Julie Steffann

Segregation at three loci explains familial and population risk in Hirschsprung disease

Hirschsprung disease (HSCR), the most common hereditary cause of intestinal obstruction, shows considerable variation and complex inheritance. Coding sequence mutations in RET, GDNF, EDNRB, EDN3 and SOX10 lead to long-segment (L-HSCR) and syndromic HSCR but fail to explain the transmission of the much more common short-segment form (S-HSCR). We conducted a genome scan in families with S-HSCR and identified susceptibility loci at 3p21, 10q11 and 19q12 that seem to be necessary and sufficient to explain recurrence risk and population incidence. The gene at 10q11 is probably RET, supporting its crucial role in all forms of HSCR; however, coding sequence mutations are present in only 40% of linked families, suggesting the importance of noncoding variation. Here we show oligogenic inheritance of S-HSCR, the 3p21 and 19q12 loci as RET-dependent modifiers, and a parent-of-origin effect at RET. This study demonstrates by a complete genetic dissection why the inheritance pattern of S-HSCR is nonmendelian.

Prenatal diagnosis of myopathy, encephalopathy, lactic acidosis, and stroke-like syndrome: contribution to understanding mitochondrial DNA segregation during human embryofetal development

Introduction: Myopathy, encephalopathy, lactic acidosis, and stroke-like (MELAS) syndrome, a maternally inherited disorder that is among the most common mitochondrial DNA (mtDNA) diseases, is usually associated with the m.3242A>G mutation of the mitochondrial tRNAleu gene. Very few data are available with respect to prenatal diagnosis of this serious disease. The rate of mutant versus wild-type mtDNA (heteroplasmy) in fetal DNA is indeed considered to be a poor indicator of postnatal outcome. Materials and methods: Taking advantage of a novel semi-quantitative polymerase chain reaction test for m.3243A>G mutant load assessment, we carried out nine prenatal diagnoses in five unrelated women, using two different fetal tissues (chorionic villi v amniocytes) sampled at two or three different stages of pregnancy. Results: Two of the five women, although not carrying m.3243A>G in blood or extra-blood tissues, were, however, considered at risk for transmission of the mutation, as they were closely related to MELAS-affected individuals. The absence of 3243A>G in the blood of first degree relatives was associated with no mutated mtDNA in the cardiovascular system (CVS) or amniocytes, and their three children are healthy, with a follow-up of 3 months–3 years. Among the six fetuses from the three carrier women, three were shown to be homoplasmic (0% mutant load), the remaining three being heteroplasmic, with a mutant load ranging from 23% to 63%. The fetal mutant load was fairly stable at two or three different stages of pregnancy in CVS and amniocytes. Although pregnancy was terminated in the case of the fetus with a 63% mutant load, all other children are healthy with a follow-up of 3 months–6 years. Conclusion: These data suggest that a prenatal diagnosis for MELAS syndrome might be helpful for at-risk families.

Pre-implantation genetic diagnosis in pulmonary arterial hypertension due to BMPR2 mutation.

To the Editors: Pulmonary arterial hypertension (PAH) is a rare and severe condition that may present as familial/heritable disease [1–3]. In recent years, there have been considerable advances in the management of PAH and disease-specific therapies have improved survival rates [3–5]. Nevertheless, PAH remains a devastating disease with progressively debilitating symptoms and high mortality even in the modern management era [6, 7]. Familial cases of PAH have been detected since the 1950s [1, 2]. Before the availability of modern genetic tools, studies of the genealogies demonstrated that familial PAH segregated as an autosomal dominant trait, thus leading to a 50% chance of inheriting the disease allele. More recently, mutations in the bone morphogenetic protein receptor 2 gene ( BMPR2 ) have been detected in 70–80% of familial cases [1, 8, 9]. The BMPR2 mutations have an incomplete but variable penetrance (on average, 10–20% of mutation carriers develop PAH, but it can be much higher in some families). A genetic anticipation phenomenon (characterised by a younger age at PAH diagnosis in subsequent generations) has been demonstrated in familial PAH, with an increased risk of cases occurring in children and young adults. Recently, we have shown that BMPR2 mutation carriers with PAH …

