Analysis of mitochondrial DNA sequences in patients with isolated or combined oxidative phosphorylation system deficiency

Background

Enzyme deficiencies of the oxidative phosphorylation (OXPHOS) system may be caused by mutations in the mitochondrial DNA (mtDNA) or in the nuclear DNA.

Objective

To analyse the sequences of the mtDNA coding region in 25 patients with OXPHOS system deficiency to identify the underlying genetic defect.

Results

Three novel non‐synonymous substitutions in protein‐coding genes, 4681T→C in MT‐ND2, 9891T→C in MT‐CO3 and 14122A→G in MT‐ND5, and one novel substitution in the 12S rRNA gene, 686A→G, were found. The definitely pathogenic mutation 3460G→A was identified in an 18‐year‐old woman who had severe isolated complex I deficiency and progressive myopathy.

Conclusions

Bioinformatic analyses suggest a pathogenic role for the novel 4681T→C substitution found in a boy with Leigh''s disease. These results show that the clinical phenotype caused by the primary Leber''s hereditary optic neuropathy mutation 3460G→A is more variable than has been thought. In the remaining 23 patients, the role of mtDNA mutations as a cause of the OXPHOS system deficiency could be excluded. The deficiency in these children probably originates from mutations in the nuclear genes coding for respiratory enzyme subunits or assembly factors.The oxidative phosphorylation (OXPHOS) system consists of five enzyme complexes composed of >70 subunits encoded by the nuclear genome and 13 subunits encoded by mitochondrial DNA (mtDNA). Both isolated and combined enzyme complex deficiencies have been reported in children with various clinical phenotypes. Defects in the OXPHOS system are common causes of inborn errors in energy metabolism, with an estimated incidence of 1 per 10 000 live births.¹ The inheritance pattern is autosomal recessive in most cases, but autosomal dominant and X‐chromosomal inheritance has also been described. Maternal inheritance points to a mutation in mtDNA as the cause of the disease.²More than 2000 human mtDNA‐coding region sequences have been reported since 2000, and about half of these sequences are from Europeans.^3,4,5,6,7,8 The total number of non‐synonymous mutations leading to an amino acid replacement in mtDNA of European origin has been estimated to be 1081, but as many as 18 100 sequences should be analysed to identify 95% of these substitutions.⁹ Sequencing of the complete mtDNA from patients with an OXPHOS system deficiency will evidently lead to the identification of novel pathogenic mutations. This approach has already yielded several novel mutations in MT‐ND genes so far, and some of them—for example, 10191T→C and 14487T→C—may not be uncommon causes of disease.^10,11

Key points

• Enzyme deficiencies of the oxidative phosphorylation (OXPHOS) system may be caused by mutations in the mitochondrial DNA (mtDNA) or in the nuclear DNA. The sequence of mtDNA‐coding region was analysed in 25 patients with OXPHOS system deficiency to identify the underlying genetic defect.
• 4681T→C, a novel substitution in MT‐ND2, was found in a patient with Leigh''s disease. Further analyses suggested a pathogenic role for this substitution.
• 3460G→A, one of the mutations causing Leber''s hereditary optic neuropathy, was identified in a patient with progressive myopathy. The finding suggests that the clinical phenotype caused by this mutation is more variable than what has been known.

There is a growing need to analyse complete mtDNA sequences with a high throughput and in a cost‐efficient manner. We analysed the entire coding region of mtDNA in 28 patients (consisting of children and young adults) with OXPHOS system deficiency using a protocol consisting of conformation‐sensitive gel electrophoresis (CSGE) of amplified mtDNA fragments and subsequent sequencing of those fragments that differed in mobility in CSGE. Obtained sequences were compared with previously reported mtDNA sequences to identify haplotype‐specific or novel variants, and to detect possible sequencing errors.¹² The quality of the sequences was confirmed by comparison of the sequences obtained using the CSGE protocol with those obtained using direct mtDNA sequencing, and by correct identification of three samples with a known pathogenic mutation. Three novel non‐synonymous substitutions and one novel rRNA substitution were detected, and their pathogenic potential was estimated on several criteria.     

Depletion of mitochondrial DNA in leucocytes harbouring the 3243A->G mtDNA mutation

Background

The 3243A→G MTTL1 mutation is the most common heteroplasmic mitochondrial DNA (mtDNA) mutation associated with disease. Previous studies have shown that the percentage of mutated mtDNA decreases in blood as patients get older, but the mechanisms behind this remain unclear.

Objectives and method

To understand the dynamics of the process and the underlying mechanisms, an accurate fluorescent assay was established for 3243A→G heteroplasmy and the amount of mtDNA in blood with real‐time polymerase chain reaction was determined. The amount of mutated and wild‐type mtDNA was measured at two time points in 11 subjects.

Results

The percentage of mutated mtDNA decreases exponentially during life, and peripheral blood leucocytes in patients harbouring 3243A→G are profoundly depleted of mtDNA.

Conclusions

A similar decrease in mtDNA has been seen in other mitochondrial disorders, and in 3243A→G cell lines in culture, indicating that depletion of mtDNA may be a common secondary phenomenon in several mitochondrial diseases. Depletion of mtDNA is not always due to mutation of a nuclear gene involved in mtDNA maintenance.The 3243A→G MTTL1 gene mutation of mitochondrial DNA (mtDNA) is the most common heteroplasmic pathogenic mtDNA mutation and is found in approximately 1 in 6000 of the general population.¹ Although first described in mitochondrial encephalomyopathy with lactic acidosis and stroke‐like episodes (MELAS), the phenotypic spectrum is extremely diverse, including isolated diabetes and deafness, hypertrophic cardiomyopathy and retinitis pigmentosa.² The clinical variability can be explained partly by tissue‐specific differences in the percentage of mutated mtDNA.^3,4Intriguingly, the percentage of mutated mtDNA is consistently lower in peripheral blood than in post‐mitotic tissues such as skeletal muscle and brain.^3,5 Serial measurements in the same subject have shown that the percentage of the 3243A→G mutation in blood decreases over time,^6,7 but the reasons for this are not clear. One possibility is that vegetative segregation in rapidly proliferating leucocyte precursors leads to high percentages of mutated mtDNA in some cells. This causes a biochemical defect of the respiratory chain, which either impairs the further proliferation of that cell lineage or leads to cell death.⁷ This would ultimately lead to a decrease in the percentage of mutated mtDNA in the daughter cells present in the peripheral blood. However, it is currently not known whether the biochemical defect is primarily because of high amounts of mutated mtDNA,⁸ low amounts of wild‐type mtDNA⁹ or a combination of both.To advance our understanding of this process, we developed and validated a highly sensitive fluorescent assay to measure the changes in heteroplasmy over time, and also measured the absolute amount of mutated and wild‐type mtDNA in 11 subjects known to harbour 3243A→G.     

