Novel SCN9A variant associated with congenital insensitivity to pain.

BACKGROUND: Congenital insensitivity to pain (CIP) is a rare autosomal recessive syndrome characterized by lack of pain perception and a wide spectrum of clinical signs such as anosmia and hyposmia. Variants in SCN9A gene are associated with CIP. We here report on a Lebanese family with three CIP patients referred for genetic investigations. METHODS AND RESULTS: Whole exome sequencing analysis revealed the presence of a novel nonsense, homozygous SCN9A pathogenic variant: SCN9A (NM_001365536.1): c.4633G > T, p.(Glu1545*) in exon 26. CONCLUSION: Our three Lebanese patients had CIP, urinary incontinence and normal olfactory function while two of them also presented with osteoporosis and osteoarthritis; this association of features has not been previously reported in the literature. We hope that this report would contribute to a better delineation of the phenotypic spectrum associated with SCN9A pathogenic variants.

BBS10 encodes a vertebrate-specific chaperonin-like protein and is a major BBS locus.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous ciliopathy. Although nine BBS genes have been cloned, they explain only 40-50% of the total mutational load. Here we report a major new BBS locus, BBS10, that encodes a previously unknown, rapidly evolving vertebrate-specific chaperonin-like protein. We found BBS10 to be mutated in about 20% of an unselected cohort of families of various ethnic origins, including some families with mutations in other BBS genes, consistent with oligogenic inheritance. In zebrafish, mild suppression of bbs10 exacerbated the phenotypes of other bbs morphants.

Ex vivo PBMC cytokine profile in familial Mediterranean fever patients: Involvement of IL-1ß, IL-1α and Th17-associated cytokines and decrease of Th1 and Th2 cytokines.

In order to clarify the inflammatory mechanism underlying familial Mediterranean fever (FMF), we aimed to evaluate the ex vivo cytokine profile of FMF patients during acute attacks and attack-free periods, and compare it with that of healthy controls. The study included 34 FMF patients, of whom 9 were studied during attack and remission and 24 healthy controls. Cytokine levels were evaluated by Luminex technology in serum and supernatants of PBMC (Peripheral Blood Mononuclear Cells) cultures with and without 24h stimulation of monocytes by LPS and T lymphocytes by anti-CD3/CD28 beads. Levels of IL-6 and TNF-α were higher in unstimulated and LPS-stimulated PBMC supernatants of FMF patients in crises compared to controls. In response to LPS stimulation, higher levels of IL-1ß and IL-1α were found in PBMC supernatants of patients during crises compared to those in remission and to controls. IFN-γ and IL-4 levels were the lowest in unstimulated and anti-CD3/CD28 stimulated PBMCs supernatants of patients during crises compared to remission and controls. The Th17 cytokines IL-17 and IL-22 were respectively higher in anti-CD3/CD28 stimulated PBMC supernatants of FMF patients during and between crises compared to controls. Amongst cytokines tested in serum, only IL-6 and TNFα were enhanced in FMF patients. The ex vivo study represents an interesting approach to evaluate cytokines' involvement in FMF. Our results suggest an ongoing subclinical inflammation and define an elevated inflammatory cytokine signature, distinctly for M694V homozygous patients. The absence of spontaneous IL-1ß release by PBMCs reflects no constitutive activation of the inflammasome in FMF physiopathology.

A homozygous missense variant in PTPN2 with early-onset Crohn's disease, growth failure and dysmorphic features in an infant: a case report.

