Does anonymous sperm donation increase the risk for unions between relatives and the incidence of autosomal recessive diseases due to consanguinity?

In France gamete donation and notably sperm donation are anonymous. It has been claimed that anonymous artificial insemination by donor (AID) could highly contribute to an increase in the level of consanguinity and the incidence of autosomal recessive diseases, due to the unions between offspring of anonymous donors, unaware of their biological kinship, with the special case of unions between half-siblings. The actual incidence of consanguinity due to AID was compared with that resulting from the two other main sources of consanguinity and recessive diseases, i.e. voluntary unions between related individuals or inadvertent unions between the offspring of a common unknown male ancestor (false paternity). From these data, we estimated that expected unions in France between half sibs per year are 0.12 between offspring of sperm donors (1.2 every 10 years) and 0.5 between offspring of common male ancestors through false paternity (5 every 10 years). More generally, the inadvertent unions between false paternity offspring are roughly four times more frequent than those resulting from anonymous AID. We estimated that in the future, when AID has been in practice for several generations, out the 820 000 annual births in France, respectively, 6 and 25 births will be consanguineous through an unknown common ancestor related to anonymous AID and to a false paternity, both of which are negligible when compared with the 1256 children born from first-degree cousins. About 672 children per year are born with a recessive genetic disease due to the panmictic risk and additional affected cases due to consanguinity would be 34.54 for first-cousin offspring, 0.33 for offspring of individuals related due to false paternity and 0.079 for offspring of individuals related due to anonymous AID. Anonymous AID would therefore be responsible for 0.46% of consanguineous births and for 0.01% of recessive diseases. Therefore, the effect of anonymous AID on half-sibling unions, consanguinity and recessive disease incidence can be regarded as marginal.

A whole-genome scan in a large family with leukodystrophy and oligodontia reveals linkage to 10q22.

Dentoleukoencephalopathies with autosomal recessive inheritance are very rare. Recently, a large inbred Syrian pedigree was reported with oligodontia in association with a degenerative neurologic condition characterized by progressive ataxia and pyramidal syndrome and abnormalities in the white matter and cortical atrophy. A whole-genome screening of this family using 382 microsatellite markers was completed, but no evidence was found of linkage to any chromosomal region. A genome-wide linkage analysis using the 260K single nucleotide polymorphism Affymetrix array was then undertaken and a maximum multipoint logarithm of the odds score of 5.66 (NPL score = 7.65) was detected on chromosome 10q22 region. This genomic interval contains 95 known genes including the Prosaposin gene (PSAP) responsible for metachromatic leukodystrophy, which was excluded. Seventeen additional candidate genes were tested and excluded. Sequencing of the whole candidate locus is in progress and should allow the identification of the causative gene in this rare disease, thereby improving the understanding of the physiopathology of this disease.

Mild forms of hypophosphatasia mostly result from dominant negative effect of severe alleles or from compound heterozygosity for severe and moderate alleles.

BACKGROUND: Mild hypophosphatasia (HPP) phenotype may result from ALPL gene mutations exhibiting residual alkaline phosphatase activity or from severe heterozygous mutations exhibiting a dominant negative effect. In order to determine the cause of our failure to detect a second mutation by sequencing in patients with mild HPP and carrying on a single heterozygous mutation, we tested the possible dominant effect of 35 mutations carried by these patients. METHODS: We tested the mutations by site-directed mutagenesis. We also genotyped 8 exonic and intronic ALPL gene polymorphisms in the patients and in a control group in order to detect the possible existence of a recurrent intronic mild mutation. RESULTS: We found that most of the tested mutations exhibit a dominant negative effect that may account for the mild HPP phenotype, and that for at least some of the patients, a second mutation in linkage disequilibrium with a particular haplotype could not be ruled out. CONCLUSION: Mild HPP results in part from compound heterozygosity for severe and moderate mutations, but also in a large part from heterozygous mutations with a dominant negative effect.

Mutations in TREM2 lead to pure early-onset dementia without bone cysts.

