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).
Background
21‐hydroxylase deficiency (21OHD) is an autosomal recessive disorder with an incidence of 1:10,000‐1:20,000 and is the result of various mutations in the CYP21A2 gene. 21OHD has been ...described in many different populations, but it has not been studied in Roma individuals so far. The aim of the study was to analyse the genotype in Roma patients with 21OHD and the prevalence of the disease in the Roma population of North Macedonia.
Methods
Molecular analysis of the nine most frequent CYP21A2 mutations in all known Roma patients with CAH in North Macedonia, relatives and healthy individuals of Roma ancestry, using the PCR/ACRS method.
Results
Ten Roma patients with 21OHD were identified, of which nine had the salt‐wasting and one had the simple virilizing form. Calculated incidence of 21OHD in the North Macedonian Roma population was 1:3375. Interestingly, 9/10 patients (90%) were homozygous for the In2G splicing mutation (293‐13A/C > G). Standard therapy with hydrocortisone and fludrocortisone had been introduced according to the guidelines. In 16 healthy relatives investigated for CYP21A2 mutations, heterozygosity for the In2G mutation was detected in 13/32 (40.6%) alleles. In 100 healthy Roma individuals, none related to the analysed families, no CYP21A2 mutations were detected.
Conclusion
The Roma population in North Macedonia had a very high incidence of classic 21OHD. Almost all patients had the severe salt‐wasting form and the In2G/In2G genotype.
Despite numerous studies in the field of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, some clinical variability of the presentation and discrepancies in the ...genotype/phenotype correlation are still unexplained. Some, but not all, discordant phenotypes caused by mutations with known enzyme activity have been explained by in silico structural changes in the 21-hydroxylase protein. The incidence of P30L mutation varies in different populations and is most frequently found in several Central and Southeast European countries as well as Mexico. Patients carrying P30L mutation present predominantly as non-classical CAH; however, simple virilizing forms are found in up to 50% of patients. Taking into consideration the residual 21-hydroxulase activity present with P30L mutation this is unexpected. Different mechanisms for increased androgenization in patients carrying P30L mutation have been proposed including influence of different residues, accompanying promotor allele variability or mutations, and individual androgene sensitivity. Early diagnosis of patients who would present with SV is important in order to improve outcome. Outcome studies of CAH have confirmed the uniqueness of this mutation such as difficulties in phenotype classification, different fertility, growth, and psychologic issues in comparison with other genotypes. Additional studies of P30L mutation are warranted.
Intron 2 Splice Mutation at CYP21 Gene in Patients with Congenital Adrenal Hyperplasia in the Republic of Macedonia Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder. In 90-95% ...of cases it results from mutations in the gene for 21-hydroxylase (CYP21, also termed CYP21A2 and P450c21). The IVS-II-656 (C/A>G) mutation leaves ~2.0% enzyme activity, and comprises 25% of the classic CYP21 deficiency alleles and 51% of alleles in the salt-wasting form. We performed direct molecular diagnosis of the IVS-II mutation in 41 Macedonian patients with different clinical forms of CAH and 55 of their healthy parents and siblings from 37 unrelated families, using the differential polymerase chain reaction/amplification created restriction site method (PCR/ACRS). The IVS-II mutation was detected in 41.5% patients (29.3% were homozygotes and 12.2% were heterozygotes). All homozygotes had a severe classical CAH phenotype (of which 91.7% were salt-wasting and 8.3% were simple virilizing). Three of the heterozygotes had a salt-wasting (SW) phenotype and were compound heterozygotes. The IVS-II mutation was also found in 30.9% of the family members (18.2% were homozygous and 12.7% were heterozygous) and none had any clinical manifestation. The frequency of the IVS-II mutation (41.5%) in these subjects was similar to that reported elsewhere.
Nonclassical congenital adrenal hyperplasia (NCAH) is an autosomal recessive imbalance in cortisol synthesis with adrenal androgen excess. Although rarely recognized in infants, it may cause ...premature adrenarche and pubarche, virilization in young women and variable symptoms in young men. It is commonly caused by mutations in CYP21A2, the gene for steroid 21-hydroxylase. Patients with the p.P30L allele tend to have pronounced evidence of androgen excess but are categorized as nonclassical. We used direct molecular detection of the p.P30L mutation in CYP21A2 in 11 Macedonian NCAH patients and in 17 members of their families using polymerase chain reaction/amplification created restriction site (PCR/ACRS) analysis and digestion with restriction enzymes. The p.P30L mutation was found in a homozygous state in seven (63.6%) and in a heterozygous state in four (36.4%) patients. Of the latter, one was also heterozygous for the p.Q318X mutation. The p.P30L mutation was found in a heterozygous state in 10 (58.8%) and in a homozygous state in one (5.9%) of the family members. These findings support a role of the p.P30L mutation in NCAH.
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