Crohn's disease (CD) is a chronic idiopathic inflammatory bowel condition that can affect any part of the gastrointestinal tract. Several hundred candidate loci or genes including PTPN2 have been reportedly associated with CD. A whole-exome sequencing (WES) was conducted in a 9-year-old Lebanese girl with a CD onset at 13 months and in both her asymptomatic parents. The analysis detected an extremely rare homozygous variant in PTPN2: c.359C>T, p.(Ser120Leu) in the patient, while both her parents were heterozygous. This variant, located in the protein tyrosine phosphatase (PTP) domain within a highly conserved amino acid, is classified as VUS according to the American College of Medical Genetics (ACMG) criteria. To evaluate the hypothetical functional consequences of the identified variant, a quantitative expression analysis of PTPN2 was performed in blood tissues of the patient, her parents, and two healthy controls. PTPN2 expression was not noted in the patient compared to her parents and the normal controls, suggesting a functional PTPN2 impairment caused by c.359C>T. This variant c.359C>T, p.(Ser120Leu) in PTPN2 has never been previously described in the literature. Our report suggests an association of PTPN2: c.359C>T with early-onset CD.

Analysis of ASS1 gene in ten unrelated middle eastern families with citrullinemia type 1 identifies rare and novel variants.

BACKGROUND: Citrullinemia type 1 (CTLN1) is a rare autosomal recessive disease caused by argininosuccinate synthetase (ASS) deficiency. Manifestations vary from the acute neonatal or "classic" form to a milder, late-onset, or "unconventional" form. To date, more than 93 variants in the ASS1 gene located on chromosome 9q43.11 (OMIM #215700) are reportedly responsible for CTLN1. Their incidence and distribution vary according to geographic origins and ethnicity, and a correlation, although not clearly delineated, has been established between the genotype and the phenotype of the disease. Though, in the Middle East, national descriptions of CTLN1 are still lacking. METHODS: A total of ten unrelated Middle Eastern families, five Lebanese, two Syrians, and three Iraqis with citrullinemia index cases, were included in this study. Upon informed consent, DNA was extracted from the whole blood of the index patients as well as their parents and siblings. Genetic analysis was carried out by Sanger sequencing of the ASS1 gene. RESULTS: Seven different variants were identified. Two novel variants, c.286C>A (p.(Pro96Thr), RNA not analyzed) in exon 5 and deletion c.685_688+6del(p.(Lys229Glyfs*4), RNA not analyzed) in exon 10, were found in one Lebanese and one Syrian family, respectively, and were correlated with early-onset and severe clinical presentation. Five other known variants: c.535T>C (p.(Trp179Arg), RNA not analyzed) in exon 8, c.787G>A (p.(Val263Met), RNA not analyzed) in exon 12, c.847G>A (p.(Glu283Lys), RNA not analyzed) in exon 13, c.910C>T (p.(Arg304Trp), RNA not analyzed) in exon 13, and c.1168G>A (p.(Gly390Arg), RNA not analyzed) in exon 15, were found in Lebanese, Syrian, and Iraqi families, and were associated with diverse clinical presentations. CONCLUSION: Two novel variants and five known variants were found in a total of ten unrelated Middle Eastern families.

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

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

The molecular basis of familial hypercholesterolemia in Lebanon: spectrum of LDLR mutations and role of PCSK9 as a modifier gene.

Autosomal dominant hypercholesterolemia (ADH), a major risk for coronary heart disease, is associated with mutations in the genes encoding the low-density lipoproteins receptor (LDLR), its ligand apolipoprotein B (APOB) or PCSK9 (Proprotein Convertase Subtilin Kexin 9). Familial hypercholesterolemia (FH) caused by mutation in the LDLR gene is the most frequent form of ADH. The incidence of FH is particularly high in the Lebanese population presumably as a result of a founder effect. In this study we characterize the spectrum of the mutations causing FH in Lebanon: we confirm the very high frequency of the LDLR p.Cys681X mutation that accounts for 81.5 % of the FH Lebanese probands recruited and identify other less frequent mutations in the LDLR. Finally, we show that the p.Leu21dup, an in frame insertion of one leucine to the stretch of 9 leucines in exon 1 of PCSK9, known to be associated with lower LDL-cholesterol levels in general populations, is also associated with a reduction of LDL-cholesterol levels in FH patients sharing the p.C681X mutation in the LDLR. Thus, by studying for the first time the impact of PCSK9 polymorphism on LDL-cholesterol levels of FH patients carrying a same LDLR mutation, we show that PCSK9 might constitute a modifier gene in familial hypercholesterolemia.