A genome-wide screen using 382 STR markers to localize and identify the gene implicated in early-onset dementia (EOD) without bone cysts in a Lebanese family with three affected subjects was conducted. A unique locus homozygous by descent at chromosome 6p21.2 locus was identified. Candidate genes were explored by fluorescent sequencing and the effect of the identified mutation was confirmed by qualitative and quantitative RT-PCR. The genetic analysis revealed a novel deletion, c.40+3delAGG, in the 5' consensus donor splice site in intron 1 of TREM2 gene which is known to be responsible for PLOSL (Polycystic Lipomembranous Osteodysplasia with Sclerosing Leukoencephalopathy) also designated as Nasu-Hakola disease. In silico analysis predicted a lower strength for the novel donor splice site. Qualitative RT-PCR revealed normal transcript while quantitative RT-PCR showed over twofold down-regulation of TREM2 transcripts. The expression profile of six genes SPP1, NEDD9, FSCN, BCL3, NFKBIA and CCL2 known as disrupted in TREM2-deficient samples was studied and showed same expression profile as TREM2-mutated samples except for CCL2 which was normally regulated. The significantly-reduced expression of TREM2 in our patients and the expression profiles of the six studied genes confirm a role for TREM2 in this distinct phenotype of EOD without bone cysts. To our knowledge, this is the first report of mutations in TREM2 causing a pure dementia.

Delayed transport of tissue-nonspecific alkaline phosphatase with missense mutations causing hypophosphatasia.

Hypophosphatasia is a rare genetic disease characterized by diminished bone and tooth mineralization due to deficient activity of tissue-nonspecific alkaline phosphatase (TNSALP). The disease is clinically heterogeneous due to different mutations in the TNSALP gene. In order to determine whether mutated TNSALP proteins may be sequestered, degraded, or subjected to delay in their transport to the cell membrane, we built a plasmid expressing a YFP-TNSALP fluorescent fusion protein allowing the observation of cellular localization in live cells by fluorescence confocal microscopy at different time points after transfection. We studied five mutants (c. 571G>A, c. 653T>C, c. 746G>T, c. 1363G>A and c. 1468A>T) exhibiting various levels of in vitro residual enzymatic activity. While the wild-type protein reached the membrane within the first 24h after transfection, the mutants reached the membrane with delays of 24, 48 or 72 h. For all of the tested mutations, accumulation of the mutated proteins, mainly in the Golgi apparatus, was observed. We concluded that reduced ALP activity of these TNSALP mutants results from structural disturbances and delay in membrane anchoring, and not from compromised catalytic activity.

[Various topics in population genetics]. / Quelques points de génétique des populations.

Through quite simple mathematic tools, population genetics affords quite simple views in biology or medicine as well as the opportunity to enlarge these views toward topics like epidemiology and public health. It is quite easy to show that every population owns, in its gene pool, a set of deleterious mutations that is maintained because it escapes the natural selection. It may also be pointed out the efficiency of consanguinity in exacerbating the frequency of genetic recessive disease, a newly arisen question in some subpopulotions in Europe. As the medicine is more and more efficient, the dysgenic effect of medicine, due to the transmission of deleterious mutations, may be estimated and we may conclude that it is not as high as some would like to exclaim. The selection has a special effect upon genes located on the X chromosome, especially for the one which is responsible for the Fabry disease.

Characterization of missense mutations and large deletions in the ALPL gene by sequencing and quantitative multiplex PCR of short fragments.

Hypophosphatasia is a rare inherited bone disorder characterized by defective bone and dental mineralization and deficiency of serum and liver/bone/kidney alkaline phosphatase activity. The disease is due to mutations in the alkaline phosphatase liver-type (ALPL) gene. Gross deletions or insertions have not previously been reported in this gene. We report here the characterization of nine novel ALPL gene mutations in a series of 8 patients affected by various forms of hypophosphatasia. The newly discovered mutations included five missense mutations (c.368C --> A, c.814C--> T, c.1196C--> T, c.1199C--> T, c.1283G--> C), two small deletions (c.797_802del, c.1044_1055del), and two large deletions. The large deletions were detected by quantitative multiplex polymerase chain reaction (PCR) of short fluorescent fragments (QMPSF). We conclude that QMPSF slightly reduces the proportion of undetected mutations in hypophosphatasia and improves genetic counselling in the affected families.

