Mutagenic efect of paternal advanced age

Summary

Increased frequency of chromosomal aberrations in children of mothers aged 35 years and older is very well known and since 1973 is an indication to investigate the fetal karyotype in cells obtained by investive method ( amniocentesis), because the genetic risk of severe affection in higher than the risk of necessary invasive method for cytogenetic investigation. Mutagenic effect of advanced paternal age is known only among geneticists (1,2,3,4) The reason is not only low absolute risk of new mutation but particularly a high number of involved genes and last but not least the limited spectrum of prenatal detection of autosomal dominant disorders by molecular genetics. Therefore the preventive methods for elimination of this risk are very limited. Only a few of them could be recognized prenatally by noninvasive methods of prenatal diagnostics, such as skeletal dysplasias, acrocephaly, limb defects

We have studied genealogical, anamnestic and clinical data of 83 patients with clinical suspection on neurocardiofaciocutaneous syndrome (NCFCs) (5,6,7). The diagnosis has not been confirmed in 29 patients, no mutation was detected in 10 investigated genes (PTPN11,SOS1, HRAS, BRAF, RAF1, MEK1, MEK 2, KRAS, NRAS, SHOC2 ). In 54 patients with autosomal dominant inherited Noonan syndrome, LEOPARD association, Costello syndrome and cardiofaciocutaneous syndrome the diagnosis was confirmed on DNA level and the biological fitness estimated in this disorder. Paternal age at conception was compared in the group of patients with familial and sporadic occurrence of Noonan and NCFC syndromes.

The clinical prognosis of this disorder is represented by biological fitness of patients. Coeficient of selection is 0,604 in Noonan and LEOPARD syndromes (29 from 48) All 6 patients with Costello and cardiofaciocutaneous syndromes are due to new mutation ( selection 1). Clinical prognosis due to severe congenital deart defects, failure to thrive in infancy, increased risk for malignancy event. another health disturbance was problematic.

We have studied paternal age at birth of child in 54 patients with autosomal dominant Neurocardiofaciocutaneous syndrome ( Noonan, LEOPARD, Costello, CFC ) with high population incidence and decreased biological fitness. High mutation rate is therefore expected. Identification of genes responsible for manifestation of this disorder enables to confirm the diagnosis and to recognize inherited and de novo mutation. Genealogy and DNA analysis of PTPN11, SOS1, HRAS, BRAF, RAF1, MEK1, MEK2, KRAS and NRAS, SHOC2 were obtained in cohort of 54 patients with NCFC syndromes and their parents. There were 48 patients with Noonan and LEOPARD syndromes. In 29 cases occurred due to mutation de novo, 19 patients inherited the mutation from one of parents ( biological fitness 0,395). All 6 patients with Costello syndrome and CFC syndrome were affected due to new mutation. DNA analysis revealed 32 mutation in PTPN11 gene, mutation in SOS1 gene was found in 10 patients, RAF1 mutation was present in 3 patients, mutation in MEK1, KRAS and NRAS genes was present in one patient each. In Costello syndrome and CFC syndrome mutation in HRAS (4 patients) and BRAF ( 2 patients) genes were detected. Genealogic data allow to analyse the parental age in group of patients with new mutation and inherited mutation. Parental age at conception of patients with Noonan syndrome due to new mutation was significantly increased in comparison to the group of fathers of Noonan patients with inherited mutation.- 38,4 years and 29,6 years resp. range 28 to 55 and 25 to 35 years resp. Maternal age was mildly increased too, 30,0 and 27,7 resp. range 24 to 42 years and 21 to 36 years resp. – but not significantly. The results support the mutagenic effect of paternal age,espec. mutation with autosomal dominant character.

Key words:
advanced parental age, mutagenic effect, chromosomal mutations, gene mutations, manifestation of new mutation, coeficient of selection, mutation rate.

Introduction

Mutations can affect whole genome ( polyploidy), individual chromosomes ( numeric aberration, aneuploidy or structural aberration as deletion, duplication, translocation, inversion, ring chromosomes) or individual genes ( gene mutation of dominant or recessive character located on autosomes or gonosomes).  Mutation can occurs in gamets ( germinal mutation transmited through generations) or in somatic cells ( somatic mutation), which resulted in mosaics with at least 2 different cell lines in the same individual. Early postzygotic mosaics disturbe ontogenesis and are manifested by malformation. Late postzygotic mosaics are manifested as tumors, or can occurr only  in cells of  gonads ( gonadal mosaics). The majority of mutation occurr in „wild“ form and result in pathogenic mutation, rare are back mutation from pathogenic form to wild form ( uniparental disomy, isodisomy, back mutation in syndromes of chromosomal instability). Mutations occurr spontaneously or are induced due to mutagenic agents.  Mutagenic effect of advanced age of mothers on occurrence of chromosomal aberrations of their fetuses ( trisomy 21 in Down syndrome, trisomy 18 in Edwards syndrome, trisomy X in superfemale syndrome and Klinefelter syndrome) is very well known in population,  and is indication for prenatal diagnosis of fetal karyotype.

Advanced age  of mother involved the fetal chromosomal mutations and normal karyotype excludes this risk. Paternal advanced age ( also more than 35 years (1,2,3,4) involves the gene mutations and relative risk is at least ten times higher than prevalence in population, but it represents absolute risk  0.5-1%o. Low absolute risk and high number of genes which could be involved and limited possibility for gene mutation detection does not offer the effective prevention.

We studied mutagenic effect of advanced paternal age in cohort of patients with new mutation responsible for manifestation of autosomal dominant Noonan syndrome ( 5.6) , with high selective coeficient (0,60) and high population prevalence ( 1:2000). Selective coeficient is lower in LEOPARD syndrome and substationally higher in Costello and CFC syndromes ( 6,7).

