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Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive degenerative disease of upper and lower motor neurons. Approximately 15% of patients display clinical features consistent with frontotemporal dementia (FTD) and 35% display milder degrees of cognitive and behavioural impairment at some stage during their illness.1 Several genes have been reported to cause both ALS and FTD. Nevertheless, it remains unclear why some patients with ALS develop cognitive impairment, while other cases, often within the same family, remain unaffected. The GBA gene (OMIM *606463) encodes glucocerebrosidase (GCase), a lysosomal enzyme that converts glucocerebroside into glucose and ceramide. Heterozygous GBA mutations increase the risk of Parkinson’s disease (PD) and the risk of cognitive impairment in patients with PD.2 It is increasingly recognised that variants in genes causing Mendelian neurodegenerative diseases may exhibit pleiotropic effects and impact the phenotypic heterogeneity of those disorders. Moreover, lysosomal dysfunction has recently been associated with both Dementia with Lewy Bodies and FTD spectrum (online supplemental table 1). Based on this, we postulated that GBA variants may influence the cognitive status of patients with ALS. ### Supplementary data [jnnp-2021-327426supp001.pdf] We examined the GBA variants’ association with the risk of cognitive impairment in 751 patients with ALS from the population-based Piemonte and Valle d’Aosta Register for ALS that had undergone both a detailed neuropsychological evaluation and a whole-genome sequencing screening.3 Patients were classified as ALS with normal cognitive function (ALS-CN), ALS-FTD and ALS with intermediate cognitive deficits. The characteristics of the study population and a detailed description of neuropsychological testing and genetic screening are reported in online supplementarl materials in the Methods section. A mutational screening of GBA exonic variants was performed and their frequencies were compared with an internal control cohort (see online supplemental methods, Subjects). To assess whether pathogenic rare variants (minor allele frequency

ObjectiveTo assess the burden of rare genetic variants and to estimate the contribution of known amyotrophic lateral sclerosis (ALS) genes in an Italian population-based cohort, we performed whole genome sequencing in 959 patients with ALS and 677 matched healthy controls.MethodsWe performed genome sequencing in a population-based cohort (Piemonte and Valle d'Aosta Registry for ALS [PARALS]). A panel of 40 ALS genes was analyzed to identify potential disease-causing genetic variants and to evaluate the gene-wide burden of rare variants among our population.ResultsA total of 959 patients with ALS were compared with 677 healthy controls from the same geographical area. Gene-wide association tests demonstrated a strong association with SOD1, whose rare variants are the second most common cause of disease after C9orf72 expansion. A lower signal was observed for TARDBP, proving that its effect on our cohort is driven by a few known causal variants. We detected rare variants in other known ALS genes that did not surpass statistical significance in gene-wise tests, thus highlighting that their contribution to disease risk in our cohort is limited.ConclusionsWe identified potential disease-causing variants in 11.9% of our patients. We identified the genes most frequently involved in our cohort and confirmed the contribution of rare variants in disease risk. Our results provide further insight into the pathologic mechanism of the disease and demonstrate the importance of genome-wide sequencing as a diagnostic tool.

BackgroundA genetic diagnosis in Amyotrophic Lateral Sclerosis (ALS) can inform genetic counselling, prognosis and, in the light of incoming gene-targeted therapy, management. However, conventional genetic testing strategies are often costly and time-consuming.ObjectiveTo evaluate the diagnostic yield and advantages of whole-genome sequencing (WGS) as a standard diagnostic genetic test for ALS.MethodsIn this population-based cohort study, 1043 ALS patients from the Piemonte and Valle d’Aosta Register for ALS and 755 healthy individuals were screened by WGS for variants in 42 ALS-related genes and for repeated-expansions in C9orf72 and ATXN2.ResultsA total of 279 ALS cases (26.9%) received a genetic diagnosis, namely 75.2% of patients with a family history of ALS and 21.5% of sporadic cases. The mutation rate among early-onset ALS patients was 43.9%, compared with 19.7% of late-onset patients. An additional 14.6% of the cohort carried a genetic factor that worsen prognosis.ConclusionsOur results suggest that, because of its high diagnostic yield and increasingly competitive costs, along with the possibility of retrospectively reassessing newly described genes, WGS should be considered as standard genetic testing for all ALS patients. Additionally, our results provide a detailed picture of the genetic basis of ALS in the general population.

