Christos Shammas

Identification and characterization of a novel X-linked AVPR2 mutation causing partial nephrogenic diabetes insipidus: A case report and review of the literature

X-linked nephrogenic diabetes insipidus (NDI) is a rare disease characterized by a malfunctioning renal response to the antidiuretic hormone arginine vasopressin (AVP) due to mutations in the AVPR2 gene. A limited number of mutations in the AVPR2 gene resulting in partial phenotype have been described so far. In this mini-review the retrospective analysis of 13 known AVPR2 mutations that have been previously shown in vitro to partially abolish AVPR2 function is described, along with a novel mutation diagnosed in a kindred with partial NDI. In the present study, a 14 year old male and his 73 year old maternal grandfather were diagnosed with partial NDI based on the clinical phenotype, the water deprivation test and the inadequate response to 1-desamino-8-d-arginine vasopressin (DDAVP) administration. Sequencing analysis of the AVPR2 gene revealed the novel missense mutation p.N317S (g.1417A > G) in both patients. This mutation was re-created by site directed mutagenesis in an AVPR2 cDNA expression vector and was functionally characterized, in terms of arginine vasopressin (AVP) and DDAVP response. AVPR2 activity of the p.N317S mutant receptor after the AVP and DDAVP administration, as assessed by cAMP production was reduced and impaired when compared to cells that expressed the wild type AVPR2 gene. In conclusion, the affected members of this family have X-linked NDI with partial resistance to AVP, due to a missense mutation in the AVPR2 gene.

Molecular Defects of the CYP21A2 Gene in Greek-Cypriot Patients with Congenital Adrenal Hyperplasia

Background/Aim: To determine the mutations in the CYP21A2 gene in Greek-Cypriots with congenital adrenal hyperplasia (CAH) and attempt a genotype-phenotype correlation. Subjects and Methods: Molecular analysis was performed by multiplex ligation-dependent probe amplification and direct sequencing of PCR products of the CYP21A2 gene in 32 CAH patients. Results: The most frequent genetic defect in the classic salt-wasting and simple virilizing forms was the IVS2-13A/C>G (55%) mutation, followed by Large lesion (20%) and in the non-classical form, the p.V281L (79.5%). Genotypes were categorized in 4 mutation groups (null, A, B and C). All 3 patients in the null group manifested the salt-wasting form and all 6 patients in mutation group A presented with the classical form. One patient in group B had the simple virilizing form and 22 patients in group C exhibited the non-classical form. Conclusion: The spectrum of mutations of the CYP21A2 gene in our population is comparable to the most common reported in similar ethnic groups. The knowledge of the ethnic specificity of the CYP21A2 mutations represents a valuable diagnostic tool for all forms of CAH.

In silico analysis of a novel MKRN3 missense mutation in familial central precocious puberty

The onset of puberty is influenced by the interplay of stimulating and restraining factors, many of which have a genetic origin. Premature activation of the GnRH secretion in central precocious puberty (CPP) may arise either from gain‐of‐function mutations of the KISS1 and KISS1R genes or from loss‐of‐function manner mutations of the MKRN3 gene leading to MKRN3 deficiency.

Endocrine profile and phenotype-genotype correlation in unrelated patients with non-classical congenital adrenal hyperplasia.

OBJECTIVES The aim of this study was to identify the molecular defect in a group of 37 unrelated Greek Cypriot patients affected by NC-CAH and evaluate the relationship between the genotype, phenotype and adrenal androgen levels. DESIGN AND METHODS Clinical evaluation, biochemical analysis of 17-OHP, Testosterone, Androstenedione, DHEA-S, direct DNA sequencing and MLPA analyses. RESULTS Eleven known mutations were identified with the p.V281L being the most predominant and observed in 68.9% of the alleles. There was no difference between the two genotypes (mild/mild and mild/severe) with clinical presentation, whereas a proportional relationship between the type of mutation and adrenal androgen levels was found. CONCLUSION The frequency of the underlying genetic defect in our patients with NC-CAH is similar to that observed in most Mediterranean populations. Although the genotype cannot solely explain the clinical expression of NC-CAH, discrimination between mild and severe alleles is crucial in antenatal diagnosis and genetic counselling.

Overview of genetic defects in endocrinopathies in the island of Cyprus; evidence of a founder effect.

