic findings, and treatment were reviewed. Two hundred sixty patients were screened for the most frequent mutations in major pancreatitis-associated genes, such as cationic trypsinogen/serine protease gene (PRSS1), serine protease inhibitor, Kazal type 1 gene (SPINK1), and cystic fibrosis transmembrane conductance regulator gene (CFTR). RESULTS: The disease onset before the age of 5 years occurred in 51 patients (group 1), the later onset in 225 patients (group 2). We found no significant discrepancies in distribution of the etiological factors between groups. The youngest patients (group 1) had more pancreatitis episodes (median 5.0 vs 3.00; P < 0.05) and underwent surgeries more frequently (25.5% vs 8.9%; P < 0.05). It could be associated with significantly longer follow-up in early onset group (median 6 vs 4 years; P < 0.05). There were no differences in nutritional status or exocrine and endocrine pancreatic function. CONCLUSIONS: Early- and later-onset pancreatitis have similar etiological factors with predominance of gene mutations. The most frequent mutation found was p.Asn34Ser (N34S) in SPINK1 gene. The clinical presentation differed in number of pancreatitis episodes and frequency of surgeries.

The clinical course of hereditary pancreatitis in children - A comprehensive analysis of 41 cases.

BACKGROUND: Available data from adult patients do not reflect natural course of hereditary pancreatitis (HP) in children. To date, no study has assessed the clinical course of HP in children. OBJECTIVE: To investigate the clinical course of HP in children and compare it to non-HP group with chronic pancreatitis (CP). METHODS: A group of 265 children with CP, hospitalized from 1988 to 2014, were enrolled in the study. Medical records of those patients were reviewed for data on presentation, diagnostic findings and treatment. All children were screened for mutations in major pancreatitis-associated genes, i.e. PRSS1, SPINK1, and CFTR. RESULTS: HP was diagnosed in 41 children (15.5%). Family history was positive in 88% of children with HP. Mutations of PRSS1 gene were found in 80% (33/41) of HP patients. We detected p.R122H, p.R122C, p.N29I, and p.E79K mutation in 34% (14/41), 27% (11/41), 12% (5/41), and 7% (3/41) of HP patients, respectively. Patients with paternal inheritance had first symptoms earlier than those with maternal inheritance (5.9 vs. 9.1 years; P < 0.05). Children with HP showed more severe changes in ERCP then those from non-HP group (2.05 Cambridge grade, vs. 1.6°; P < 0.05). ESWL was performed more frequently in HP group (12.2% vs. 3.1%; P < 0.05). There was no difference in age of disease onset (7.98 vs. 8.9 years; NS), pancreatic duct stenting (46.3% vs. 33%; NS), or number of surgical interventions (12.2% vs. 14.3%; NS) between both groups. CONCLUSIONS: Children with HP reveal significantly more severe clinical presentation of the disease than non-HP patients, despite the same age of onset.

Gene conversion between cationic trypsinogen (PRSS1) and the pseudogene trypsinogen 6 (PRSS3P2) in patients with chronic pancreatitis.

Mutations of the human cationic trypsinogen gene (PRSS1) are frequently found in association with hereditary pancreatitis. The most frequent variants p.N29I and p.R122H are recognized as disease-causing mutations. Three pseudogene paralogs in the human trypsinogen family, including trypsinogen 6 (PRSS3P2), carry sequence variations in exon 3 that mimic the p.R122H mutation. In routine genetic testing of patients with chronic pancreatitis, we identified in two unrelated individuals similar gene conversion events of 24-71 nucleotides length between exon 3 of the PRSS1 (acceptor) and PRSS3P2 (donor) genes. The converted allele resulted in three nonsynonymous alterations c.343T>A (p.S115T), c.347G>C (p.R116P), and c.365_366delinsAT (p.R122H). Functional analysis of the conversion triple mutant revealed markedly increased autoactivation resulting in high and sustained trypsin activity in the presence of chymotrypsin C. This activation phenotype was identical to that of the p.R122H mutant. In addition, cellular secretion of the triple mutant from transfected HEK 293T cells was increased about twofold and this effect was attributable to mutation p.R116P. Our observations confirm and extend the notion that recombination events between members of the trypsinogen family can generate high-risk PRSS1 alleles. The pathogenic phenotype of the novel conversion is explained by a unique combination of increased trypsinogen activation and secretion.