Clinical Utility Gene Card for: incontinentia pigmenti

1.5 Mutational spectrum IP is a rare X-linked genodermatosis, characterized by typical skin alterations, the hallmarks of the disease, and, in addition, by other neuroectodermal defects affecting the eyes, the nails, the hair, the teeth, and the central nervous system (CNS). The clinical diagnosis of IP is based on the presence of dermatological lesions that develop in four successive, sometimes overlapping, characteristic stages that start shortly after birth with an inflammatory vesicular rash (stage1), followed by verrucous lesions (stage2). The third stage is marked by the appearance of a skin area displaying hyperpigmentation that at the fourth stage becomes patches of atrophic hypopigmented skin. In addition, IP females have heterogeneous and often severe clinical signs including ophthalmological (strabismus, cataracts, optic atrophy, retinal vascular pigmentary abnormalities, microphthalmia), odontological, (partial anodontia, delayed dentition, cone/peg-shaped teeth, impactions) and neurological defects (seizures, spastic paralysis, motor, and mental retardation, microcephaly).1 IP is a genomic disorder inherited as an X-linked dominant trait. IP is generally lethal in males while heterozygous females survive owing to functional mosaicism.1 All cases of IP are due to mutations in NEMO (nuclear-factor-kappa-B essential modulator)/IKBKG gene located in Xq28 region, and the mutation detection rate in IP is around 80%. IKBKG, encodes the regulatory subunit of the IkB kinase complex required for nuclear factor-kB (NF-kB) activation.2,3 Mutations in different domains of protein may produce different effects on NF-kB activation by reducing or abolishing the response after stimulations. Noteworthy, some IKBKG hypomorphic mutations, affecting the zinc finger (ZF) domain of the NEMO protein and reducing but do not eliminating NF-kB activation, were found in surviving male patients. These males are affected by a different disease, named hypohidrotic ectodermal dysplasia-associated with severe immunodeficiency (EDA-ID HED-ID OMIM#300291) or occasionally associated with osteopetrosis and lymphoedema (OL-EDA-ID).4,5 The most frequent mutation in IP (70%) is a recurrent exon 4_10 deletion (NEMOexon4_10del) due to non allelic homologous recombination that occurs between two repeats (MER67B) located in intron 3 and downstream exon 10, causing the removal of the entire genomic region from exon4 to 10.6,7 Missense, nonsense, deletions, and insertions have been reported in addition to gross rearrangements.8 With the exception of a tract of cytosines in exon 10 that appears to be prone to mutations in IP/HED-ID, no mutational hotspots or common point mutations are seen. To date, 53 different mutations (from large deletions to single amino-acid substitutions) affecting IKBKG have been reported: 7 gross deletions, 27 frameshift, 11 nonsense, four missense, one is an in-frame deletion of one codon, two are splice-site mutations, and one is a nonstop mutation.6–8 No evident genotype–phenotype correlation is apparent from comparison of patients with different loss-of-function mutations. The majority of mutations are ‘private’ to specific families. The rate of de novo mutations is about 65%.

Parental mosaicism is a pitfall in preimplantation genetic diagnosis of dominant disorders

PCR amplification on single cells is prone to allele drop-out (PCR failure of one allele), a cause of misdiagnosis in preimplantation genetic diagnosis (PGD). Owing to this error risk, PGD usually relies on both direct and indirect genetic analyses. When the affected partner is the sporadic case of a dominant disorder, building haplotypes require spermatozoon or polar body testing prior to PGD, but these procedures are cost and time-consuming. A couple requested PGD because the male partner suffered from a dominant Cowden syndrome (CS). He was a sporadic case, but the couple had a first unaffected child and the non-mutated paternal haplotype was tentatively deduced. The couple had a second spontaneous pregnancy and the fetus was found to carry the at-risk haplotype but not the PTEN mutation. The mutation was present in blood from the affected father, but at low level, confirming the somatic mosaicism. Ignoring the possibility of mosaicism in the CS patient would have potentially led to selection of affected embryos. This observation emphasizes the risk of PGD in families at risk to transmit autosomal-dominant disorder when the affected partner is a sporadic case.

Single-sperm analysis for recurrence risk assessment of spinal muscular atrophy.

With the detection of a homozygous deletion of the survival motor neuron 1 gene (SMN1), prenatal and preimplantation genetic diagnosis (PGD) for spinal muscular atrophy has become feasible and widely applied. The finding of a de novo rearrangement, resulting in the loss of the SMN1 gene, reduces the recurrence risk from 25% to a lower percentage, the residual risk arising from recurrent de novo mutation or germline mosaicism. In a couple referred to our PGD center because their first child was affected with SMA, the male partner was shown to carry two SMN1 copies. An analysis of the SMN1 gene and two flanking markers was performed on 12 single spermatozoa, to determine whether the father carried a CIS duplication of the SMN1 gene on one chromosome and was a carrier, or if the deletion has occurred de novo. We showed that all spermatozoa that were carriers of the ‘at-risk haplotype’ were deleted for the SMN1 gene, confirming the carrier status of the father. We provide an original application of single germ cell studies to recessive disorders using coamplification of the gene and its linked markers. This efficient and easy procedure might be useful to elucidate complex genetic situations when samples from other family members are not available.

Diagnostic génétique sur l’embryon humain de fécondation in vitro : faits, perspectives et limites

Le diagnostic genetique sur embryon humain issu de fecondation in vitro est envisageable par voie invasive en prelevant l’embryon au stade clive ou blastocyste. L’objectif peut etre de realiser un diagnostic genetique lorsqu’un couple est a risque de transmettre une maladie genetique – diagnostic preimplantatoire (DPI) – mais egalement de realiser un depistage d’aneuploidies (PGS, pour preimplantation genetic screening) pour ameliorer la performance de la fecondation in vitro. Alors que dans le cas des maladies genetiques la pratique d’un diagnostic est justifiee, il persiste un doute quant aux reelles ameliorations que procure le depistage. Les recentes publications sur le PGS applique au stade de blastocyste rapportent des resultats attractifs et relancent le debat sur sa possible utilisation en France. Par ailleurs, certains essaient de correler des marqueurs morphologiques, cinetiques et environnementaux au statut genetique de l’embryon. Les resultats sont prometteurs et aboutiront peut-etre a la mise au point d’un depistage non invasif.