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 tRNA^leu 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.Mitochondrial DNA (mtDNA) mutations cause a wide range of serious genetic diseases with maternal inheritance. Most of these defects result in a progressive disabling neurological syndrome with premature death. Among them, the myopathy, encephalopathy, lactic acidosis, and stroke‐like (MELAS) syndrome (OMIM: 540 000) is one of the most common and serious conditions. MELAS syndrome is mainly caused by the m.3243A>G mutation in the mitochondrial tRNALeu gene¹ (Genbank NC001 807), which produces a generalised dysfunction of the mitochondrial respiratory chain. The clinical features are highly variable, not only in terms of age at onset and severity of symptoms but also in relation to the specific organs associated.² Many patients present with mellitus diabetes, deafness, cardiomyopathy, external ophthalmoplegia and skeletal myopathy, variously associated in different degrees.³ The interfamilial and intrafamilial variability of the clinical phenotype is classically related to the properties of mtDNA segregation: heteroplasmy, “threshold” effect, mitochondrial “bottleneck” and variation in the tissue distribution⁴.Owing to the severity of the disease, the high risk of recurrence in siblings and the absence of efficient treatment, couples at risk of transmitting the m.3243A>G mutation often ask for prenatal diagnosis. However, very few data are so far available with respect to the prenatal diagnosis of MELAS syndrome.^4,5 Thus, whether prenatal assessment of the fetal mutant load (rate of mutant v wild‐type mtDNA) is a good predictive marker for the postnatal clinical outcome remains a matter of debate. In the few available reports, imprecise assessment of the fetal mutant load, most often studied in a single fetal tissue, hampers consideration of this issue. We report here a sensitive novel technical approach, designed to quantify as accurately as possible the mutant load in a given tissue. The results of prenatal analyses in a series of nine cases are detailed.     

Screening of calpain-3 autolytic activity in LGMD muscle: a functional map of CAPN3 gene mutations

Background

The diagnosis of calpainopathy is obtained by identifying calpain‐3 protein deficiency or CAPN3 gene mutations. However, in many patients with limb girdle muscular dystrophy type 2A (LGMD2A), the calpain‐3 protein quantity is normal because loss‐of‐function mutations cause its enzymatic inactivation. The identification of such patients is difficult unless a functional test suggests pursuing a search for mutations.

Materials and methods

A functional in vitro assay, which was able to test calpain‐3 autolytic function, was used to screen a large series of muscle biopsy specimens from patients with unclassified LGMD/hyperCKaemia who have previously shown normal calpain‐3 protein quantity.

Results

Of 148 muscle biopsy specimens tested,17 samples (11%) had lost normal autolytic function. CAPN3 gene mutations were identified in 15 of 17 patients (88%), who account for about 20% of the total patients with LGMD2A diagnosed in our series.

Conclusions

The loss of calpain‐3 autolytic activity is highly predictive of primary calpainopathy, and the use of this test as part of calpainopathy diagnosis would improve the rate of disease detection markedly. This study provides the first evidence of the pathogenetic effect of specific CAPN3 gene mutations on the corresponding protein function in LGMD2A muscle and offers new insights into the structural–functional relationship of the gene and protein regions that are crucial for the autolytic activity of calpain‐3.Limb girdle muscular dystrophies (LGMDs) comprise a clinically and genetically heterogeneous group of diseases usually characterised by progressive muscle weakness and wasting of pelvic and shoulder girdles. LGMD type 2A (LGMD2A, MIM 253600) was the first form of LGMD to be mapped and molecularly characterised, probably because it is the most frequent.^1,2,3,4,5 LGMD2A is caused by mutations in the CAPN3 gene (MIM 114240) that encodes for a non‐structural protein, the enzyme called calpain‐3.¹ Calpain‐3 is the muscle‐specific member of a family of Ca²⁺‐dependent proteases, which are supposed to play a part in many intracellular processes, including cell motility, apoptosis, differentiation and cell cycle regulation, by modulating the biological activity of their substrates through limited and strictly controlled proteolysis. Calpain‐3 is composed of four functional domains, and has three exclusive sequence inserts (NS, IS1 and IS2). The activation of calpain‐3 depends on phospholipids and Ca²⁺ ions,^6,7 takes place after unknown stimuli, and results in partial autolytic degradation.^8,9,10,11,12The molecular diagnosis of calpainopathy is complex because of the variability of clinical phenotypes, the effort required to identify point mutations in a relatively large gene, and incomplete sensitivity and specificity of calpain‐3 protein analysis on muscle biopsy specimen.^{13,14,15,16,17,18,19}An increasing number of studies have reported patients with LGMD2A whose diagnosis had been obtained by mutation identification despite normal levels of calpain‐3 protein in their muscle biopsy specimen.^4,15,20,21 As calpain‐3 is an enzyme, some mutations may not cause a reduction in protein content but its functional inactivation. Clinicians are increasingly aware that although the number of patients with LGMD2A showing normal calpain‐3 levels is marked, the identification of such cases is difficult. In patients with abnormal calpain‐3 protein levels, the effort required to search for mutations is justified because it will almost certainly confirm the LGMD2A diagnosis.¹⁶ Conversely, in patients with normal calpain‐3 protein content, an alternative diagnosis is usually pursued. For this reason, many patients with LGMD2A remain undiagnosed, unless a search is conducted for the CAPN3 gene mutation even if protein results are not indicative. To overcome this problem, we have developed a functional in vitro assay that is able to test the calpain‐3 autolytic activity in muscle samples,¹⁵ and showed that the loss of this function is associated with specific CAPN3 gene mutations.We report the results of extensive use of this functional assay as a screening tool in muscle biopsy specimens from patients with unclassified LGMDs and normal calpain‐3 protein quantity. Besides identifying patients with loss‐of‐function mutations, which would improve the rate of detection of the disease, we aimed at designing a functional map of the mutations and the protein regions involved in the autolytic activity to determine how single mutations exert their deleterious effect on the mutant protein.     