Transthyretin (TTR) amyloidosis (ATTR) is either an inherited condition or a non hereditary disease due to misfolding of wild-type (WT) TTR. Amyloid deposits can be mainly detected in nerves in the inherited form and in myocardium in the acquired variant. Renal involvement has been described only in the Val30Met mutation of the familial form and is thought to be extremely rare in the WT TTR. However, ATTR is multi-organ disease, and even in the WT forms, apparently limited to the heart, carpal tunnel syndrome and lumbar or cervical spine amyloid deposition have been described. A series of 4 cases of biopsy-proven renal TTR amyloid deposition is reported in the present paper. We describe 2 WT ATTR patients, 1 patient with c.424G>A (p.(Val142Ile)) mutation of the TTR gene, and 1 patient with Val30Met mutation of the TTR gene. In all patients, the biopsy showed the presence of amyloid deposits with different distribution (#1 pericapsular, #2–3 vessels, #4 vessels, interstitium of medulla and cortex, and tubular basement membrane). The use of immunohistochemistry has enabled the identification of TTR, and identify the precursor protein. The possibility of kidney involvement in TTR amyloidosis should be taken into account in patients with renal impairment and unexplained cardiomyopathy and/or neuropathy. This is even of greater interest to the elderly for the possible confounding co-existence of plasma cell dyscrasia that could lead the clinician, in the presence of renal amyloid deposits, to misdiagnose AL amyloidosis and undertake inappropriate treatments.

Purpose Neuropathological data suggest that ALS with SOD1 mutations (SOD1-ALS) is a distinct form of ALS. We evaluated brain metabolic changes characterizing SOD1-ALS as compared to sporadic ALS (sALS), employing 18fluorodeoxyglucose-positron-emission tomography (18F-FDG-PET). Methods We included 18 SOD1-ALS patients, 40 healthy controls (HC), and 46 sALS patients without mutations in SOD1, TARDBP, FUS, and C9ORF72, randomly selected from 665 subjects who underwent brain 18F-FDG-PET at diagnosis between 2008 and 2019 at the ALS Centre of Turin. We excluded patients with frontotemporal dementia. We used the full factorial design in SPM12 to evaluate whether differences among groups exist overall. In case the hypothesis was confirmed, group comparisons were performed through the two-sample t-test model of SPM12. In all the analyses, the height threshold was P < 0.001 (P < 0.05 FWE-corrected at cluster level). Results The full factorial design resulted in a significant main effect of groups. We identified a relative hypometabolism in sALS patients compared to SOD1-ALS cases in the right precentral and medial frontal gyrus, right paracentral lobule, and bilateral postcentral gyrus. SOD1 patients showed a relative hypermetabolism as compared to HC in the right precentral gyrus and paracentral lobule. As compared to HC, sALS patients showed relative hypometabolism in frontal, temporal, and occipital cortices. Conclusion SOD1-ALS was characterized by a relative hypermetabolism in the motor cortex as compared to sALS and HC. Since promising, targeted, therapeutic strategies are upcoming for SOD1-ALS, our data support the use of PET to study disease pathogenesis and to track its course in clinical trials, in both asymptomatic and symptomatic mutation carriers.