Familial Mediterranean fever (FMF) in Lebanon and Jordan: a population genetics study and report of three novel mutations.

Familial Mediterranean fever (FMF) is an autosomal recessive disease mostly frequent in Mediterranean populations. Over 50 mutations have been identified in the gene responsible for the disease, MEFV. The present study reports the frequencies of MEFV mutations in 558 Lebanese and 55 Jordanian FMF patients and points out the severity of the M694V frequently observed mutation among these patients. Three novel mutations, T177I, S108R and E474K were also identified in the Lebanese group. An excess of homozygotes and a deficit of heterozygotes were observed in both samples when compared to the expected number of observed genotypes under the Hardy-Weinberg hypothesis. Homozygotes for M694V and M694I were still in excess in the Lebanese group of patients, even after consanguinous homozygotes were removed, or population structure was considered. This excess is therefore neither due to consanguinity nor to subgroups in the Lebanese population, but rather to more remote consanguinity or to a selection bias favoring the census of these genotypes. The fact that FMF female patients were less censed than male patients may be due to the greater resistance of females to pain and to the possibility of confusing abdominal and gynecological pain. The phenotypic heterogeneity of the FMF could then originate both from genetic causes like allelic heterogeneity or modulating genes, and cultural background facing the physiological consequences of genotypes at risk.

Genome-wide inbreeding estimation within Lebanese communities using SNP arrays.

Consanguineous marriages have been widely practiced in several global communities with varying rates depending on religion, culture, and geography. In consanguineous marriages, parents pass to their children autozygous segments known as homozygous by descent segments. In this study, single-nucleotide polymorphisms were analyzed in 165 unrelated Lebanese people from Greek Orthodox, Maronite, Shiite and Sunni communities. Runs of homozygosity, total inbreeding levels, remote consanguinity, and population admixture and structure were estimated. The inbreeding coefficient value was estimated to be 1.61% in offspring of unrelated parents over three generations and 8.33% in offspring of first cousins. From these values, remote consanguinity values, resulting from genetic drift or recurrent consanguineous unions, were estimated in offspring of unrelated and first-cousin parents to be 0.61 and 1.2%, respectively. This remote consanguinity value suggests that for any unrelated marriages in Lebanon, the mates could be related as third cousins or as second cousins once removed. Under the assumption that 25% of marriages occur between first cousins, the mean inbreeding value of 2.3% may explain the increased incidence of recessive disease in offspring. Our analysis reveals a common ancestral population in the four Lebanese communities we studied.

Involvement of the modifier gene of a human Mendelian disorder in a negative selection process.

BACKGROUND: Identification of modifier genes and characterization of their effects represent major challenges in human genetics. SAA1 is one of the few modifiers identified in humans: this gene influences the risk of renal amyloidosis (RA) in patients with familial Mediterranean fever (FMF), a Mendelian autoinflammatory disorder associated with mutations in MEFV. Indeed, the SAA1 alpha homozygous genotype and the p.Met694Val homozygous genotype at the MEFV locus are two main risk factors for RA. METHODOLOGY/PRINCIPAL FINDINGS: HERE, WE INVESTIGATED ARMENIAN FMF PATIENTS AND CONTROLS FROM TWO NEIGHBORING COUNTRIES: Armenia, where RA is frequent (24%), and Karabakh, where RA is rare (2.5%). Sequencing of MEFV revealed similar frequencies of p.Met694Val homozygotes in the two groups of patients. However, a major deficit of SAA1 alpha homozygotes was found among Karabakhian patients (4%) as compared to Armenian patients (24%) (p = 5.10(-5)). Most importantly, we observed deviations from Hardy-Weinberg equilibrium (HWE) in the two groups of patients, and unexpectedly, in opposite directions, whereas, in the two control populations, genotype distributions at this locus were similar and complied with (HWE). CONCLUSIONS/SIGNIFICANCE: The excess of SAA1alpha homozygotes among Armenian patients could be explained by the recruitment of patients with severe phenotypes. In contrast, a population-based study revealed that the deficit of alpha/alpha among Karabakhian patients would result from a negative selection against carriers of this genotype. This study, which provides new insights into the role of SAA1 in the pathophysiology of FMF, represents the first example of deviations from HWE and selection involving the modifier gene of a Mendelian disorder.