Schimke immuno-osseous dysplasia: a clinicopathological correlation

Background

Schimke immuno‐osseous dysplasia (SIOD) is a fatal autosomal recessive disorder caused by loss‐of‐function mutations in swi/snf‐related matrix‐associated actin‐dependent regulator of chromatin, subfamily a‐like 1 (SMARCAL1).

Methods

Analysis of detailed autopsies to correlate clinical and pathological findings in two men severely affected with SIOD.

Results

As predicted by the clinical course, T cell deficiency in peripheral lymphoid organs, defective chondrogenesis, focal segmental glomerulosclerosis, cerebral ischaemic lesions and premature atherosclerosis were identified. Clinically unexpected findings included a paucity of B cells in the peripheral lymphoid organs, emperipolesis‐like (penetration of one cell by another) abnormalities in the adenohypophysis, fatty infiltration of the cardiac right ventricular wall, pulmonary emphysema, testicular hypoplasia with atrophy and azospermia, and clustering of small cerebral vessels.

Conclusions

A regulatory role for the SMARCAL1 protein in the proliferation of chondrocytes, lymphocytes and spermatozoa, as well as in the development or maintenance of cardiomyocytes and in vascular homoeostasis, is suggested. Additional clinical management guidelines are recommended as this study has shown that patients with SIOD may be at risk of pulmonary hypertension, combined immunodeficiency, subcortical ischaemic dementia and cardiac dysfunction.The osteochondrodysplasias are a heterogeneous group of inherited disorders of skeletal growth causing disproportionate short stature. Of the 230 distinct osteochondrodysplasias,² several have been associated with nephrotic syndrome or immunodeficiency.² Schimke immuno‐osseous dysplasia (SIOD), an autosomal recessive panethnic multisystem osteochondrodysplasia, is characterised by dysmorphism,^3,4 spondyloepiphysial dysplasia,^5,6 T cell immunodeficiency^3,5 and nephrotic syndrome.^{3,5,7,8,9,10,11} Less penetrant features include hypothyroidism,³ migraine‐like headaches,¹² cerebral ischaemia^7,13 and enteropathy.^14,15,16,17 Stroke, severe opportunist infections, bone marrow failure, complications of renal failure or an undefined pulmonary disease,³ result in premature mortality in most patients during childhood to early adolescence; however, a few patients remain alive in their third and fourth decades of life.¹⁸SIOD is caused by biallelic loss‐of‐function mutations in swi/snf‐related matrix‐associated actin‐dependent regulator of chromatin, subfamily a‐like 1 (SMARCAL1).¹⁹ Previous studies have shown that SMARCAL1 encodes a protein homologous to the SNF2 family of chromatin remodelling proteins.^20,21 However, the mechanism by which SMARCAL1 mutations cause SIOD remains unknown. Hypotheses on the pathophysiology have considered SIOD to be an autoimmune disorder,^5,14 a connective tissue disorder,^16,22,23 a vascular endothelial disorder,^7,8,24 a metabolic disorder affecting chondrocyte and T cell differentiation,^5,16 or a cellular proliferation disorder.^3,19Murine Smarcal1 is expressed in tissues equivalent to those implicated as affected by clinical symptoms.²⁵ From this observation and clinical data, we have hypothesised that SIOD arises autonomously in each cell type. To determine whether disease occurs in each of these tissues as hypothesised and to elucidate further the pathophysiology of SIOD and potential functions for SMARCAL1, we undertook a detailed analysis of autopsy samples from two patients with SIOD and correlated the findings with their clinical course. Although secondary effects may confound interpretation of some findings, we find that the loss of functional SMARCAL1 has tissue‐specific effects on cellular proliferation, development and maintenance, and uncovered previously unknown pathological features relevant for management of patients with SIOD.     

Expansion of the genotypic and phenotypic spectrum in patients with KRAS germline mutations

Background

Noonan syndrome, cardio‐facio‐cutaneous syndrome (CFC) and Costello syndrome constitute a group of developmental disorders with an overlapping pattern of congenital anomalies. Each of these conditions can be caused by germline mutations in key components of the highly conserved Ras‐MAPK pathway, possibly reflecting a similar pathogenesis underlying the three disorders. Germline mutations in KRAS have recently been identified in a small number of patients with Noonan syndrome and CFC.

Methods and results

260 patients were screened for KRAS mutations by direct sequencing. Overall, we detected KRAS mutations in 12 patients, including three known and eight novel sequence alterations. All mutations are predicted to cause single amino acid substitutions. Remarkably, our cohort of individuals with KRAS mutations showed a high clinical variability, ranging from Noonan syndrome to CFC, and also included two patients who met the clinical criteria of Costello syndrome.