Amyotrophic lateral sclerosis (ALS) is known to represent a collection of overlapping syndromes. Various classification systems based on empirical observations have been proposed, but it is unclear to what extent they reflect ALS population substructures. We aimed to use machine-learning techniques to identify the number and nature of ALS subtypes to obtain a better understanding of this heterogeneity, enhance our understanding of the disease, and improve clinical care.In this retrospective study, we applied unsupervised Uniform Manifold Approximation and Projection [UMAP]) modelling, semi-supervised (neural network UMAP) modelling, and supervised (ensemble learning based on LightGBM) modelling to a population-based discovery cohort of patients who were diagnosed with ALS while living in the Piedmont and Valle d'Aosta regions of Italy, for whom detailed clinical data, such as age at symptom onset, were available. We excluded patients with missing Revised ALS Functional Rating Scale (ALSFRS-R) feature values from the unsupervised and semi-supervised steps. We replicated our findings in an independent population-based cohort of patients who were diagnosed with ALS while living in the Emilia Romagna region of Italy.Between Jan 1, 1995, and Dec 31, 2015, 2858 patients were entered in the discovery cohort. After excluding 497 (17%) patients with missing ALSFRS-R feature values, data for 42 clinical features across 2361 (83%) patients were available for the unsupervised and semi-supervised analysis. We found that semi-supervised machine learning produced the optimum clustering of the patients with ALS. These clusters roughly corresponded to the six clinical subtypes defined by the Chiò classification system (ie, bulbar, respiratory, flail arm, classical, pyramidal, and flail leg ALS). Between Jan 1, 2009, and March 1, 2018, 1097 patients were entered in the replication cohort. After excluding 108 (10%) patients with missing ALSFRS-R feature values, data for 42 clinical features across 989 patients were available for the unsupervised and semi-supervised analysis. All 1097 patients were included in the supervised analysis. The same clusters were identified in the replication cohort. By contrast, other ALS classification schemes, such as the El Escorial categories, Milano-Torino clinical staging, and King's clinical stages, did not adequately label the clusters. Supervised learning identified 11 clinical parameters that predicted ALS clinical subtypes with high accuracy (area under the curve 0·982 [95% CI 0·980-0·983]).Our data-driven study provides insight into the ALS population substructure and confirms that the Chiò classification system successfully identifies ALS subtypes. Additional validation is required to determine the accuracy and clinical use of these algorithms in assigning clinical subtypes. Nevertheless, our algorithms offer a broad insight into the clinical heterogeneity of ALS and help to determine the actual subtypes of disease that exist within this fatal neurodegenerative syndrome. The systematic identification of ALS subtypes will improve clinical care and clinical trial design.US National Institute on Aging, US National Institutes of Health, Italian Ministry of Health, European Commission, University of Torino Rita Levi Montalcini Department of Neurosciences, Emilia Romagna Regional Health Authority, and Italian Ministry of Education, University, and Research.For the Italian and German translations of the abstract see Supplementary Materials section.

Objective: To investigate the impact of a novel heterozygous FUS mutation in the acceptor splice site of intron 14 (c.1542 ��� 1 g > t) on protein expression in Peripheral Blood Mononuclear Cells (PBMC) from a familial ALS patient. Methods: PBMC were isolated for mRNA analysis (cDNA synthesis, sequencing and one-step RT-PCR), Western Immunoblot (WI), and Immunofluorescence (IF). Results: cDNA analysis revealed the skipping of exon 15 and a premature stop codon at c.228. RT-PCR showed reduced FUS mRNA by more than half compared to a healthy control (HC) and an ALS patient without genetic mutations (wtALS). In WI FUS band intensity in the proband was 30���50% compared to HC and wtALS. An antibody expected to detect only the wild-type protein did not reveal any reduction of FUS band intensity compared to the other antibodies. IF showed no difference among HC, wtALS, and the proband. Discussion: The reduction of FUS mRNA and protein in PBMC suggests the absence of the truncated protein, probably due to nonsense-mediated decay, leading to loss of function.