Familial Mediterranean Fever in Lebanon: founder effects for different MEFV mutations.

Haplotype analysis of 376 Familial Mediterranean Fever (FMF) patients and 100 controls from Lebanon was performed using 4 microsatellite loci to study founder effects for the five most frequent mutations within the MEFV gene (M694V, M694I, V726A, M680I and E148Q). Each of these mutations was associated with a particular haplotype that was less frequent among controls, confirming that they have probably arisen from unique mutation events and that the carrier chromosomes derived from a common ancestor. The estimated ages of the most recent common ancestor for each of the 5 mutations, using the ESTIAGE program, were 7000, 8500, 15000, 23000 and 30000 years for M694V, M694I, V726A, M680I and E148Q, respectively. Varying the mutation rate at one of the markers led to younger age estimates, but the mutation E148Q remained the oldest one. Comparison of haplotype distributions among the different Lebanese religious groups confirmed that Muslim sub-populations (Shiites and Sunnites) as well as Christian ones, including Armenians who were formerly settled in the South-Eastern part of Asia Minor (Cilicia), are all derived from an ancient common ancestral population in which most of the MEFV mutations were already present with their respective associated haplotypes.

Hypophosphatasia: molecular testing of 19 prenatal cases and discussion about genetic counseling.

OBJECTIVE: We studied hypophosphatasia (HP) mutations in 19 cases prenatally detected by ultrasonography without familial history of HP. We correlated the mutations with the reported ultrasound signs, and discussed genetic counseling with regard to the particular dominantly inherited prenatal benign form of HP. METHOD: The coding sequence of the tissue nonspecific alkaline phosphatase (TNSALP) gene was analyzed by DNA sequencing, and 3D modeling was used to locate the mutated amino acids with regard to the functional domains of TNSALP. RESULTS: Although reported ultrasound signs were heterogeneous, two mutated alleles were found in 18 of the 19 cases studied, indicating recessive transmission of the disease. Functional domains of TNSALP were affected by 74% of missense mutations. In all the cases, including one with only a heterozygous mutation, molecular, biological, and familial data do not corroborate the hypothesis of prenatal benign HP. The mutation c.1133A>T observed in the prenatal benign form of HP and common in USA was not found in this series. CONCLUSION: The results point out the prenatally detectable allelic heterogeneity of HP. The nature of the detected mutations and the evidence of recessive inheritance do not support these cases being affected with prenatal benign HP.

Characterization of 11 novel mutations in the tissue non-specific alkaline phosphatase gene responsible for hypophosphatasia and genotype-phenotype correlations.

Hypophosphatasia is an inherited disorder caused by mutations in the bone alkaline phosphatase gene. We report here 11 new mutations responsible for hypophosphatasia. Four of them were deletions or insertions resulting in frameshift, two affected a donor splice site and five were missense mutations. Site-directed mutagenesis and transfection experiments of missense mutations showed that the mutations resulted in loss of most enzymatic activity, confirming the disease-causing role of these mutations. Analysis of the 3D model of tissue non-specific alkaline phosphatase showed that among the five missense mutations, one affected a residue in the crown domain and four affected residues located in the calcium-binding region. Alignment of the protein sequences of the calcium-binding region from 11 species showed that the four residues coordinating the calcium ion and the residues affected by the missense mutations described here are conserved in vertebrates. Together, our results confirm the functional role of the calcium site and suggest that its function is likely to be specific to vertebrates.