Conclusion

Our findings reinforce the picture of a clustered distribution of disease associated KRAS germline alterations. We further defined the phenotypic spectrum associated with KRAS missense mutations and provided the first evidence of clinical differences in patients with KRAS mutations compared with Noonan syndrome affected individuals with heterozygous PTPN11 mutations and CFC patients carrying a BRAF, MEK1 or MEK1 alteration, respectively. We speculate that the observed phenotypic variability may be related, at least in part, to specific genotypes and possibly reflects the central role of K‐Ras in a number of different signalling pathways.Noonan syndrome (OMIM 163950), cardio‐facio‐cutaneous syndrome (CFC; OMIM 115150) and Costello syndrome (OMIM 218040) are distinct entities that share a common pattern of congenital anomalies, including typical heart defects, overlapping craniofacial dysmorphisms, short stature and a variable degree of mental retardation. Discrimination between the three conditions is based mainly on distinct clinical features such as dry hyperkeratotic skin and hair abnormalities in patients with CFC,¹ and redundant and loose skin with deep palmar and plantar creases as well as a coarse facial appearance in those with Costello syndrome.² In addition, mental development is more severely impaired in CFC and Costello syndrome whereas Noonan syndrome is usually associated with minor cognitive deficits or even normal intelligence.³ While patients with Noonan syndrome and CFC have no or only a slightly increased risk of tumour development, the incidence of tumours in Costello syndrome has been estimated to be 7–21%.⁴ Although various attempts have been undertaken to develop standardised diagnostic criteria for these entities,^5,6 considerable overlap exists and in some instances a patient''s phenotype cannot be clearly assigned to one of these conditions.Missense mutations in PTPN11 were first identified in patients with Noonan syndrome⁷ and subsequently have been shown to account for almost 50% of cases.^8,9PTPN11 encodes the protein tyrosine phosphatase SHP‐2 which relays growth signals from activated tyrosine kinase receptors to other signalling molecules, particularly Ras (reviewed by Neel et al¹⁰). Noonan syndrome causing PTPN11 mutations have been thought to result in gain‐of‐function of SHP‐2 and cause deregulation of Ras dependent signalling cascades.¹¹ Heterozygous germline mutations in KRAS were reported to occur in a minority of patients with Noonan syndrome¹² and CFC,^12,13 shortly after mutations in HRAS had been detected in the majority of individuals with Costello syndrome.¹⁴ Moreover, mutations in BRAF, MEK1 and MEK2, encoding proteins involved in Ras downstream signalling, were shown to cause CFC syndrome.^13,15 Taken together, the current data suggest that germline missense mutations in the aforementioned genes culminate in deregulated Ras‐MAPK signalling that most likely represents the common pathogenetic basis of this group of developmental disorders.^16,17Ras isoforms encoded by the three genes KRAS, HRAS and NRAS represent highly conserved signal transduction molecules. They act as molecular switches through cycling between an active GTP bound and an inactive GDP bound state,¹⁸ and in their active form they interact with a variety of downstream effector proteins.¹⁹RAS genes have long been known as proto‐oncogenes mutated in various types of human cancers (reviewed by Bos²⁰). The majority of these oncogenic RAS mutations affect amino acid residues G12, G13 and Q61 and cause Ras to accumulate in the active GTP bound state by impairing intrinsic GTPase activity and conferring resistance to GTPase activating proteins (GAPs).²⁰ Germline HRAS mutations associated with Costello syndrome almost exclusively affect codons 12 and 13 and are identical to somatic alterations identified in cancer,¹⁴ hence explaining the high risk of tumour development in Costello syndrome. In contrast, KRAS mutations described to date in patients with Noonan syndrome/CFC are distinct from those found in malignancies. Similar to the concept of activating PTPN11 mutations in Noonan syndrome and malignancies,¹¹KRAS mutations associated with Noonan syndrome or CFC might give rise to mutant proteins with a relatively mild gain‐of‐function which are tolerated in the germline as well as during embryonic development. Specifically, KRAS mutations identified in Noonan syndrome patients include V14I and T58I whereas P34R and G60R were found in CFC patients.^12,13 Mutations in KRAS exon 6, causing amino acid alterations in the C terminal portion of isoform B, such as D153V and V152G, were found to be associated with a severe Noonan syndrome or CFC phenotype.^12,13,21 It has been proposed that all mutations lead to stabilisation of K‐Ras in the active conformation, most likely by different gain‐of‐function mechanisms.^12,21Here we report the results of KRAS mutation screening in a large cohort of patients with Noonan syndrome and related disorders.     

Detection of BRAF V600E activating mutation in papillary thyroid carcinoma using PCR with allele-specific fluorescent probe melting curve analysis

Background

A single hotspot mutation at nucleotide 1799 of the BRAF gene has been identified as the most common genetic event in papillary thyroid carcinoma (PTC), with a prevalence of 29–83%.

Aims

To use a PCR assay to molecularly characterise the BRAF activating point mutation in a series of PTC and benign thyroid cases and correlate the mutation results with histological findings.

Methods

Formalin‐fixed paraffin‐embedded (FFPE) sections were evaluated for the BRAF V600E mutation using LightCycler PCR with allele‐specific fluorescent probe melting curve analysis (LCPCR).

Results

42 (37 PTC; 5 benign) surgical tissue samples were analysed for the BRAF V600E activating point mutation. Using LCPCR and direct DNA sequencing, the BRAF mutation was identified in 23/37 (62.2%) PTC FFPE samples. DNA sequencing results demonstrated confirmation of the mutation.