To determine whether the neuropsychological profiles of patients with amyotrophic lateral sclerosis (ALS) with (ALSC9+) and without (ALSC9-)The study population includes 741 patients with ALS who were consecutively diagnosed at the Turin ALS expert center in the 2010-2018 period and who underwent both cognitive/behavioral and genetic testing. Patients' neuropsychological patterns were compared (1) at the same degree of cognitive and behavioral deficit according to the revised ALS-Frontotemporal Dementia Consensus Criteria and (2) at the same level of motor impairment according to the King staging system.Despite being about 7 years younger, ALSC9+ patients had significantly lower scores in tests exploring executive function and verbal memory both when classified as cognitively normal and when diagnosed in the intermediate cognitive categories. Considering the clinical perspective, ALSC9+ patients showed significantly lower scores compared to ALSC9- patients at King stage 1 and 3 in almost all the examined neuropsychological domains; at King stage 2, ALSC9+ patients were more severely affected only in the verbal memory domain. Behavioral function was comparably impaired in the 2 cohorts.ALSC9+ patients show a different neuropsychological profile compared to ALSC9- patients, being more impaired in executive functions and verbal memory domains at all King stages. Verbal memory emerged as a particularly vulnerable function in ALSC9+, with worse performances even when patients were still classified as cognitively normal.

Background: Non-invasive prenatal diagnosis has found application in a limited number of genetic diseases due to the difficulty in detecting a few copies of fetal mutated sequences in the presence of a large excess of wild-type maternal alleles, even in the case of single-base mutations. Methods: We developed conditions for the enrichment of fetal mutated alleles in maternal plasma based on CO-amplification at lower denaturation temperature-PCR (COLD-PCR). In particular, we applied a full COLD-PCR protocol to the identification of a p.A87_G92del mutation in the TWIST1 gene causing craniosynostosis in a couple at risk for the disease. Results: The use of the COLD-PCR protocol coupled with direct sequencing enabled correct identification of the fetal paternally inherited mutated allele, in accordance with the result obtained on DNA extracted from chorionic villi. Conclusions: COLD-PCR proved to be a simple and powerful tool for the identification of minority mutated alleles even in the case of a moderately large deletion (18 bp) and confirmed to be very suitable for non-invasive prenatal diagnosis of a variety of genetic diseases.

By DNA sequence analysis we identified two new strain types and five novel sporadic variations among 25 isolates of Pneumocystis carinii f. sp. hominis obtained from 19 human immunodeficiency virus-positive patients. Of these, 13 were infected with a single strain and 6 were coinfected. Fifteen different combination types were identified among the 18 strains for which complete molecular typing was accomplished.

Vaccine-based strategies are currently under investigation as a means of inducing tumour-specific immune responses and improving the clinical outcome of multiple myeloma (MM) patients in remission after high-dose chemotherapy and peripheral blood progenitor cell (PBPC) infusion. The immune competence of these patients was investigated by determining the overall diversity of the T-cell receptor (TCR) repertoire in the peripheral blood (PB) and bone marrow (BM). The average time after transplantation was 13 months. The clonality and reciprocal usage of BV gene segments (TCRBV repertoire) was estimated at the cDNA level and membrane protein expression. The TCRBV repertoire of MM was severely disrupted compared with age-matched normal donors. On average, one-third of the total repertoire in both the PB and the BM consisted of T cells expressing oligoclonal TCRbeta transcripts. Flow cytometry showed an increased frequency of abnormally expanded BV subfamilies at both sites. BV expansions were predominantly CD8+ and had the phenotype of antigen-experienced memory T cells as well as T cells with the naive phenotype. Oligoclonality was not restricted to phenotypically expanded BV subfamilies, but also involved normally represented BV subfamilies. The TCR repertoire of MM in remission was then compared with monoclonal gammopathy of undetermined significance (MGUS) and MM patients at diagnosis. The degree of TCR diversity was similar in age-matched normal donors and MGUS, but progressively decreased from MGUS to MM at diagnosis and then to MM in remission. These data indicate that: (1) there is a long-lasting and severe disruption of TCR diversity after high-dose chemotherapy and PBPC infusion, and (2) the extent of TCR disruption may affect the clinical outcome of vaccine-based strategies delivered at the stage of minimal residual disease.