FokI vitamin D receptor gene polymorphism and serum 25-hydroxyvitamin D in patients with cardiovascular risk.

INTRODUCTION: The biological actions of vitamin D are mediated through vitamin D receptor (VDR). Numerous single-nucleotide polymorphisms (SNPs) in the VDR gene have been identified, and some have been associated with cardiovascular disease (CVD) risk factors. This study aims to evaluate the association of five SNPs in the VDR gene with 25-hydroxyvitamin D (25[OH]D) levels in patients with at least one CVD risk factor. MATERIAL AND METHODS: Genomic DNA was sequenced using standard Sanger methods for five VDR SNPs (BsmI rs1544410; ApaI rs7975232; Cdx2 rs11568820; TaqI rs731236; FokI rs2228570) in 50 Mediterranean subjects having hypovitaminosis D with at least one documented CVD risk factor, aged 18 years or more. The collected variables were serum levels of (25[OH]D), HbA1c, fasting plasma glucose, triglycerides, LDL cholesterol, and total cholesterol. RESULTS: BsmI, ApaI, and TaqI were moderately to highly intercorrelated. Cdx2 was less frequent than expected. With respect to the number of mutations in FokI, levels of (25 [OH]D) were 11.2 ±5.5 ng/ml in the absence of mutations, 12.6 ±4.7 ng/ml in the presence of one mutation, and 16.5 ± 5.5 ng/ml in the presence of two mutations. CONCLUSIONS: FokI polymorphism is more frequent in subjects with cardiovascular risk factors than in the general Caucasian population.

The added value of WES reanalysis in the field of genetic diagnosis: lessons learned from 200 exomes in the Lebanese population.

BACKGROUND: The past few decades have witnessed a tremendous development in the field of genetics. The implementation of next generation sequencing (NGS) technologies revolutionized the field of molecular biology and made the genetic information accessible at a large scale. However, connecting a rare genetic variation to a complex phenotype remains challenging. Indeed, identifying the cause of a genetic disease requires a multidisciplinary approach, starting with the establishment of a clear phenotype with a detailed family history and ending, in some cases, with functional assays that are crucial for the validation of the pathogenicity of a mutation. METHODS: Two hundred Lebanese patients, presenting a wide spectrum of genetic disorders (neurodevelopmental, neuromuscular or metabolic disorders, etc.), sporadic or inherited, dominant or recessive, were referred, over the last three and a half years, to the Medical Genetics Unit (UGM) of Saint Joseph University (USJ). In order to identify the genetic basis of these diseases, Whole Exome Sequencing (WES), followed by a targeted analysis, was performed for each case. In order to improve the genetic diagnostic yield, WES data, generated during the first 2 years of this study, were reanalyzed for all patients who were left undiagnosed at the genetic level. Reanalysis was based on updated bioinformatics tools and novel gene discoveries. RESULTS: Our initial analysis allowed us to identify the specific genetic mutation causing the disease in 49.5% of the cases, in line with other international studies. Repeated WES analysis enabled us to increase the diagnostics yield to 56%. CONCLUSION: The present article reports the detailed results of both analysis and pinpoints the contribution of WES data reanalysis to an efficient genetic diagnosis. Lessons learned from WES reanalysis and interpretation are also shared.

Pitfalls of homozygosity mapping: an extended consanguineous Bardet-Biedl syndrome family with two mutant genes (BBS2, BBS10), three mutations, but no triallelism.