Conclusions

Detection of BRAF‐activating mutations in PTC suggests new approaches to management and treatment of this disease that may prove worthwhile. Identification of the BRAF V600E activating mutation in routine FFPE pathology samples by a rapid laboratory method such as LCPCR could have significant value.Although thyroid cancer represents only 1% of all human malignancies, it accounts for more than 90% of all endocrine cancers.¹ The incidence of thyroid cancer has risen in the United States, from a rate of 3.6 per 100 000 in 1973 to 8.7 per 100 000 in 2002.² The increase in thyroid cancer incidence can be attributed primarily to an increase in the incidence of papillary thyroid carcinoma (PTC). For the time period 1973 to 2002, the incidence of PTC increased from 2.7 to 7.7 per 100 000, a 2.9‐fold increase.² In a report of 15 700 patients in the United States, overall survival rates, corrected for age and sex, were 98% for PTC, 92% for follicular carcinoma, 80% for medullary carcinoma, and 13% for anaplastic carcinoma.³ Among the most curable of cancers, PTC tends to remain localised in the thyroid gland, but in time it may metastasise to regional lymph nodes and, less commonly, to the lungs. Peak incidence of PTC is in the fifth decade of life and it occurs nearly three times more frequently in women than in men.⁴A single hotspot mutation at nucleotide 1799 of the BRAF gene has been identified as the most common genetic event in PTC, with a prevalence of 29–83%.^{5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24} Activating BRAF mutations may be an important event in the development of PTC. This mutation had been formerly termed T1796A, based on the NCBI GenBank nucleotide sequence NM 004333, which missed a codon (three nucleotides) in exon 1 of the BRAF gene. With the correct version of the NCBI GenBank nucleotide sequence NT 007914 available, this BRAF mutation is now designated T1799A.²⁵ This thymine (T) to adenine (A) transversion mutation (T→A) results in the substitution of valine with glutamate in codon 600 (V600E, formerly V599E) and converts BRAF into a dominant transforming protein that causes constitutive activation of the mitogen‐activated protein kinase (MAPK) pathway, independent of RAS activation.²⁶ Amino acid 600 lies in the kinase domain of BRAF, and the V600E mutation makes the enzyme more active than wild‐type BRAF. The resulting protein shows increased kinase activity that can transform NIH3T3 cells.²⁶ This suggests that therapy with RAF kinase inhibitors may be of use in this disease. If the response to RAF kinase inhibition is dependent on the presence of an activated BRAF protein, it will be necessary to evaluate cases of PTC for the presence or absence of mutations.The BRAF V600E activating point mutation appears to be highly specific for PTC, with no benign or other well‐differentiated thyroid neoplasm having been found to harbour this mutation. Moreover, some studies have suggested that BRAF mutation may serve as a novel prognostic biomarker that predicts poor clinicopathological outcomes, helping to identify patients who should undergo more aggressive clinical follow‐up.^7,9,27 While these preliminary reports are somewhat controversial^{17,18,21,28,29,30} and additional studies are required to establish the efficacy of this marker, these findings suggest that BRAF mutation detection may serve as a useful tool for diagnosis, management and treatment of PTC.LightCycler PCR with allele specific fluorescent probe melting curve analysis (LCPCR) has been used successfully to detect BRAF activating point mutations in PTC.⁷ Recently, we reported on the clinical utility of this method for detecting BRAF mutations in a series of indeterminate thyroid fine needle aspirate (FNA) cytology samples.³¹ Based on the detection of either a fluorescent reporter probe or double‐stranded DNA‐binding dyes, “real‐time” PCR instruments, such as the LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) can provide quantitative information regarding target nucleic acid sequences. The LightCycler is a microvolume fluorimeter integrated with a thermal cycler. Using post‐amplification melting curve analysis, the LightCycler instrument can also be used to differentiate alleles for the purpose of determining sequence variations or point mutations. In this report, we used LCPCR to molecularly characterise the BRAF activating point mutation in a series of PTC and benign thyroid cases and correlate the mutation results with histological findings.     

New VMD2 gene mutations identified in patients affected by Best vitelliform macular dystrophy

Purpose

The mutations responsible for Best vitelliform macular dystrophy (BVMD) are found in a gene called VMD2. The VMD2 gene encodes a transmembrane protein named bestrophin‐1 (hBest1) which is a Ca²⁺‐sensitive chloride channel. This study was performed to identify disease‐specific mutations in 27 patients with BVMD. Because this disease is characterised by an alteration in Cl⁻ channel function, patch clamp analysis was used to test the hypothesis that one of the VMD2 mutated variants causes the disease.

Methods

Direct sequencing analysis of the 11 VMD2 exons was performed to detect new abnormal sequences. The mutant of hBest1 was expressed in HEK‐293 cells and the associated Cl⁻ current was examined using whole‐cell patch clamp analysis.

Results

Six new VMD2 mutations were identified, located exclusively in exons four, six and eight. One of these mutations (Q293H) was particularly severe. Patch clamp analysis of human embryonic kidney cells expressing the Q293H mutant showed that this mutant channel is non‐functional. Furthermore, the Q293H mutant inhibited the function of wild‐type bestrophin‐1 channels in a dominant negative manner.

Conclusions

This study provides further support for the idea that mutations in VMD2 are a necessary factor for Best disease. However, because variable expressivity of VMD2 was observed in a family with the Q293H mutation, it is also clear that a disease‐linked mutation in VMD2 is not sufficient to produce BVMD. The finding that the Q293H mutant does not form functional channels in the membrane could be explained either by disruption of channel conductance or gating mechanisms or by improper trafficking of the protein to the plasma membrane.Best disease, also called Best vitelliform macular dystrophy (BVMD), is a bilateral, progressive disease of the retinal pigment epithelium (RPE) leading to decreased visual acuity. Best disease has an autosomally dominant transmission, but the penetrance is incomplete and its expression is highly variable.^1,2,3 Although Best disease is the second most common form of juvenile macular degeneration, with an onset usually before 15 years of age, only about 1% of all cases of macular degeneration can be attributed to Best disease.⁴ However, the degree of central vision impairment and the age of onset of symptoms varies widely,^5,6,7 even among members of the same family.On fundus examination, the central macula has a transient “egg yolk”‐like appearance measuring between one and two disc areas in size. During angiographic examination, a blockade of the choroidal fluorescence by vitelliform material was observed.⁸ This means the lesion is an abnormal accumulation of lipofuscin‐like material in front of the choroid and within and beneath the RPE, but not within the neural retina. At the vitelliform stage, visual acuity is surprisingly good or slightly subnormal. The lesion evolves through several stages over many years (scrambled egg stage, cyst stage, pseudohypopyon stage, atrophic stage, see classification by Mohler and Fine⁹), with visual acuity usually decreasing when disintegration of the yellowish material has been observed (scrambled egg stage).⁸ When atrophic changes take place, visual acuity may drastically be reduced. Infrequently, in some patients, the lesion may degenerate, resulting in the development of subretinal haemorrhage with identifiable or unidentifiable choroidal neovascular membranes.^10,11,12,13 Some people without any subretinal macular deposit but who carry causative BVMD mutations may never experience a noticeable decline of central vision.About 10% of affected eyes have multifocal lesions in the extrafoveal region. Furthermore, lesions similar to those seen in Best disease may occur in patients with adult vitelliform macular dystrophy (AVMD).^14,15 In some cases, similar conditions are caused by mutations of other genes, such as the peripherin/RDS gene.¹⁶The differential diagnosis of BVMD from other macular dystrophies is most effectively made by measuring the electrooculogram (EOG),^17,18 whereas full‐field electroretinograms are usually normal in patients with BVMD.¹ Although for years the EOG has been considered as the main functional test to define BVMD and was used especially for the detection of non‐manifesting carriers of the mutated VMD2 gene, recent studies have reported normal EOG recordings in patients with BVMD.^6,7,19 Thus, it seems that a normal EOG alone may not unequivocally exclude non‐manifesting carriers. A complete clinical examination of patients combined with molecular genetics studies of the VMD2 gene is mandatory for adequate counselling of the families. In both affected patients and carrier patients, EOG often shows an abnormal light‐peak/dark‐trough ratio. Abnormal EOG responses can be recorded in asymptomatic patients, sometimes long before the appearance of any clinical manifestations. Multifocal electroretinography (mfERG) shows variable central function loss depending on the stage of the disease and has become an important tool in assessing the function of the remaining macular cones.^20,21The mutations responsible for Best disease are found in a gene called VMD2. It encodes a transmembrane protein named bestrophin‐1 (hBest1). The protein is located in the basolateral plasma membrane of RPE cells.²² Bestrophin is a member of the RFP‐TM family of proteins, so named for their highly conserved arginine, phenylalanine, proline (RFP) motif.^23,24,25 Bestrophin contains several domains that are highly conserved between species.²³ Patch clamp studies of bestrophin and other RFP‐TM family members heterologously overexpressed in cell culture have suggested that bestrophin is a Ca²⁺‐sensitive chloride channel.^26,27,28Some mutations in the VMD2 gene have also been associated with some cases of bull''s‐eye maculopathy²⁹ and of AVMD.^29,30,31,32In this study, we identify six new, independent, disease‐specific mutations in patients with BVMD and their families, and in isolated patients. One of these mutations (Q293H) found in a large family from the west part of France is particularly severe. Patch clamp analysis of human embryonic (HEK) cells expressing the Q293H mutant bestrophin‐1 shows that this mutant channel is non‐functional. Furthermore, the Q293H mutant inhibits the function of wild‐type bestrophin‐1 channels in a dominant negative manner. These findings support the idea that BVMD is a chloride channelopathy.     