The pathophysiology and clinical severity of beta-thalassemia are related to the degree of alpha/non-alpha-chain imbalance. A triplicated alpha-globin gene locus can exacerbate effects of excess alpha-chains caused by a defective beta-globin gene, although this is not observed in all cases. Extensive studies on this condition are lacking. We report a group of 17 patients who are heterozygous for both the alpha alpha alpha(anti-3.7) allele and a mutation in the beta-globin gene cluster. Their clinical phenotypes varied: six had mild anemia with microcytosis and hypochromia, while 11 had more severe anemia with splenomegaly requiring splenectomy (three cases) and blood transfusions (four cases). Different phenotypes were also evident in the presence of the same beta-thalassemia mutation: in one family, two individuals had the same alpha- and beta-globin genotypes but presented with different hematologic phenotypes. In addition, the complex interaction involving a triplicated alpha-globin gene, beta39- and delta+27-thalassemia mutations is studied in a family with two siblings presenting with hemolytic anemia, normal Hb A2 and increased Hb F. Analysis of this series of patients suggests that additional genetic determinants play a role in modulating phenotypic expression in individuals with identical alpha- and beta-globin genotypes. Interaction with a triplicated alpha-gene can play a role in the clinical presentation of patients with defective beta-globin gene expression and should be considered in the diagnosis of atypical cases.

Hereditary hemochromatosis (HFE) is an inherited disorder whose gene lies in the proximity of the histocompatibility antigen (HLA) class I region, on 6p21.3. Despite efforts in refining the HFE region, a number of informative DNA markers, linked to the disease locus and amenable to use in an assay based on the polymerase chain reaction (PCR) is available. The gene content of this region is high, and the HFE gene has not so far been identified. We have used a strategy based on PCR protocols potentially able to detect both polymorphisms and expressed sequences. This approach has been applied to a 700-kb stretch (approximately) of DNA corresponding to the insert of a Centre d'Etude du Polymorphisme Humain yeast artificial chromosome (225 B1) of the possible candidate region. Five new polymorphisms have been detected among 20 specific fragments isolated. Four of them are tightly linked to the HFE locus. Because of the strong linkage disequilibrium with the disease demonstrated by these markers, they could represent starting points for the identification and characterization of the HFE gene. The remaining non-polymorphic fragments, being amplifiable and in most cases linked to NotI sites, may be useful starting points for the generation of a genomic contig of band 6p21.3 and for gene identification.

Mutations in the Cu/Zn superoxide dismutase (SOD1), transactive response (TAR)-DNA binding protein (TARDBP) and fused in sarcoma (FUS) genes account for approximately one third of familial amyotrophic lateral sclerosis (ALS) cases. Mutations in these genes have been found in 1 to 2% of apparently sporadic cases. We present the first case of an ALS patient carrying a de novo missense mutation of the FUS gene (c.1561C>T, p.R521C). This report highlights the importance of screening ALS patients, both familial and sporadic, for FUS mutations and also suggests that de novo mutations is a relevant mechanism underlying sporadic neurodegenerative disease.