The extensive genetic heterogeneity of Bardet-Biedl syndrome (BBS) is documented by the identification, by classical linkage analysis complemented recently by comparative genomic approaches, of nine genes (BBS1-9) that account cumulatively for about 50% of patients. The BBS genes appear implicated in cilia and basal body assembly or function. In order to find new BBS genes, we performed SNP homozygosity mapping analysis in an extended consanguineous family living in a small Lebanese village. This uncovered an unexpectedly complex pattern of mutations, and led us to identify a novel BBS gene (BBS10). In one sibship of the pedigree, a BBS2 homozygous mutation was identified, while in three other sibships, a homozygous missense mutation was identified in a gene encoding a vertebrate-specific chaperonine-like protein (BBS10). The single patient in the last sibship was a compound heterozygote for the above BBS10 mutation and another one in the same gene. Although triallelism (three deleterious alleles in the same patient) has been described in some BBS families, we have to date no evidence that this is the case in the present family. The analysis of this family challenged linkage analysis based on the expectation of a single locus and mutation. The very high informativeness of SNP arrays was instrumental in elucidating this case, which illustrates possible pitfalls of homozygosity mapping in extended families, and that can be explained by the rather high prevalence of heterozygous carriers of BBS mutations (estimated at one in 50 in Europeans).

Heterozygous and homozygous mutations in PITX3 in a large Lebanese family with posterior polar cataracts and neurodevelopmental abnormalities.

PURPOSE: The PITX3 gene, which codes for a homeobox bicoidlike transcription factor is responsible for dominant cataract and anterior segment mesenchymal dysgenesis in humans. In the current study, a family with autosomal dominant posterior polar cataract (PPC) and a PITX3 mutation that cosegregates with the disease was examined. Also studied were two siblings who were homozygous for the PITX3 mutation who had microphthalmia and significant neurologic impairment. METHODS: A genome-wide screen, linkage analysis in the PITX3 chromosomal region 10q25, haplotype analysis, and sequencing of the PITX3 gene were performed on 28 affected and 14 unaffected member of a three-generation Lebanese family. RESULTS: Genome-wide linkage analysis showed a lod score of 3.56 at theta = 0.00 on chromosome 10 at area q25. Analysis of the haplotypes and phenotypes confined the disease locus to a region on 10q25 between the markers D10S1239 and D10S1268. A candidate gene, PITX3, maps to that region. Sequencing of the PITX3 gene revealed a heterozygous G deletion mutation in 25 of the 42 family members. In addition, two siblings from a consanguineous marriage were found to be homozygous for the deletion. CONCLUSIONS: This is the first report of homozygous PITX3 mutations in humans. The phenotype in these individuals highlights the role of PITX3 in ocular and central nervous system (CNS) development.

Familial Mediterranean fever in the Syrian population: gene mutation frequencies, carrier rates and phenotype-genotype correlation.

BACKGROUND: Familial Mediterranean fever (FMF) is an autosomal recessive disease mainly affecting particularly Arabs, Non-Ashkenazi Jews, Armenians, and Turks. It is an autoinflammatory periodic disorder characterized by febrile and painful attacks due to inflammation involving the serosal membranes in the abdomen, chest or joints. Over 50 mutations have been identified in the MEFV gene responsible for FMF. OBJECTIVE: To identify the distribution and the frequency of the MEFV gene mutations in Syrian FMF patients and population and perform a genotype/phenotype correlation in the patients' cohort. PATIENTS AND METHODS: The study was carried out on 83 clinically diagnosed Syrian FMF patients and 242 healthy subjects. The tested individuals were screened for the most common five MEFV mutations (M694V, M694I, M680I, V726A and E148Q) by restriction fragment length polymorphism. Sequencing of exon 10 was performed only for the patients' DNA where just one or no mutation was detected. RESULTS AND DISCUSSION: Of the 83 patients studied, 74 (89%) were positive either for one, two or three mutations and nine (11%) had no mutations detected. Of those positive for mutations, 25 were homozygous, 30 were compound heterozygotes, three had complex alleles, and 16 patients had only one mutation. The M694V, V726A, M694I, M680I and E148Q mutations accounted for 45.8%, 26%, 13.9%, 4.8% and 6% of the alleles, respectively. The carrier rate in the Syrian population for the tested mutations was 17.5%, E148Q being the most common mutation, followed by V726A and M694V. The severity of the disease and development of amyloidosis seem to have an association with M694V, the most common mutation in Syrian FMF patients.

