C. Le Maréchal

A NOVEL DFNB1 DELETION ALLELE SUPPORTS THE EXISTENCE OF A DISTANT CIS-REGULATORY REGION THAT CONTROLS GJB2 AND GJB6 EXPRESSION

Wilch E, Azaiez H, Fisher RA, Elfenbein J, Murgia A, Birkenhäger R, Bolz HJ, da Silva‐Costa SM, del Castillo I, Haaf T, Hoefsloot L, Kremer H, Kubisch C, Le Marechal C, Pandya A, Sartorato EL, Schneider E, Van Camp G, Wuyts W, Smith RJH, Friderici KH. A novel DFNB1 deletion allele supports the existence of a distant cis‐regulatory region that controls GJB2 and GJB6 expression.

Mitochondrial DNA A3243G mutation involved in familial diabetes, chronic intestinal pseudo-obstruction and recurrent pancreatitis.

AIMS To report on a family with five members who carry the A3243G mutation in mitochondrial tRNA for leucine 1 (MTTL1) and present with diabetes, chronic intestinal pseudo-obstruction (CIPO) and recurrent pancreatitis, and to screen for this mutation in a cohort of 36 unrelated patients with recurrent pancreatitis. METHODS The mutation was quantified in several tissue samples from patients. Respiratory chain activity was studied in muscle biopsies and fibroblast cultures. In addition, the thymidine phosphorylase gene (TP) involved in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) and three genes involved in chronic pancreatitis - PRSS1, SPINK1 and CFTR - were sequenced in affected patients. Finally, the MTTL1 gene was examined in 36 unrelated patients who had recurrent pancreatitis, but no mutations in the PRSS1 and SPINK1 genes. RESULTS Heteroplasmy for the mtDNA A3243G mutation was found in all tissue samples from these patients, but no mutations were found in the genes coding for thymidine phosphorylase, PRSS1, SPINK1 and CFTR. Also, none of the 36 unrelated patients with recurrent pancreatitis were carrying any MTTL1 mutations. CONCLUSION The mtDNA A3243G mutation associated with the gastrointestinal manifestations observed in the affected family should be regarded as a possible cause of CIPO and unexplained recurrent pancreatitis. However, the mutation is probably only weakly involved in cases of isolated recurrent pancreatitis.

The experience of extended blood group genotyping by next-generation sequencing (NGS): investigation of patients with sickle-cell disease.

Patients suffering from haemoglobinopathies may be treated by red blood cell (RBC) transfusion on a regular basis and then exposed to multiple antigens with a recurrent, potential risk of alloimmunization routinely prevented by extended RBC antigen cross‐matching. While time‐consuming and labour‐intensive serological analyses are the gold standard for RBC typing, genotyping by current high‐throughput molecular tools, including next‐generation sequencing (NGS), appears to offer a potent alternative.

Copy number variations in chronic pancreatitis

In 1996, shortly after a locus for hereditary pancreatitis had been mapped to chromosome 7q35, an apparent gain-of-function missense mutation, p.R122H, in the cationic trypsinogen gene (PRSS1) was identified. Thereafter, the search for chronic pancreatitis-associated genetic factors has been largely focused on one form of genetic variation, namely, single nucleotide substitutions (SNSs). Only very recently has another type of genetic variation – copy number variations (CNVs) – been found to cause the disease. First, we identified duplication and triplication of an ∼605 kb segment on chromosome 7q35 in French white patients with hereditary or idiopathic chronic pancreatitis. These alterations increased the copy number of PRSS1 as well as PRSS2, which encodes anionic trypsinogen. Second, we characterized a hybrid trypsinogen gene, in which exons 1 and 2 were derived from PRSS2 and exons 3 to 5 from PRSS1. Interestingly, this hybrid gene had two independent gain-of-function effects: increased trypsinogen gene copy number and it contained the p.N29I pancreatitis-causing missense mutation. Lastly, we identified two loss-of-function copy number mutations (deletions) in the SPINK1 gene, which encodes pancreatic secretory trypsin inhibitor (PSTI). Particularly, in one family with chronic pancreatitis, deletion of the complete SPINK1 gene was co-inherited with a CFTR missense mutation (p.L997F), revealing another layer of complexity between CNV and SNS interactions in the determination of a given disease phenotype. These findings represent a further demonstration of how studies of CNVs have altered the landscape of genetic research in the past few years and offer a fresh glimpse into the exciting realm of human CNVs.