AIM Hereditary endocrinopathies in Cyprus exhibit evidence of a founder effect and display the influence of past migration patterns. The genetic frequency and mutation pattern of a specific disorder of sex development (DSD), which is classified as 46,XX DSD or 46,XY DSD, and the non-classic form of congenital adrenal hyperplasia (NC-CAH) outline a type of genetic drift. RESULTS Not only the high prevalence of the NC-CAH p.V281L mutation but also the rarity of CAH large lesions present a genetic diversity similar to that observed in the Middle Eastern countries. In addition, both the high frequency of the 5-alpha steroid reductase deficiency (5αSRD) IVS1-2A>G mutation and the carrier frequency of the 17-beta hydroxysteroid dehydrogenase 3 (17β-HSD-3) p.R80Q mutation are good examples of a founder effect. p.R80Q can be considered a founder mutation, even though it has been identified in patients of Dutch, Brazilian, and Portuguese origin. This has led to the speculation that it has a Phoenician origin. Phoenicians as ancient traders migrated around 750 BC from present day Syria, Lebanon, and Israel toward Portugal, Spain, and also to nearby Cyprus. While the 5αSRD IVS1-2A>G mutation has already been extensively reported in Turkish patients, it is very common in the Eastern Mediterranean region. CONCLUSION This short article portrays clearly, through specific endocrine genetic disorders, the past migration trends in Cyprus that shaped the present-day gene pool of the Greek-Cypriot population.

Familial Mediterranean fever associated with MEFV mutations in a large cohort of Cypriot patients.

Familial Mediterranean fever (FMF) is caused by mutations in the MEFV gene and the spectrum of mutations among Greek–Cypriots with FMF‐related symptoms was examined. Sequence analysis for exons 2, 3, 5, and 10 of the MEFV gene was performed in a cohort of 593 patients. A total of 70 patients carried mutations in the homozygote or compound heterozygote state, 128 were identified with one MEFV mutation and 395 had no mutations. Of the 268 identified alleles, p.Val726Ala (27.61%) was the most frequent followed by p.Met694Val (19.40%). The missense mutations p.Arg761His (3.73%) and p.Ala744Ser (2.24%) were identified as the rarest. An interesting finding is the high frequency (18.28%) of the complex p.Phe479Leu–p.Glu167Asp that was identified in 49 of the mutated alleles. The MEFV genotypes did not follow a binomial distribution and proved not to satisfy the HWE (P < 0.001). The high percentage (66.61%) of patients with unidentified mutations could be due to mutations in the rest of the coding or noncoding MEFV gene or due to mutations in other genes that are also causing Hereditary Recurrent Fevers. Results from this work indicate the high incidence of FMF in Cyprus and describe the spectrum of the mutations which occur in the country.

Epidemiology of type 1 diabetes mellitus in Cyprus: rising incidence at the dawn of the 21st century.

OBJECTIVEThe incidence of Type 1 diabetes mellitus (T1DM) in Greek-Cypriot children aged less than 15 years between 1990 and 2009 was examined along with gender differences concerning the age of onset and the seasonal variation at manifestation of the disease.DESIGNAll newly diagnosed cases of T1DM in children less than 15 years old were registered with the capture-recapture method from 1990 until 2009.RESULTSThe overall mean annual incidence during these 20 years is 12.46 per 100,000. A comparison of the incidence between the two decades (1990– 1999 vs 2000–2009) indicated a rising trend, from 10.80 per 100,000 person-years during the first decade to 14.44 per 100,000 person-years during the second decade. There was an overall male predominance (M/F: 1.05), which is in agreement with the male predominance in the population less than 15 years of age, except for the group who manifested T1DM at ages 10–15 years where females prevail. The percentage of children who developed T1DM at ages 0–5 years in the total T1DM population increased in the second decade (26.4% vs 19.0%), and significantly more children were diagnosed during the cold months as opposed to the warm months (p<0.001).CONCLUSIONThe incidence of T1DM in Cyprus is rising. The identification of causative environmental factors will theoretically explain this phenomenon and new preventive strategies can therefore potentially be developed.