Objectives Molecular biological testing for genetic diseases has grown rapidly, but speed, accuracy, specificity, sensitivity, throughput, and cost become more important as large scale screening is considered. This is a pilot study of an assay for the simultaneous detection of up to 31 cystic fibrosis mutations in a multicentre population based screening of 4476 Italian newborns. Methods The assay is a polymerase chain reaction, followed by an oligonucleotide ligation assay (PCR/OLA) and finally a sequence coded separation. It allows the detection of up to 31 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Guthrie spots were used as a source of template DNA. Results 144 carriers were detected during the analysis of 4476 samples, which translates into a carrier frequency of 1/31.1. Forty two carriers were detected from 1341 samples in Pavia (1/31.9), 53 from 1574 in Turin (1/29.7), and 49 from 1561 in San Giovanni Rotondo (1/31.8). Fifteen different mutations were detected, the most common being ΔF508 (0.625). Other common mutations included G542X (16 of 144), which was particularly common in southern Italy (14 of 49), N1303K (8 of 144), and R117H (8 of 144), detected only in the northern centres. Conclusions PCR/OLA is a robust, accurate, user friendly method for cystic fibrosis screening of newborns using blood spots in a semiautomated way at a low cost per mutation (0.8 Euro).

Ataxia is a frequently reported symptom in prion diseases (PD) and it is characteristic of Gerstmann-Straussler-Scheinker syndrome (GSS), a genetic PD mainly related to the P102L mutation in the PRNP gene. Our aim was to screen for the P102L and other six known PRNP gene mutations (P105L, A117V, Y145X, E200K, D202N, and V210I) a group of 206 consecutive patients diagnosed with adult-onset cerebellar ataxia of unknown origin. The patients, negative for the most common acquired and genetic forms, were analyzed using a combination of restriction endonuclease digestion and pyrosequencing; eight, affected by ataxia and cognitive dysfunction, were also sequenced for the PRNP gene. One patient resulted to be heterozygous for the P102L mutation. Retrospectively, the clinical picture was consistent with a "classical" GSS phenotype. In conclusion, the screening for the P102L mutation, or even the sequencing of the PRNP gene should be taken in consideration in patients with late-onset ataxia (>50 years).

The presence of fetal DNA in maternal plasma can be exploited to develop new procedures for non-invasive prenatal diagnosis. Tests to detect 7 frequent beta-globin gene mutations in people of Mediterranean origin were applied to the analysis of maternal plasma in couples where parents carried different mutations. A mutant enrichment amplification protocol was optimized by using peptide nucleic acids (PNAs) to clamp maternal wild-type alleles. By this approach, 41 prenatal diagnoses were performed by microelectronic microchip analysis, with total concordance of results obtained on fetal DNA extracted from chorionic villi. Among these, 27/28 were also confirmed by direct sequencing and 4 by pyrosequencing.

A role for metal-mediated oxidative stress in the pathogenesis of amyotrophic lateral sclerosis (ALS) was proposed in 1994 in the first studies of familial ALS mutant superoxide dismutase 1, and interference with iron homoeostasis is now postulated.1 The HFE gene on chromosome 6 is a mean corpuscular haemoglobin class I-like molecule related to iron regulation. Mutations in the coding region cause hereditary haemochromatosis, a common autosomal recessive disorder of iron metabolism that leads to iron overload in adulthood. Recent reports on HFE mutations in ALS showed contradictory results. Two studies described a higher prevalence of the HFE mutations in ALS than in the control group, and one study did not find any difference between the patients with ALS and the control group.2–4 We analysed a series of Italian patients with ALS to investigate whether mutations in the HFE gene could represent a risk factor for ALS. A total of 149 sporadic Italian patients with ALS (mean (standard deviation (SD)) age 59.4 (9.7) years) according to El Escorial criteria for clinically definite or probable ALS were consecutively recruited to this study. Control samples were obtained from 168 healthy people, matched by age (difference of 5 years), sex and …