Correlation between clinical severity in patients with Rett syndrome with a p.R168X or p.T158M MECP2 mutation, and the direction and degree of skewing of X-chromosome inactivation

Introduction

Rett syndrome (RTT) is an X‐linked dominant neurodevelopmental disorder that is usually associated with mutations in the MECP2 gene. The most common mutations in the gene are p.R168X and p.T158M. The influence of X‐chromosome inactivation (XCI) on clinical severity in patients with RTT with these mutations was investigated, taking into account the extent and direction of skewing.

Methods

Female patients and their parents were recruited from the UK and Australia. Clinical severity was measured by the Pineda Severity and Kerr profile scores. The degree of XCI and its direction relative to the X chromosome parent of origin were measured in DNA prepared from peripheral blood leucocytes, and allele‐specific polymerase chain reaction was used to determine the parental origin of mutation. Combining these, the percentage of cells expected to express the mutant allele was calculated.

Results

Linear regression analysis was undertaken for fully informative cases with p.R168X (n = 23) and p.T158M (n = 20) mutations. A statistically significant increase in clinical severity with increase in the proportion of active mutated allele was shown for both the p.R168X and p.T158M mutations.

Conclusions

XCI may vary in neurological and haematological tissues. However, these data are the first to show a relationship between the degree and direction of XCI in leucocytes and clinical severity in RTT, although the clinical utility of this in giving a prognosis for individual patients is unclear.Rett syndrome (RTT) is an X‐linked dominant neurodevelopmental disorder, usually caused by mutations in the methyl‐CpG‐binding protein 2 (MECP2, OMIM#300005) gene. Mutations often arise at CpG hotspots,¹ and the most common mutations in MECP2 found in RTT cases are p.T158M and p.R168X (RettBASE, http://mecp2.chw.edu.au/). RTT has a wide clinical variability in terms of its severity.² Studies investigating the association between genotype and phenotype were originally quite inconsistent in their findings. However, with larger studies and increasing numbers of publications, evidence for definite relationships between genotype and phenotype is becoming clearer.³ Apparent differences in study results often occurred because of the use of different means of classifying and recording clinical severity. Additionally, the effects of X‐chromosome inactivation (XCI) status and other epigenetic influences on MECP2 function are likely to have a real influence on the variation in phenotype associated with specific mutations.XCI occurs early in embryogenesis at the blastula stage, and is usually a random process.^4,5 The inactive X chromosome is determined as cells become pluripotent; once this has happened, lineages derived from each of these cells will all have the same X chromosome inactivated through a process of methylation. Some studies have shown that there is an increased tendency for skewing of XCI in lymphocytes in RTT when compared with age‐matched controls, and that this usually confers a protective effect.^6,7,8 The most striking clinical examples of the effects of XCI in RTT are seen in twins with disparate severity^9,10 and in healthy carrier mothers with skewed XCI (presumed favourable) with affected daughters.^{11,12,13,14,15}In this study, we adopted a new approach to investigate genotype–phenotype relationships in RTT by exploring the association between clinical severity and the proportion of active mutated allele for the two common MECP2 mutations, p.R168X and p.T158M.     

Three single-nucleotide polymorphisms in LPA account for most of the increase in lipoprotein(a) level elevation in African Americans compared with European Americans

Background

The extent which universally common or population‐specific alleles can explain between‐population variations in phenotypes is unknown. The heritable coronary heart disease risk factor lipoprotein(a) (Lp(a)) level provides a useful case study of between‐population variation, as the aetiology of twofold higher Lp(a) levels in African populations compared with non‐African populations is unknown.

Objective

To evaluate the association between LPA sequence variations and Lp(a) in European Americans and African Americans and to determine the extent to which LPA sequence variations can account for between‐population variations in Lp(a).

Methods

Serum Lp(a) and isoform measurements were examined in 534 European Americans and 249 African Americans from the Choices for Healthy Outcomes in Caring for End‐Stage Renal Disease Study. In addition, 12 LPA variants were genotyped, including 8 previously reported LPA variants with a frequency of >2% in European Americans or African Americans, and four new variants.

Results

Isoform‐adjusted Lp(a) level was 2.23‐fold higher among African Americans. Three single‐nucleotide polymorphisms (SNPs) were independently associated with Lp(a) level (p<0.02 in both populations). The Lp(a)‐increasing SNP (G‐21A, which increases promoter activity) was more common in African Americans, whereas the Lp(a)‐lowering SNPs (T3888P and G+1/inKIV‐8A, which inhibit Lp(a) assembly) were more common in European Americans, but all had a frequency of <20% in one or both populations. Together, they reduced the isoform‐adjusted African American Lp(a) increase from 2.23 to 1.37‐fold(a 60% reduction) and the between‐population Lp(a) variance from 5.5% to 0.5%.