Duplication of exons 3–10 of the HSD17B3 gene: A novel type of genetic defect underlying 17β-HSD-3 deficiency

The clinical, biochemical and genetic features of a Cypriot origin male of non-consanguineous parents due to 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD-3) deficiency are presented. The patient, currently a 10 old male, was referred to our clinic because of ambiguous genitalia at birth. Gonads were palpable in the inguinal canal bilaterally and no Müllerian structures identified on pelvic ultrasound. Chromosomal analysis showed an apparently normal male 46,XY karyotype. Diagnosis of 17β-HSD-3 deficiency in the newborn was suspected based on biochemical findings, following human chorionic gonadotrophin (hCG) stimulation test. Sequence analysis and real time PCR along with MLPA identified the patient with a novel 11.96 kb duplication that spans exons 3-10 of the HSD17B3 gene and extends from intron 2 to intron 10 in compound heterozygosity with the known p.R80Q missense mutation leading to 17β-HSD-3. In conclusion, 17β-HSD-3 deficiency was diagnosed in this patient based on endocrinologic evaluation and confirmed with genetic analysis of the HSD17B3 gene. The novel large duplication spanning exons 3-10 of the HSD17B3 gene that we report here in compound heterozygosity with the known p.R80Q leads to 17β-HSD-3 deficiency presenting as 46,XY Disorder of Sex Development. Following diagnosis and appropriate genetic counselling, the patient was raised a boy and successfully underwent surgical correction of crytptorchidism and hypospadias.

Tackling the pathogenesis of RNA nuclear retention in myotonic dystrophy.

DM1 (myotonic dystrophy type I) is a common form of muscular dystrophy that affects mainly adults. It is a disease that belongs to the group of defective RNA export diseases, since a major part of the pathogenic mechanism of the disease is the retention of the mutant transcripts in the cell nucleus. The presence of an expanded CUG trinucleotide repeat in the 3′‐UTR (3′‐untranslated region) of the DMPK (myotonic dystrophy protein kinase) gene causes the attraction of RNA‐binding proteins by the nuclear‐located mutant transcripts. As a result of the occupation of the RNA‐binding proteins, there is defective mis‐splicing of several cellular transcripts. This is believed to be a major pathogenic mechanism of the disease and any attempt to repair the activities of the RNA‐binding proteins or target the mutant transcripts should be beneficial for the patients. Certain approaches have been described in the literature and they demonstrate progress in various directions. The purpose of the present review is to summarize the successful attempts to tackle the pathogenesis caused by nuclear retention of mutant transcripts in myotonic dystrophy and to discuss the possible gains from such approaches.

Evidence of digenic inheritance in autoinflammation-associated genes

Familial Mediterranean fever (FMF) has traditionally been considered as a monogenic autosomal recessive disorder caused by mutations in the MEFV gene with highest incidence among Mediterranean populations. In a considerable number of patients with typical FMF, only one MEFV mutation was identified and the possibility that more than one autoinflammatory gene may be responsible for their disease was investigated. In the present study, an extensive search for possible mutations in three hereditary recurrent fever (HRF) genes was performed in 128 MEFV heterozygous Greek–Cypriots clinically diagnosed based on their phenotype with FMF-like disease from a previous study. Sequence analysis was performed for MVK, TNFRSF1A and NLRP3 genes which is also known to cause HRFs. In total, three patients were identified with heterozygous mutations and a second mutation in an autoinflammatory gene. Two patients carried a MEFV mutation and a NLRP3 mutation, and an additional third carried a MEFV mutation and a TNFRSF1A mutation. Patient 1 carried MEFV p.[Val726Ala] (NM_000243.2:c.2177T >C) and NLRP3 p.[Val198Met] (NM_001243133.1:c.592G >A) variants and patient 2 carried MEFV p.[Glu148Gln] (NM_000243.2:c.442G >C) variant which is of uncertain significance and NLRP3 p.[Arg176Trp] (NM_001243133.1:c.526C >T). Lastly, patient 3 was identified to carry MEFV p.[Met694Val] (NM_000243.2:c.2080A >G) and TNFRSF1A p.[Arg121Gln] (NM_001065.3:c.362G >A) variants. The results from this study indicate that screening of genes known to cause HRFs in patients already identified with a single MEFV mutation, can reveal quite rare but potentially causative mutational combinations at different loci. Such interaction provide further evidence for possible locus–locus interactions and phenotypes resulting from digenic inheritance.