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

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

Contribution of copy number variants (CNVs) to congenital, unexplained intellectual and developmental disabilities in Lebanese patients.

BACKGROUND: Chromosomal microarray analysis (CMA) is currently the most widely adopted clinical test for patients with unexplained intellectual disability (ID), developmental delay (DD), and congenital anomalies. Its use has revealed the capacity to detect copy number variants (CNVs), as well as regions of homozygosity, that, based on their distribution on chromosomes, indicate uniparental disomy or parental consanguinity that is suggestive of an increased probability of recessive disease. RESULTS: We screened 149 Lebanese probands with ID/DD and 99 healthy controls using the Affymetrix Cyto 2.7 M and SNP6.0 arrays. We report all identified CNVs, which we divided into groups. Pathogenic CNVs were identified in 12.1% of the patients. We review the genotype/phenotype correlation in a patient with a 1q44 microdeletion and refine the minimal critical regions responsible for the 10q26 and 16q monosomy syndromes. Several likely causative CNVs were also detected, including new homozygous microdeletions (9p23p24.1, 10q25.2, and 8p23.1) in 3 patients born to consanguineous parents, involving potential candidate genes. However, the clinical interpretation of several other CNVs remains uncertain, including a microdeletion affecting ATRNL1. This CNV of unknown significance was inherited from the patient's unaffected-mother; therefore, additional ethnically matched controls must be screened to obtain enough evidence for classification of this CNV. CONCLUSION: This study has provided supporting evidence that whole-genome analysis is a powerful method for uncovering chromosomal imbalances, regardless of consanguinity in the parents of patients and despite the challenge presented by analyzing some CNVs.

Non-syndromic recessive deafness in Jordan: mapping of a new locus to chromosome 9q34.3 and prevalence of DFNB1 mutations.

Non-syndromic recessive deafness (NSRD) is the most commonly encountered form of hereditary hearing loss. The majority of NSRD cases in the Mediterranean area are linked to the DFNB1 locus (the connexin 26 GJB2 gene). Unrelated NSRD patients issued from 68 Jordanian families, were tested for mutations of the GJB2 gene by sequencing. Sixteen per cent of the families tested were linked to the DFNB1 locus. The 35delG was the only GJB2 mutation detected in these families. One of these families, presenting with four affected members and not linked to the gene, was subjected to a genome-wide search and was found to be mapped to 9q34.3 with a multipoint lodscore of 3.9. One candidate gene in the interval, coding for the chloride intracellular channel 3, CLIC3, was tested and excluded. The identification of a new NSRD locus, DFNB33, in one Jordanian family, shows the wide genetic heterogeneity that characterizes hearing impairment and the genetic diversity in Middle-Eastern populations.

Amyloidosis in familial Mediterranean fever patients: correlation with MEFV genotype and SAA1 and MICA polymorphisms effects.

BACKGROUND: Familial mediterranean fever (FMF) is a recessively inherited disease characterized by recurrent crises of fever, abdominal, articular and/or thoracic pain. The most severe complication is the development of renal amyloidosis. Over 35 mutations have been discovered so far in the gene responsible for the disease, MEFV. This article aims at determining a correlation between the MEFV genotype and the occurrence of amyloidosis in FMF patients, in addition to the study of the modifying effects of the SAA1 (type 1 serum amyloid A protein) and MICA (Major Histocompatibility Complex (MHC) class-I-chain-related gene A) genes on this severe complication. METHODS: Fourteen MEFV mutations were screened and the SAA1 and MICA polymorphisms tested in 30 FMF patients with amyloidosis and 40 FMF patients without amyloidosis. RESULTS: The M694V and V726A allelic frequencies were, respectively, significantly higher and lower in the group with amyloidosis, compared to the control FMF group. The beta and gamma SAA1 alleles were more frequently encountered in the group without amyloidosis, whereas the alpha allele was significantly more observed in FMF patients with amyloidosis (p < 0.025). All the MICA alleles were encountered in both patients' groups, but none of them was significantly associated with amyloidosis. CONCLUSIONS: The results suggest a protective effect of the SAA1 beta and gamma alleles on the development of amyloidosis and show the absence of a MICA modifying effect on amyloidosis development. Testing these polymorphisms on a larger sample will lead to more definite conclusions.