Noonan syndrome were  recognized as entity 1963 (5) and characterized by facial dysmorphy (antimongoloid palpebral fissures, hypertelorism, ocular ptosis, epikantes, strabism, anomaly of auriculs), severe cardial anomalies ( valvular pulmonal stenosis, hypertrophic cardiomyopathy, septal defects, patent ductus arteriosus,  peripheral pulmonal anomalies), hypogenitalism, cryptorchism, chest anomaly ( pectus carinatum, pectus infundibulare) failure to thrive in infancy, later growth retardation.  The majority of patients have curly hair, pterygium coli and low hair line. Mental development is in 80% of patients normal. Speech development is delayed and voice horse.  The patients have macrocephaly, neurosensoric hearing loss, small mammilas, high palate, scapulatae alatae, cubiti valgi, kyfoscoliosis, supranumeric cervical ribs, edemas of hands and feet in infancy due to dysplastic lymphatic vessels, atypical dermatoglyphic patterns. Clinical  prognosis  was not good, because only 40- 50% of patients have had offsprings. The mortality due to cardiac malformation, failure to thrive, infections abd malignancies was high. The chance to find a partner was  worse because of inatractive face, small stature, hypogenitalism, hearing and mental defect contributed to the genetic letality (6) The selection is according of Hardy-Weinberg law compensated by fresh mutation, mutation rate is high.  The neurocardiofaciocutaneous syndromes show  genetic as well as molecular heterogenity. At present time are identified 9 genes ( 6,7,8).

Material

In 1999 was identified PTPN11 gen, which is responsible for manifestation of Noonan phenotype. The possibility of confirmation of diagnosis and detection of new or inherited mutation by DNA analysis of parents was open. We have studied the cohort of 83 patients with clinical diagnosis of Noonan syndrome, LEOPARD syndrome, Costello and CFC syndromes (prenatal and perinatal  anamnesis, professional anamnesis of parents, their exposition to the mutages and teratogenes).  In all families we obtained genealogy for at least 4 generations and with agreement the EDTA blood was obtained from patient, his parents and siblings.  DNA probes were sent within  interuniversity cooperation to Institute of Human Genetics in Magdeburg because 10 years ago was the DNA diagnosis  of Noonan syndrome not available in Czech Republic. During 10 years were identified further genes, responsible for Noonan syndrome spectrum – RAF1, MEK1, MEK 2, HRAS, NRAS, KRAS, BRAF, SOS1 RIT1.  which were  analysed in cohort of our patients.

Metod

Genomic DNA was extrahed from EDTA blood samples. In all DNA probes were sequenced 9 genes as described in papers 9, 10. Following genes were investigated PTPN11 located on 12q34.13, SOS1 on 2p22, RAF1 on 3p25, KRAS on 12p12.1, NRAS on 1p13.2, BRAF on 7q34, HRAS on 11p15.5 and MEK1 ( MAP2K1) on 15q21. Gene RIT1 was idetified only in 2013 and the investigation was not yet made.

Results

We have obtained genealogical, anamnestic and clinical  data and DNA probes from 83 patients with clinical diagnosis Noonan syndrome. Mutation of one of investigated genes was confirmed in 54 patients , 65%.  The frequency of individual gene with mutation is given in Tab. 1

Tab 1. Frequency of mutated genes in patients with confirmed diagnosis NCFC syndromes

DNA analysis in the families with confirmed diagnosis we have identified 19 inherited mutation ( 39,5%) and in 35 patients was the mutation de novo ( 60,5%).  No contact with mutagenes was given in 57 children with NCFC. In 26 adult patients were the se data unavailable.  We have analysed the parental age in group of inherited as well as new mutation. The parents  of patients with new mutation were older, mean age of fathers was 38,4 years and the mothers 30,9 years. The paternal age of patients with inherited NCFC syndromes was in  average 29,6 years and the maternal age was 27,1 years. The parental age of patients without confirmed NCFC diagnosis was 29,6 years ( fathers) and 24,5 years ( mothers). The difference was significant only in group of fathers of patients with de novo mutation. ( P = 0,01 according Student t test). Proportion  new mutations is untill now so high as 65% ( biological fitness 0,35) and show relative severe clinical prognosis in patients with Noonan, Costello and CFC syndromes.

Tab. 2. Mean age of parents at birth of patients with NCFC syndromes

Discussion

Fresh new gene mutations are often mentioned in children of older fathers with autosomal dominant diseases as neurofibromatosis of Recklinghausen, Marfan syndrome, Tuberous sclerosis or gonosomal recessive  Duchenne muscular dystrophy and Alport syndrome ( 1,2,3,4).  Feasibility of confirmation of diagnosis on DNA level   makes possible the study of   parental age of  neurocardiofaciocutaneous syndromes with high seletive coeficient and therefore high mutation rate ( 5,6, 7,8).  We have found that until now is the biologicaL fitness  0,35  in NCFC.  The phenotype of NCFC is characteristic as is shown in 65% of confirmed patients  with clinical suspection Noonan syndrome. Nevertheless  the Noonan syndrome is underdiagnosed in our country because according the population prevalence 1:2000 newborns is expected 50 patients born  yearly  and in our cohort ( in this period only we have had possibility to study the mutation in NCFC syndromes spectrum) only 3-5 patients were  born  in one year.

Increased   genetic risk of  poit mutation de novo  due to advanced paternal age is not preventable by medical methods. The effective prevention of new mutation is paternity  bevor 35 years of age.

Acknowledge

We wish to thank  the families of patients with Noonan syndrome for kind cooperation

References

in Czech version