Conclusions

Multiple low‐prevalence alleles in LPA can account for the large between‐population difference in serum Lp(a) levels between European Americans and African Americans.Most genetic association studies focus on within‐population, rather than between‐population, differences in disease susceptibility. Although between‐population variation accounts for only 5–15% of human genetic diversity,¹ it is invoked increasingly to explain differences in disease risk and drug response across populations.^2,3 It is unknown whether such differences result from universally common alleles, which are hypothesised to contribute to within‐population risk variation for common diseases,^4,5 or from alleles that are population specific or nearly so. The allelic spectrum of between‐population variation has important implications, as identifying susceptibility alleles that are population specific may require strategies different from those identifying alleles that are common in all populations.The coronary heart disease risk factor lipoprotein(a) (Lp(a)) level is twofold higher in African than in non‐African populations,^6,7 and provides a useful case study of between‐population variation. The cardiovascular pathogenicity of Lp(a) is established best in Caucasians^8,9 and probably involves the low‐density lipoprotein‐bound plasminogen homologue apolipoprotein(a) (apo(a)), may increase low‐density lipoprotein delivery¹⁰ and may inhibit plasminogen‐mediated thrombolysis.¹¹ The apo(a) gene (LPA; MIM 152200) explains about 90% and 80% of Lp(a) level variance in European American¹² and African American¹³ populations, respectively. A genomically unusual 5.6‐kb variable tandem repeat in LPA encodes the KIV‐2 units, whose number determines apo(a) isoform size and explains half of the LPA effect on Lp(a) level.^12,14 The inverse association between isoform size and Lp(a) level probably reflects impaired cellular secretion of larger isoforms, which has been observed in vitro.¹⁵Isoform distributions are similar across populations, and do not explain higher African Lp(a) levels.⁷ Eight LPA variants (all but one are single‐nucleotide polymorphisms (SNPs)) have been associated with the isoform‐adjusted Lp(a) level in Europeans^{16,17,18,14,19,20} or Africans.^21,18 Some investigators have speculated that unidentified trans‐acting^16,22 or environmental²³ factors may explain the between‐population difference, as none of these have been shown to contribute substantially to higher African Lp(a) levels. . However, a substantial contribution of LPA to the between‐population difference cannot be excluded and provides the simplest explanation.Previous analyses have not considered multiple LPA variants simultaneously or comprehensively, and the extensive linkage disequilibrium across the gene^15,18,14,19 is expected to confound single‐locus effect estimates. Also, few studies included both European and African or African American populations, precluding direct quantification of LPA variant contributions to the between‐population difference. We report the results of a simultaneous analysis of multiple LPA variants, isoforms and Lp(a) level in a cohort of European Americans and African Americans.     

IRAK4 and NEMO mutations in otherwise healthy children with recurrent invasive pneumococcal disease

Background

About 2% of childhood episodes of invasive pneumococcal disease (IPD) are recurrent, and most remain unexplained.

Objective

To report two cases of otherwise healthy, unrelated children with recurrent IPD as the only clinical infectious manifestation of an inherited disorder in nuclear factor‐κB(NF‐κB)‐dependent immunity.

Results

One child carried two germline mutations in IRAK4, and had impaired cellular responses to interleukin (IL)1 receptor and toll‐like receptor (TLR) stimulation. The other child carried a hemizygous mutation in NEMO, associated with a broader impairment of NF‐κB activation, with an impaired cellular response to IL‐1R, TLR and tumour necrosis factor receptor stimulation. The two patients shared a narrow clinical phenotype, associated with two related but different genotypes.

Conclusions

Otherwise healthy children with recurrent IPD should be explored for underlying primary immunodeficiencies affecting the IRAK4‐dependent and NEMO‐dependent signalling pathways.The known inherited risk factors for invasive pneumococcal disease (IPD) in children include sickle‐cell disease¹ and primary immunodeficiencies (PIDs).² PIDs include defects in the classic complement activation pathway, defects in carbohydrate‐specific antibody responses, congenital asplenia and the more recently discovered IRAK4 and NEMO deficiencies.² These defects impair a step in the process leading to the phagocytosis of opsonised bacteria by splenic macrophages. NEMO and IRAK4 deficiencies also involve an impaired mucosal and systemic inflammation response, due to the impaired activation of nuclear factor‐κB (NF‐κB) by microbes and cytokines.^3,4,5,6 Although the risk of IPD in children with these PIDs is well known, the fraction of patients with IPD cases associated with a PID in the general population is unknown. This fraction is, however, thought to be small, because PIDs are usually associated with multiple infectious diseases, whereas IPD mostly affects otherwise healthy children. Thus, most cases of IPD remain unexplained genetically and immunologically, at least partly because they are not investigated.We hypothesisd that isolated IPD may be due to inborn errors in immunity to infection in an unexpectedly large fraction of children.⁷ We first focused on recurrent IPD,⁸ which is arbitrarily defined as two episodes of IPD occurring at least 1 month apart, whether caused by the same or different serotypes or strains. Recurrent IPD occurs in at least 2% of patients in most series, making IPD the most important known risk factor for subsequent IPD.^{8,9,10,11,12,13,14,15,16,17,18} Most series of recurrent cases of IPD have included mostly adults, many with overt underlying conditions, such as HIV infection, organ failure, or cancer.^{8,17,18,19,20} The situation is much less clear for recurrent IPD in children. The risk factors for this condition identified to date in paediatric patients are sickle‐cell disease^12,21 and congenital^22,23 or acquired¹⁴ cerebrospinal fluid leaks, specifically predisposing the patient to recurrent meningitis. In a North American series, six children with recurrent IPD had sickle‐cell disease, whereas the recurrence remained idiopathic in the other 10 children.¹² In a Swiss series, two children had a history of basal skull fracture, whereas the remaining eight were idiopathic.¹⁴A high proportion of children with idiopathic recurrent IPD are likely to have undetected PIDs. The diagnosis of these PIDs is difficult, because PIDs are rare and diverse, and because immunological investigations, and assays of antibody response in particular, are difficult to perform in children aged <2 years. Several PIDs probably associated with an IPD have not yet been discovered.² Only a few cases of children with PIDs and isolated recurrent IPD have been reported. They include children with congenital asplenia,^24,25,26,27 complement pathway deficiencies,^{28,29,30,31,32,33,34,35} X‐linked agammaglobulinaemia³⁶ and selective antipolysaccharide antibody deficiency.³⁷ Transient IgG2 deficiency has also been described in one child.³⁸ Isolated recurrent IPD has been reported in a single patient with a mild form of X‐linked recessive anhydrotic ectodermal dysplasia with immunodeficiency bearing a NEMO mutation,³⁹ but not in patients with autosomal recessive IRAK4 deficiency, or in patients with mutations in NEMO but no developmental phenotype. Autosomal recessive IRAK4 deficiency and X‐linked recessive NEMO deficiencies correspond to two recently described PIDs that affect NF‐κB‐mediated immunity and cause a relatively broad susceptibility to infections. We report here two otherwise healthy boys with isolated, recurrent IPD and inherited NEMO and IRAK4 defects. These patients displayed none of the other known infectious phenotypes associated with these disorders.     