Next‐generation sequencing for blood group genotyping

In the field of immunohematology, blood group genotyping has become a very common approach for routine purpose to complement the gold standard serological studies, and to further guarantee the safety of patients. It has more recently explored the major advances in the available technologies for molecular analysis, including next‐generation sequencing (NGS). NGS, known as second‐generation technologies in its current format, has revolutionized the field of molecular genetics for the past decade. For the very recent years, a few pilot studies using various commercial platforms have paved the way towards blood group genotyping by NGS in both transfusion and obstetric medicine. Advantages and limitations have been reported, and future trends for improvements have been clearly identified. Third‐generation sequencing, which is interestingly characterized by the production of long reads that have been shown to cover up to several dozen kilobases, has also become available in the meantime. This novel technology shows great promises for clinical applications in the very next future, most importantly by resolving the challenging issue of characterizing gene conversion events occurring in genes of the Rh and MNS blood group systems. By considering recent and future advances in these technologies and their related bioinformatics resources, it is tempting to suggest that a routine, NGS‐based strategy will be soon available for blood group genotyping with high accuracy at a low cost. Clinical utility and clinical applications of this technology remain to be evaluated.

Exercise testing‐based algorithms to diagnose McArdle disease and MAD defects

As exercise intolerance and exercise‐induced myalgia are commonly encountered in metabolic myopathies, functional screening tests are commonly used during the diagnostic work‐up. Our objective was to evaluate the accuracy of isometric handgrip test (IHT) and progressive cycle ergometer test (PCET) to identify McArdle disease and myoadenylate deaminase (MAD) deficiency and to propose diagnostic algorithms using exercise‐induced lactate and ammonia variations.

Weak D type 1, 2 and 3 subtype alleles are rare in the Western French population

Dear Sir, In the complex and polymorphic Rh blood group system, expression of the D antigen, carried by the transmembrane protein RhD, is of major clinical interest in both transfusion and obstetric medicine. Among the multiple rare variant alleles in the RHD gene reported so far in the Rhesus Base reference database (Wagner & Flegel, 2014), the missense weak D type 1, 2 and 3 alleles, which are characterised by single-nucleotide polymorphisms (SNPs) c.809T>G, c.1154G>C and c.8C>G, respectively (Wagner et al., 1999), have long been known to be the most common variant alleles in the Caucasian population, with a cumulative frequency typically >60%. Caucasian population-specific strategies have therefore been implemented by European and Australian immunohematology laboratories to screen these alleles in priority. Approaches include commercial methods based on technologies such as real-time polymerase chain reaction (PCR) or microarray, as well as in-house-developed tests. Since the discovery of the molecular basis of these weak D alleles (Wagner et al., 1999), other rare alleles on the same respective background, namely ‘subtype’ alleles, which are characterised respectively by additional single-nucleotide variations in the RHD gene, have been reported (Table 1). As little is known about (i) the frequency of the subtypes in the French population and (ii) the risk of alloimmunisation in weak D recipients carrying those alleles, we thought about genotyping specifically the subtype variants in weak D type 1, 2 and 3 allele carriers. The main purpose of this work is to provide molecular data about the distribution of the subtype variant alleles in the Western French population, with the subsequent aim of adapting the routine genotyping strategy based on the output data, if necessary. Subsets of consecutive weak D type 1 (n= 256), 2 (257) and 3 (32) samples referred to our laboratory between 2012 and 2014 and previously characterised by a simple routine approach (Fichou et al., 2013) were selected for the study. RHD exons (1, 3 and 5 in weak D type 1 samples; 2 and 6 in weak D type 2 samples; and 2 in weak D type 3 samples) were PCR-amplified and sequenced with primers described previously in the same conditions (Fichou et al., 2013). Sequencing data were aligned and analysed with Sequencher v5.0 (Gene Codes Corporation, Ann Arbor, MI, USA). In the samples genotyped as weak D type 1, only the RHD(V238L, V270G) allele, which was first described in the