The aim of this work was to develop advanced and accessible protocols for noninvasive prenatal diagnosis of genetic diseases. We are evaluating different technologies for mutation detection, based on fluorescent probe hybridization of the amplified product and pyrosequencing, a technique that relies on the incorporation of nucleotides in a primer-directed polymerase extension reaction. In a previous investigation, we have already proven that these approaches are sufficiently sensitive to detect a few copies of a minority-mutated allele in the presence of an excess of wild-type DNA, In this work, in order to further enhance the sensitivity, we have employed a mutant enrichment amplification strategy based on the use of peptide nucleic acids (PNAs). These DNA analogues bind wild-type DNA, thus interfering with its amplification while still allowing the mutant DNA to become detectable. We have synthesized different PNAs, which are highly effective in clamping wild-type DNA in the beta-globin gene region, where four beta-thalassemia mutations are located (IVSI.110, CD39, IVSI.1, IVSI.6) plus HbS. The fluorescence microchip readout allows us to monitor the extent of wild-type allele inhibition, thus facilitating the assessment of the optimal PNA concentration.

Hereditary hemochromatosis (HH) is a disorder of iron metabolism that leads to iron overload in middle age and can be caused by homozygosity for the C282Y mutation in the HFE gene. Preliminary studies have estimated the frequency of this mutation at 0.5-1% in Italy, but this has not been verified on a large sample. We analyzed 1,331 Italian newborns for the C282Y mutation in the HFE gene using dried blood spots (DBS) from the Neonatal Screening Center in Turin, Italy. The mutation was assessed using a semi-automatable 5'-nuclease assay (TaqMan technology). We detected 55 heterozygotes and no homozygotes in our sampling, resulting in an overall frequency of 2.1% +/- 0.6 for the C282Y allele. Differences in allele frequency were observed, and ranged from 2.7% +/- 1.3 in samples from Northern Italy, to 1.7% +/- 0.9 in samples from Central-Southern Italy. The low frequency of the at-risk genotype for iron overload suggests that genetic screening for HFE in Italy would not be cost effective. The present study, in addition to defining C282Y frequency, documents detection of the major HFE mutation on routine DBS samples from neonatal screening programs using a semi-automatable, rapid, reliable, and relatively inexpensive approach.

Hereditary Hemochromatosis (HC) is the primary form of iron overload, due to a genetic defect of iron absorption, which is especially frequent in Caucasians. Affected subjects may develop in midlife liver cirrhosis, hearth failure and arrhytmias, diabetes and other endocrinopathies, skin pigmentation, arthropathies and increased susceptibility to hepatocellular carcinoma. Since excess iron may be effectively removed by phlebotomies, early diagnosis is important to prevent disease complications (1,2). HC is the most common autosomic recessive disorder in Northern-European populations (3). The frequency of homozygotes approximates 3–5 per 1000 (1–4). Clinical symptoms occur more frequently in males than in females. The latter, although genetically affected, may not express the disease during the fertile age due to physiological iron losses.

We evaluate the relation between genotype and phenotype in 47 Italian male patients with homozygous genetic hemochromatosis (GH). Phenotype evaluation was based on the ratio of amount of iron removed (IR) by phlebotomy and age (IP/age). Patients were divided in two classes of phenotype expression: class I included 26 patients with less severe iron overload (IR/age ≤0.33) and class II included 21 patients with a more marked one (IR/age >0.33). Genetic variability was assessed by haplotype analysis combining alleles at HLA-B, D6S265, HLA-A, and D6S105 loci. A common ancestral haplotype carrying D6S265-1, HLA-A3, and D6S105-8 alleles was present in 13 of 52 (25%) chromosomes in class I and in 24 of 42 (57%) chromosomes in class II (P = .0027). Homozygotes and heterozygotes for the ancestral haplotype had higher iron indices than patients carrying two haplotypes other than the ancestral one. Seven of the eight patients homozygous for the ancestral haplotype were in class II, heterozygotes were equally distributed between the two classes, whereas 14 of 18 carriers of other haplotype combinations were in class I. Our results suggest that the gene defect linked to the ancestral haplotype is the result of a single, severe mutation. The high variability of phenotype expression in heterozygotes for the ancestral haplotype could be accounted for the contribution of the mutation carried by the second haplotype. Combination of different mutations could be responsible for the variable degrees of iron overload found in patients with GH.