A comprehensive strategy for the subtyping of patients with Fanconi anaemia: conclusions from the Spanish Fanconi Anemia Research Network

Background

Fanconi anaemia is a heterogeneous genetic disease, where 12 complementation groups have been already described. Identifying the complementation group in patients with Fanconi anaemia constitutes a direct procedure to confirm the diagnosis of the disease and is required for the recruitment of these patients in gene therapy trials.

Objective

To determine the subtype of Fanconi anaemia patients in Spain, a Mediterranean country with a relatively high population (23%) of Fanconi anaemia patients belonging to the gypsy race.

Methods

Most patients could be subtyped by retroviral complementation approaches in peripheral blood T cells, although some mosaic patients were subtyped in cultured skin fibroblasts. Other approaches, mainly based on western blot analysis and generation of nuclear RAD51 and FANCJ foci, were required for the subtyping of a minor number of patients.

Results and conclusions

From a total of 125 patients included in the Registry of Fanconi Anaemia, samples from 102 patients were available for subtyping analyses. In 89 cases the subtype could be determined and in 8 cases exclusions of common complementation groups were made. Compared with other international studies, a skewed distribution of complementation groups was observed in Spain, where 80% of the families belonged to the Fanconi anaemia group A (FA‐A) complementation group. The high proportion of gypsy patients, all of them FA‐A, and the absence of patients with FA‐C account for this characteristic distribution of complementation groups.Fanconi anaemia is a rare hereditary recessive disease characterised by developmental abnormalities, bone marrow failure and predisposition to cancer, mainly acute myeloid leukaemia.¹ To date, 12 complementation groups have been reported (FA‐A, B, C, D1, D2, E, F, G, I, J, L and M) and 11 associated genes have already been identified: FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG/XRCC9, BRIP1/FANCJ, FANCL and FANCM/Hef.^{2,3,4,5,6,7,8,9,10,11,12,13,14} In dividing cells or in cells exposed to DNA damage, eight Fanconi anaemia proteins (FANCA/B/C/E/F/G/L/M) form a Fanconi anaemia core complex, necessary for the monoubiquitination of FANCD2.^2,5,15 In contrast with these Fanconi anaemia proteins, FANCD1 and FANCJ are not involved in FANCD2 monoubiquitination, indicating that these proteins participate downstream of FANCD2 in the FA/BRCA pathway.^2,7Because of the overlap in the phenotype and molecular pathways between the different chromosome fragility syndromes, Fanconi anaemia subtyping constitutes an invaluable approach to confirm the diagnosis of the disease.^16,17 Additionally, in the case of patients with FAD1, subtyping analysis allows the identification of BRCA2 mutation carriers, characterised by an increased risk of developing breast, ovarian and other types of cancers.¹⁸ Fanconi anaemia subtyping also facilitates mutation screening studies and therefore the identification of mutations with particular pathogenic effects. In addition to the above‐mentioned applications, subtyping is essential before enrolling a patient with Fanconi anaemia in a gene therapy trial.Progress in the cloning of Fanconi anaemia genes enabled the identification of mutations in specific Fanconi anaemia genes by means of DNA sequencing approaches or other methods.¹⁹ The large number and complexity of some Fanconi anaemia genes and their mutations, together with the necessity of verifying the pathogenicity of each new mutation, implies that subtyping of patients with Fanconi anaemia by mutational analysis is often time consuming and laborious. The possibility of reverting the phenotype of Fanconi anaemia cells by the transfer of functional Fanconi anaemia genes has been recently proposed as an efficient approach for identifying the pathogenic genes that account for the disease in patients with Fanconi anaemia.^20,21 A different Fanconi anaemia subtyping approach is based on the western blot analysis of FANCD2.²² By means of the observation of the ubiquitinated (FANCD2‐L) and non‐ubiquitinated (FANCD2‐S) forms of the protein FANCD2, it is possible to predict pathogenic mutations in proteins upstream or downstream of FANCD2.²³ In the case of patients belonging to rare complementation groups such as FAD1 or FA‐J, approaches based on the formation of RAD51 or BRIP1 nuclear foci are also highly informative in identifying their complementation group.²⁴With the purpose of determining the prevalence of the different Fanconi anaemia complementation groups in Spain, we conducted an extensive subtyping study of Fanconi anaemia in this Mediterranean country. In addition to a predominantly caucasian population, a relatively large population of about 500 000 gypsies also live in Spain. In this population, the incidence of recessive syndromes is high, owing to the high rates of consanguinity.²⁵ This study will allow us to identify potential differences in the distribution of Fanconi anaemia subtypes due to geographical and ethnic characteristics, and will also allow us to conduct further mutation studies within the population of patients subtyped for Fanconi anaemia. Additionally, our subtyping study will facilitate the enrolling of patients with Fanconi anaemia in clinical gene therapy trials aimed at the genetic correction of their haematopoietic stem cells.