[von Hippel-Lindau syndrome: molecular diagnosis of two Lebanese families and analysis of the genotype-phenotype correlation]. / La maladie de von Hippel-Lindau: Etude moléculaire de deux families libanaises et analyse de la corrélation génotype-phénotype.

The von Hippel-Lindau syndrome (VHL) is a dominantly transmitted hereditary disorder associating multisystemic tumors affecting mainly the central nervous system, the kidneys, the pancreas, as well as pheochromocytomas. Mutations of the tumor suppressor gene VHL on chromosome 3 are responsible for the disease. This article reports for the first time the study of two Lebanese VHL affected families, presenting particularly hemangioblastomas of the central nervous system. Two different mutations of the VHL gene, S65W and F76S, respectively identified in the two families, confirmed the clinical diagnosis of the patients. Molecular diagnosis was then performed for at risk members of these families. This article reveals the importance of molecular diagnosis for suspected patients and of presymptomatic diagnosis for at risk members, especially that a close follow-up of carriers allows an early detection of tumors and prevents the metastasis stage, the most common cause of death of these patients.

Prevalance of BRCA1 and BRCA2 mutations in familial breast cancer patients in Lebanon.

Breast cancer is the most prevalent malignancy in women in Western countries, currently accounting for one third of all female cancers. Familial aggregation is thought to account for 5-10 % of all BC cases, and germline mutations in BRCA1 and BRCA2 account for less of the half of these inherited cases. In Lebanon, breast cancer represents the principal death-causing malignancy among women, with 50 % of the cases diagnosed before the age of 50 years.In order to study BRCA1/2 mutation spectra in the Lebanese population, 72 unrelated patients with a reported family history of breast and/or ovarian cancers or with an early onset breast cancer were tested. Fluorescent direct sequencing of the entire coding region and intronic sequences flanking each exon was performed.A total of 38 BRCA1 and 40 BRCA2 sequence variants were found. Seventeen of them were novel. Seven confirmed deleterious mutations were identified in 9 subjects providing a frequency of mutations of 12.5 %. Fifteen variants were considered of unknown clinical significance according to BIC and UMD-BRCA1/BRCA2 databases.In conclusion, this study represents the first evaluation of the deleterious and unclassified genetic variants in the BRCA1/2 genes found in a Lebanese population with a relatively high risk of breast cancer.

Familial Mediterranean fever in a large Lebanese family: multiple MEFV mutations and evidence for a Founder effect of the p.[M694I] mutation.

Familial Mediterranean fever (FMF) is an autoinflammatory autosomal recessive disease characterized by recurrent fever crises and serous inflammation. The MEFV gene responsible for the disease was identified on chromosome 16, and 5 of the mutations discovered so far in the gene are most frequently encountered in FMF patients: p.[M694V], p.[V726A], p.[M680I] and p.[M694I] in exon 10, and p.[E148Q] in exon 2. The present work describes multiple MEFV mutations and the corresponding haplotypes for 31 FMF patients as well as 32 "healthy" individuals of a large consanguineous Lebanese family. The DNAs were screened for MEFV mutations, and determination of the corresponding haplotypes was performed for all individuals by genotyping 4 microsatellites surrounding the gene. Five different mutations were detected in this one family, which is unexpected in such a genetic isolate. A phenotypic variability was also observed. The haplotype carrying the p.[M694I] allele, detected in all the family branches, was well conserved and therefore seems to be the ancestral one.